Annual Review of Immunology Volume 26 2008

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Annual Review of Immunology

Annu. Rev. Immunol. 2008.26:1-28. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:1-28. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Contents

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Doing What I Like

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K. Frank Austen Department of Medicine, Harvard Medical School, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, Massachusetts 02115; email: [email protected]

Annu. Rev. Immunol. 2008. 26:1–28

Key Words

First published online as a Review in Advance on September 17, 2007

slow reacting substance of anaphylaxis, mast cell, alternative complement pathway, cysteinyl leukotrienes, mast cell progenitors

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090339 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0001$20.00

Abstract I have spent my entire professional life at Harvard Medical School, beginning as a medical student. I have enjoyed each day of a diverse career in four medical subspecialties while following the same triad of preclinical areas of investigation—cysteinyl leukotrienes, mast cells, and complement—with occasional translational opportunities. I did not envision a career with a predominant preclinical component. Such a path simply evolved because I chose instinctively at multiple junctures to follow what proved to be propitious opportunities. My commentary notes some of the highlights for each area of interest and the mentors, collaborators, and trainees whose counsel has been immensely important at particular intervals or over an extended period.

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EARLY CAREER DETERMINANTS

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In 1946, after I graduated from high school, I was hospitalized with paralytic poliomyelitis. Although I was spared respiratory involvement, I experienced a loss and gradual recovery of strength in my extremities. I vividly recall the lumbar puncture on admission, the hospital-based management of the muscular discomfort with “hot packs,” and exercising with my mother’s cooking weights after discharge. The hospitalization prevented my entry into Amherst College with my class but did lead to one spring and three summer semesters at Akron University. I concentrated on the physical sciences at Akron University and ﬁnished Amherst College on schedule. It was this illness that prompted me to envision a career in medicine. Thus, I set serious academic goals for my Amherst years, whereas during high school, my studies had been secondary to sports. I majored in both chemistry and biology, as biochemistry did not have department status. My honors thesis, entitled “The Structure and Synthesis of Certain Uracil Analogs,” was directed to development of dietary inhibitors of Tetrahymena geleii. My remembrances of my student years at Harvard Medical School (HMS) are highlighted by three very different events. One was a contact from the Admissions Committee via a professor of gross anatomy to ask whether the selection of my brother, W. Gerald Austen, a senior at MIT majoring in mechanical engineering, for the next entering class would negatively affect my academic performance. Gerry and I shared a dormitory room for the next three years and pooled our advance yearly tuition to purchase a car in the fall, which we sold in a timely manner in the spring. Our competitive natures maximized our individual performances. The second event was a rather complex episode in which I and some old friends from Amherst College acquired a sheep from the Biophysics Department late one evening, which we then

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slipped into the dormitory and introduced during intermission at the annual ﬁrst-year dance. At 3:00 am, a security ofﬁcer knocked on my door with an offer to forget what had occurred if I would quietly collect and return the sheep to its campus location. The third and most important remembrance relates to my exposure to Cliff Barger of the Physiology Department. He involved students in meaningful laboratory experimentation that required both technical skill and the ability to understand and analyze the observations being recorded. The experiments led students to an understanding of certain principles of renal physiology. Many years later, when I was preparing a presidential address to the American Association of Immunologists, I borrowed what I had learned from Dr. Barger about homeostasis. The talk, entitled “Homeostasis of Effector Systems Which Can Also Be Recruited for Immunologic Reactions,” noted the remarkable array of negative regulators for the inﬂammatory functions of the complement pathway as well as those for activated cell types in IgE-mediated reactions (1). In 1954, after completing medical school, I became one of 12 interns in internal medicine at the Massachusetts General Hospital (MGH). In contrast to my colleagues, I had never heard of the NIH, and thus, I was the only one to enter into the Berry Plan to meet my two years of obligated military service. At the end of my internship year, there was a major outbreak of poliomyelitis in the Boston area, and the two hospitals that conventionally accepted affected patients reached saturation. At that point, the MGH decided to ﬁll a major medical need by accepting these patients. As there was no one at the MGH with particular expertise in the management of patients with poliomyelitis, Dr. Walter Bauer, Chief of Medicine, asked two of the newly minted assistant residents, Jan Koch-Weser and me, to take responsibility for managing what he assumed would be a small polio service. In a relatively short time, the MGH had hundreds of admissions, and we became

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responsible for those patients cared for in iron lungs. No one else was permitted to write orders for these patients. Patients and their families were somewhat encouraged by the fact that I had had polio, whereas I was most distressed by the substantial mortality rate of individuals approximately my own age. While using positive pressure to force the ﬂow of air into the lungs of a patient with poliomyelitis in the interval when the linens were being changed in the iron lung, I noted an improvement in his skin color and blood pressure, suggesting better oxygenation. By comparing an arterial oxygen determination made with the patient inside the machine, which applied negative pressure to suck air in versus that during a positive pressure period, I noted that the patient had marked hypoxia while in the iron lung. This condition had not been recognized owing to the limited facial exposure and congestion from the collar around the patient’s neck when in the iron lung. By removing patients from the iron lung and breathing them with a positive pressure method, we prevented any further deaths from vascular collapse. As a result, I published a paper in the New England Journal of Medicine (NEJM ) (2) while a house ofﬁcer and learned that dogma based on existing knowledge could be incorrect. The dogma was that these patients died of midbrain damage from the virus, whereas the real cause was inadequate lung function. The latter was correctly termed a ventilation-perfusion pulmonary mismatch many years later by Jeffrey Drazen when he presented me with the Kober Medal from the American Association of Physicians (3). A second NEJM paper with two microbiologists focused on the intercurrent respiratory infections in bulbospinal poliomyelitis (4). While on the neurology service later in the year, I recognized that individuals with pulmonary insufﬁciency and CO2 necrosis had not only a disorder of consciousness but also a tremor, termed asterixis, and papilledema. This insight arose from my reading about hypoxia while on the polio service and led to another publication in the NEJM (5).

Dr. Bauer had arranged for me to be assigned to direct a Rheumatology Service at Walter Reed Army Hospital during my obligated military service. However, during basic training, my credentials were reviewed, and the decision at Fort Sam Houston based on my three NEJM publications was that I should proceed to the Walter Reed Army Institute of Research. I was immensely fortunate to be assigned to Elmer L. Becker, MD, PhD, who had discovered that the ﬁrst component of guinea pig complement was an enzyme with esterase activity. He introduced me to the discipline of immunology and to the view that immunologic reactions initiate biochemical events. At the same time, I was required to meet my second set of orders to the hospitalbased Rheumatology Service and studied the effects on patients of aspirin at a dose that uncoupled oxidative phosphorylation and raised the metabolic rate (6, 7). On returning to the MGH as a medical resident in l958, I received my ﬁrst NIH grant, which provided me with technical support to shift the studies begun with Dr. Becker to human serum (8). Dr. Becker provided the recommendation essential for acceptance into the immunology laboratory directed by John Humphrey at the National Institute for Medical Research in Mill Hill, England, and the NIH provided a fellowship. I decided to join that laboratory because I hoped to develop a program to learn how immunologic reactions initiated biochemical events that, in turn, had biologic implications. It occurred to me that the immunologic release of histamine from mast cells (MCs) might afford the in vitro model that I sought. John Humphrey was isolating rat MCs from the peritoneal cavity with density gradients, and his colleague, Walter Brocklehurst, was studying the antigeninduced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from sensitized guinea pig lung. That histamine release was a biochemical process rather than an exchange with external cations seemed likely based on the reported calcium and temperature dependence of this reaction. An www.annualreviews.org • Doing What I Like

MC: mast cell SRS-A: slow reacting substance of anaphylaxis

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unanticipated bonus was my introduction to SRS-A, a material that constricted guinea pig airways in an isolated heart-lung preparation and was not inhibited by antihistamines. I knew that antihistamines were efﬁcacious for treating allergic rhinitis but not bronchial asthma, and I had the instinctive feeling that SRS-A might be a mediator of airways constriction in bronchial asthma. Brocklehurst had developed the ﬁrst bioassay to measure both histamine and SRS-A in the diffusate of antigen-challenged guinea pig lung fragments. Histamine, which elicited distinct rapid contractions of guinea pig ileal smooth muscle in an organ bath, was measured ﬁrst. Then the ileal muscle was rendered unresponsive to histamine with an antihistamine, and the samples were reassessed for their content of SRS-A, which elicited a slow, progressive contraction. These studies led to three publications with Dr. Brocklehurst in the Journal of Experimental Medicine (9–11). The work with John Humphrey on isolated rat MCs resulted in a collaboration with another visitor, Herbert Rapp (12), who felt that I should have yet another fellowship to learn formal immunochemistry in the laboratory of Manfred Mayer at Johns Hopkins. In July 1961, I returned to the MGH to serve one year as Dr. Bauer’s chief medical resident. During that year, the physician responsible for the subspecialty of infectious diseases had a sabbatical, and Dr. Bauer assigned me the additional task of answering the consults in infectious diseases. He observed that I had had experience in managing microbial infections in patients with bulbospinal polio and understood the host through immunology. He added that Louis Weinstein, a distinguished infectious diseases clinician elsewhere in Boston, would make weekly rounds with me to facilitate my education and to advise on difﬁcult cases. After completing the chief residency, I had a period with Manfred Mayer and Herb Rapp in the Microbiology Department at Johns Hopkins during which we demonstrated the enzymatic activity of the ﬁrst complement component (C1) on one of its natu-

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C: complement (used only with numbers to indicate components)

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ral substrates, the second component (C2), by immunochemical techniques (13). This work was particularly rewarding since during my time with Elmer Becker, the Mayer group had openly doubted the acetyltyrosine esterase activity Becker had reported for C1. My departure from Hopkins after only six months was somewhat precipitous. Walter Bauer had entered into pulmonary failure from emphysema, and he asked me to share the responsibility for his care with his personal physician. To the extent that Dr. Bauer could talk when we unplugged the tracheotomy, he focused on issues of academic medicine, my career development, and my family. He left me his desk, which I treasure in my home. After three years as a faculty member of the Infectious Disease group, I was asked by Robert Ebert, Chairman of Medicine, to become chief of the Pulmonary Unit. The rationale was that I was Board-certiﬁed in allergy and immunology, had experience in infectious diseases, had some knowledge of pulmonary function from my polio duties, and had a laboratory interest pertinent to bronchial asthma. The Pulmonary Unit was housed in new facilities and appreciably increased our laboratory space. The space contained a new cyclotron, a machine I did not understand and never used, for a physician-scientist who never appeared. My new responsibilities to pulmonary medicine energized my interest in one human disease, bronchial asthma, and reinforced my goal to study SRS-A.

BECOMING A DEPARTMENT In 1966, when Robert Ebert was appointed dean of HMS, he suggested that my immunology program would beneﬁt if I moved closer to the HMS campus as physician-inchief of the Robert B. Brigham Hospital (RBBH), a small institution entirely committed to the subspecialty of rheumatology. By now, I had been clinically active in infectious diseases and in pulmonary diseases at the MGH and in rheumatology during my service at the Walter Reed Army Hospital,

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although my only subspecialty certiﬁcation was in allergy and immunology. I requested support to recruit two colleagues with different research backgrounds in immunology so that we could offer a coherent and broadly based training program. John R. David and I had been house ofﬁcers together at the MGH and had encountered each other again when we were postdoctoral fellows in England, albeit at different institutions. His expertise was in cell-mediated immunity, whereas mine was in antibody-mediated events. Peter Schur, who had also trained with Elmer Becker during his military service and then with Henry Kunkel, conducted clinical research, whereas John David and I were working with in vitro systems. The goal for the three of us in 1966 was to form a cohesive, yet technically diverse program through which to facilitate our individual research and improve our postdoctoral mentoring. The RBBH allocated all its research space to our program and had a patient base that allowed us to engage in clinical teaching as well as research. As John David, Peter Schur, and I not only shared common training experiences but also had the same goals for the department, its administrative structure was ﬂat. In the early 1970s, I was invited to take a chairmanship and historical chair of medicine at a school that at one time would have been my life’s goal. To my own astonishment, I made a decision that profoundly changed my career path. I found that my commitment to my personal research and my enjoyment of our developing department were so great that I preferred to remain at the RBBH. I would not again seriously consider becoming a chairman or accepting another major administrative task elsewhere. My decision to remain at the RBBH was driven by the knowledge that becoming a proper chairman would end my opportunity to unravel the aspects of the inﬂammatory response that seemed so intriguing and yet approachable. In 1974, our Rheumatology and Immunology program at the RBBH was granted departmental appointing status at HMS for ap-

proximately 20 years. This decision meant that promotions and appointments could be discussed directly with the HMS dean and further assured the development of our enterprise. Although we had signiﬁcant clinical and teaching responsibilities, they were elective. Thus, we had an optimal amount of time for research. However, as we were not a line department within the structure of HMS, we also had no budget from the school and were almost entirely responsible for our own funding. Thus, the department was dynamic and was supported almost entirely by the peer review process. We enjoyed a continued inﬂux of outstanding trainees, who progressed to faculty rank and then were recruited away at higher rank to ﬁne schools and teaching hospitals. We began to attract individuals with PhDs who were interested in the integrated biology of the inﬂammatory response. Over time, the MD faculty was enriched with fulltime clinical scholars and the laboratory faculty with PhD-trained investigators who often had received their postdoctoral training in the department. This combination of faculty improved our shared and ever-evolving technology and added to the quality of the training program. While at the MGH, I had begun to collaborate with Albert L. Sheffer in the study of patients with hereditary angioedema (HAE) due to a lack of a functional inhibitor of the ﬁrst component of complement (ClINH). After my appointment to the RBBH, we continued to study the roles of the complement system in HAE and the MC in patients with physical allergies. Indeed, the triad of complement, MC, and SRS-A research favored translational research in allergy and asthma over other medical applications. Thus, we added a training program in allergy and immunology and, in 1971, were funded as one of the seven initial Asthma and Allergic Diseases Centers conceived by the NIAID. At the same time, with the history and focus of the RBBH on the medical and surgical care of patients with arthritic diseases, we had a training grant in rheumatology www.annualreviews.org • Doing What I Like

HAE: hereditary angioedema

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and a Clinical Research Center supported by the NIAMD. HMS appointed me the ﬁrst incumbent of the Theodore Bevier Bayles Professorship in 1972; and in 1974, I was elected to the National Academy of Sciences and the American Academy of Arts and Sciences. In the background during the 1970s was the merger of the RBBH with the Peter B. Brigham Hospital (PBBH) and the Boston Hospital for Women (BHW). I chaired a committee composed of two physicians and one administrator from each of the hospitals to work out the new bylaws. There was also much to do in the community to acquire a Certiﬁcate of Need so that construction could proceed for what is now known as the Brigham and Women’s Hospital (BWH). Although our department beneﬁted greatly from being the focus of the RBBH, we sought the merger because of concern that a subspecialty hospital built on the need for in-patient care would not thrive with the changing paradigms and increasing complexity of medical and surgical care. As our three laboratories continued to expand, we began to appreciate that the beneﬁts from the cohesion of our setting were perhaps mitigated by its off-site location. Again, Dean Ebert played a key role in my life. He suggested that the RBBH and the BHW gift HMS the matching funds needed to build the Seeley G. Mudd Building on the HMS campus. The cost of our space would be discounted, and we would have state-of-theart new laboratories on the quadrangle adjacent to the new medical center encompassing the merged hospitals. Hence, in 1977, after 11 years at the RBBH, we moved to the campus of HMS. With the transfer of our clinical activities to the BWH, we retained our status as a separate appointing Department of Medicine at HMS, but for clarity we were designated as the Department of Rheumatology and Immunology within the BWH. In 1987, Dean Dan Tosteson funded the K. Frank Austen Professorship of Medicine with a gift from David A. Jones and the Humana Foundation. After a typical, somewhat

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long HMS search, Michael Brenner, MD, was appointed to the K. Frank Austen Professorship in 1991. In 1995, our department became a division of the Department of Medicine of HMS at the BWH with Dr. Brenner as chair. Over three decades, our program had grown from four full-time and seven parttime faculty members with ﬁve postdoctoral fellows to 55 full-time and 6 part-time faculty members with 45 fellows. Two years later, we moved from the Seeley G. Mudd Building to an expanded site in the Dana-Farber Cancer Institute. Michael Brenner added “Allergy” to the name of the division, and from 1995 onward I have focused on the formal development of that program with Al Sheffer. As I did previously for rheumatology, this effort has meant developing the needed laboratory-based and clinically oriented faculty directly from our training program. As the Bayles Chair was named for a distinguished RBBH rheumatologist, it was appropriate to transfer that chair to Dr. Brenner and for me to take a new chair directed to inﬂammation and respiratory diseases.

THE LABORATORY TRIAD: SRS-A/CYSTEINYL LEUKOTRIENES, MAST CELLS, AND COMPLEMENT As I was learning about SRS-A from Walter Brocklehurst and MCs from John Humphrey, Rodney Porter and others were unraveling the structure of antibody through protein chemistry. Perhaps because of my four years of training in internal medicine, I believed that interventions for the outcome or inﬂammatory side of immune injury would precede any development of therapies for the recognition side. As I also wished to maintain a clinical life, I felt that it would be best to work in areas relevant to a clinical question. Only later did I recognize the additional requirement that these questions needed to be addressed with state-of-the-art technology. Happily, because I entered these research areas at such an

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early point, I could add the needed technology in a workable time frame that also allowed for a clinical life. Indeed, the latter stimulated my bench research. The three inﬂammatory components—mast cells, cysteinyl leukotrienes, and complement—that retained my interest all intersect with one another, providing occasional insights into parts of an integrated host inﬂammatory response. Nonetheless, the Study Section that awarded me a 10-year merit-based grant included the message that I could not in the future aggregate three strong areas into a single RO1. Below are my observations on each area, from the early years to the present.

THE CYSTEINYL LEUKOTRIENES (SRS-A) We now know that SRS-A, an antihistamineresistant constrictor of smooth muscle that is released from antigen-challenged guinea pig and human lung, is a mediator of bronchial asthma. We also know that the substance originally termed SRS-A was in fact composed of three cysteinyl leukotrienes (cysLTs): the biosynthetic intracellular product, leukotriene C4 (LTC4 ), and its extracellular metabolites, LTD4 and LTE4 . A limited number of cell types including MCs, eosinophils, basophils, and monocyte/macrophages possess the full biosynthetic pathway for LTC4 . Arachidonic acid released from the outer nuclear membrane by cytosolic phospholipase A2 (cPLA2 /type IV PLA2 ) in the presence of 5-lipoxygenase (5-LO) activating protein (FLAP) is converted sequentially by 5LO into 5-hydroperoxy-eicosatetraenoic acid (5HPETE) and LTA4 . LTA4 is then conjugated to reduced glutathione (GSH) to form LTC4 by LTC4 synthase (LTC4 S), an integral outer nuclear membrane protein. After carrier-mediated export of LTC4 , the cleavage removal of glutamic acid and then glycine from the GSH moiety provides the metabolites, LTD4 and LTE4 . The functions of the cys-LTs, which extend well beyond their historical smooth muscle–constricting action on

airways and microvasculature to include a range of leukocyte responses, are mediated by two known receptors, the CysLT1 and CysLT2 receptors. The structural deﬁnition of SRS-A as a conjugate of a cysteine-containing peptide and a metabolite of arachidonic acid, termed LTC4 , by Robert (Bob) Murphy with Sven Hammarstrom and Bengt Samuelsson (14) in 1979 profoundly changed the direction of my work. Through collaboration with E.J. Corey of the Chemistry Department of Harvard University, we immediately had synthetic LTC4 and then its metabolites, LTD4 and LTE4 , for pharmacologic studies in animals and humans. The use of these products to standardize an analytic reverse phase–high performance liquid chromatography (RP-HPLC) system allowed the identiﬁcation of LTC4 , LTD4 , and LTE4 in complex biologic mixtures; the characterization of their extracellular inactivation; the puriﬁcation of native human LTC4 S from lung; and, most important, the construction of a novel assay for expression cloning of human LTC4 S. As we had previously addressed such areas as the cell sources and immunoglobulin (Ig) classes involved in SRS-A generation and the physicochemical characteristics (with Robert Murphy) and pharmacologic actions (with Jeffrey Drazen) of SRS-A, our studies with SRSA/cys-LTs can be divided into those before and those after the structure was known.

cys-LT: cysteinyl leukotriene LTC4 S: leukotriene C4 synthase CysLT1 and CysLT2 receptors: receptors for the cysteinyl leukotrienes PCA: passive cutaneous anaphylaxis

SRS-A My initial studies with Brocklehurst demonstrating various common biochemical requirements for the antigen-induced release of SRS-A and histamine from actively sensitized guinea pig lung (9–11) did not reveal the responsible Ig class or cellular source. With Kurt Bloch, we found that haptenspeciﬁc guinea pig IgG1, but not IgG2, sensitized guinea pig lung fragments for DNPBSA-elicited histamine and SRS-A release and also mediated a passive cutaneous anaphylaxis (PCA) reaction implicating the MC www.annualreviews.org • Doing What I Like

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(15). A second Ig class, rat IgE, was shown to passively sensitize the rat peritoneal cavity for DNP-BSA-elicited histamine and SRSA release, and that response was lost with MC depletion (16). Deﬁnitive studies linking MCs and IgE to SRS-A generation were conducted in monkey lung fragments, with Teruko and Kimishige Ishizaka, using isolated IgE myeloma or atopic serum for passive sensitization followed by challenge with anti-IgE (17). Rat IgGa, which also prepared the peritoneal cavity for antigen-induced SRS-A generation and was produced more easily than IgE, was used routinely to generate product for characterization (18). As agonists other than SRS-A contracted the guinea pig ileum in an antihistamine-resistant manner, we considered it critical to produce the material immunologically. Because solubility studies revealed SRS-A to have both polar and nonpolar characteristics, we recruited Bob Murphy from the Department of Chemistry at MIT to help with a structural analysis. Robert (Bob) Orange produced heroic amounts of SRS-A from hundreds of rats and conducted the chromatography, while Bob Murphy used gas chromatography, gas chromatographymass spectroscopy, and high-resolution mass spectroscopy to identify the contaminants remaining after each puriﬁcation step and to assess the ﬁnal product from their combination. Rat and human SRS-A behaved in an identical fashion, with the ﬁnal product being an acidic, low-molecular-weight (400–1400 MW) moiety bioactive at a nanogram level or less (19). Although the moiety was not suitable for conventional mass spectrometric analyses, spark source mass spectroscopy demonstrated the sulfur atom to be more abundant in the bioactive samples, a ﬁnding conﬁrmed by electron probe analysis (20). The inactivation of SRS-A by arylsulfatases but not by a range of other enzymes and the ability of SRS-A to inhibit the cleavage of synthetic substrates of the enzyme were puzzling since the electrophoretic mobility and isoelectric point of SRS-A did not suggest a sulfate residue. In

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retrospect, the triene structure of the cys-LTs was read as an aromatic ring in the inhibition studies of arylsulfatases by SRS-A, and a contaminant was responsible for the inactivation (21). Nonetheless, the partial puriﬁcation and characterization of SRS-A as a sulfur-enriched substance and the ﬁnding of Charles Parker’s group that labeled arachidonic acid could be incorporated into SRS-A (22) provided background for the generation of double-labeled SRS from mastocytoma cells stimulated with calcium ionophore. Indeed, Murphy and colleagues needed to break the sulfur bridge between the lipid backbone and peptide adduct to characterize the lipid component as a triene (14). In a study with Robert Lewis, another long-time colleague, we used the puriﬁcation scheme to identify the cellular biosynthesis of bioactive SRS-A before its release from human MCs (23). These ﬁndings predicted the export pathway for newly synthesized LTC4 described 15 years later. Human eosinophils loaded with LTA4 at 4◦ C formed but did not release LTC4 until the temperature was raised, thereby demonstrating an energy-dependent export pathway (24). When the effects of intravenously administered SRS-A, histamine, prostaglandin (PG) F2α, and bradykinin on lung function in guinea pigs were compared, SRS-A markedly reduced dynamic compliance with minimal effect on pulmonary resistance, whereas the reverse was true for the other agonists (25). These ﬁndings, which indicated that SRS-A had a unique preference for peripheral airways, were further examined by comparing the in vitro contractile activity of SRS-A with histamine for guinea pig tracheal spirals and parenchymal strips. Whereas the effect of histamine concentration was equal for contractions of both smooth muscle preparations, the sensitivity of the tracheal spirals to SRS-A was 100-fold less than that of the parenchymal strips. These ﬁndings with puriﬁed SRS-A predicted the novel selectivity of LTC4 and the other cys-LTs for peripheral airways when inhaled by human volunteers.

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Cysteinyl Leukotrienes LTC4 , LTD4 , and LTE4 On the basis of the composition of the SRS activity released by ionophore stimulation of mouse mastocytoma cells (14), E.J. Corey synthesized candidate stereoisomers with various sulfur-linked peptide adducts. In parallel bioassays of chemical moieties and the MCgenerated product by the Samuelsson group, LTC4 was established as 5(S)-hydroxy-6(R) glutathionyl-7, 9-trans, 11, 14-cis eicosatetraenoic acid (26). Corey provided our group with authentic LTC4 and related structures so that we could characterize biologic SRS-A generated from the rat peritoneal cavity and from human lung fragments. After a ﬁnal RPHPLC step (19, 25), the SRS-A prepared from both sources had two activity peaks for contraction of the guinea pig ileum in units/pmol that were equivalent to LTC4 and LTD4 (27). LTD4 had been identiﬁed as the product of cleavage removal of glutamic acid from the glutathionyl adduct of LTC4 by Piper and colleagues (28). Two of ﬁve puriﬁed rat SRS-A preparations contained a previously undescribed activity peak by RP-HPLC that eluted after LTC4 and LTD4 and also exhibited a triene UV absorbance spectrum at 280 nm. The elution time corresponded to a candidate structure in which the glycine had been cleaved from LTD4 and left only the cysteinyl adduct, which we termed LTE4 . Synthetic and biologic LTE4 had the same speciﬁc contractile activity for guinea pig ileum and parenchymal strips (29). However, LTE4 was a log more potent than LTD4 in contracting tracheal spirals and a log less potent in contracting parenchymal strips (30). The possibility of two separate cys-LT receptors had already arisen because an antagonist, FPL 55712, blocked the low-dose, high-afﬁnity parenchymal strip contractions produced by LTD4 but not the contractions initiated by LTC4 or high-dose LTD4 (31). Furthermore, after a contraction of parenchymal strips elicited by LTE4 but not LTC4 or LTD4 , followed by full relaxation, the response of the strip to

histamine was augmented due to the induced generation of a prostanoid (30), suggesting even a third response pathway. Because the cys-LTs are mediators generated during a local inﬂammatory response, we assessed the impact of the respiratory burst elicited in polymorphonuclear leukocytes (PMN) by phorbol myristate acetate (PMA) on their function, immunoreactivity, and integrity. By RP-HPLC with synthetic standards, each cys-LT was converted to a subclass-speciﬁc S-diastereoisomeric sulfoxide without function but with retained immunoreactivity and then, with disruption of the sulfur bridge, to an unreactive common end product, 6-trans-LTB4 . The metabolic inactivation was replicated with hydrogen peroxide, myeloperoxidase, and chloride anion, indicating an attack on the sulfur moiety by hypochlorous acid. Dose-response studies revealed LTE4 to be the most stable cys-LT in the presence of this inactivating pathway (32). Thus, in an integrated setting, the aggregate cellular effects of the cys-LTs reﬂect their relative individual stability and differential potency for expressed receptors on target cells.

LTC4 Synthase and the cys-LT Receptors With deﬁnition of LTC4 as the only biosynthetic intracellular cys-LT, we turned to isolation of the responsible enzyme, LTC4 S. Partial puriﬁcation of LTC4 S solubilized from rat basophilic leukemia-1 cells or guinea pig lung revealed that it was an integral membrane protein that lacked the substrate speciﬁcity for xenobiotics characteristic of the glutathione S-transferases (GSTs) (33, 34). Homogeneous 18-kDa human LTC4 S was solubilized and isolated from 60 billion KG-1 cells, and an Nterminal sequence of 22 amino acids and an internal sequence of 14 residues were obtained (35, 36). For expression cloning, Bing Lam developed a high-throughput ﬂuorescencelinked competitive immunoassay for detection of LTC4 generation by Cos cells www.annualreviews.org • Doing What I Like

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transfected with a cDNA library from KG-1 cells. The full-length cDNA that encoded the LTC4 S activity included the residues identiﬁed by amino acid sequencing of the puriﬁed protein (36). The N-terminal two-thirds of LTC4 S was 44% identical to FLAP at the protein level and 52% identical for the corresponding nucleotides. Both are integral perinuclear membrane proteins, and genomic cloning revealed that their intron/exon junctions align identically, suggesting their evolution by gene duplication (37). By ﬂuorescence in situ hybridization, the gene for human LTC4 S is on chromosome 5q35 distal to the gene cluster for the cytokines that are central to the Th2 phenotype implicated in asthmatic and allergic inﬂammation. The cDNA for mouse LTC4 S is 87% homologous to the human cDNA (38), and genomic cloning revealed identical intron/exon boundaries. The mouse LTC4 S gene is located in a region syntenic to human 5q35 on chromosome 11 near to the Th2 gene cluster (39). The LTC4 S gene is the only one among the recently deﬁned superfamily of membraneassociated proteins involved in eicosanoid and glutathione metabolism located on a chromosome with a Th2 gene cluster. That Th2 cytokines can regulate the pathway for cys-LT generation was recognized using human MCs derived from cord blood by culture in stem cell factor (SCF), interleukin (IL)-6, and IL-10 in studies with Joshua Boyce. Such MCs are deﬁcient in LTC4 S, and priming them with IL-4 induced steady-state transcripts, protein, and function for LTC4 S but did not increase transcripts or protein for other pathway components such as cPLA2 , FLAP, or 5-LO (40). In addition, priming these MCs with IL-3 or IL-5 translocated 5LO to the outer nuclear membrane for FLAPdependent generation of LTA4 , thereby optimizing LTC4 generation when IL-4 was also present. IL-4 priming also enhanced the capacity of the cys-LTs to induce chemokine and cytokine expression by culture-derived human MCs. The cys-LTs did not cause exocytosis, and their action involved two receptors

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IL: interleukin

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inasmuch as a CysLT1 receptor antagonist blocked induction of IL-5 but not IL-8 (41). Although site-directed mutagenesis of human LTC4 S has identiﬁed residues for substrate binding and the integrated conjugation function for GSH and LTA4 (42), crystallography is critical for a mechanistic model. Thus, human LTC4 S was overexpressed in ﬁssion yeast, puriﬁed to apparent homogeneity, and grown in 2D crystals in sheets and vesicles for electron crystallography. Projection maps at 4.5 and 7.5 A˚ show that the enzyme is a trimer and that each monomer contains at least four alpha helices that insert into the membrane (43). Analysis of the atomic structure of LTC4 S at 3.3-A˚ resolution by X-ray crystallography has revealed that the monomer has four transmembrane alpha helices and forms threefold symmetric trimers as a unit with functional domains across each interface. The residues conjugating GSH at C6 and generating a hydroxyl group at C5 are on opposing monomers (44). To examine the functions of the cys-LTs in complex models of inﬂammation, mouse strains with targeted disruption of LTC4 S and each of the known cys-LT receptors, CysLT1 and CysLT2 , were developed and extensively backcrossed by Yoshihide Kanaoka. Disruption of the LTC4 S gene abrogated the capacity of tissues to conjugate LTA4 methyl ester with GSH, establishing the dominant role of this enzyme rather than GSTs in LTC4 biosynthesis (45). IgE-dependent PCA in the ear of the LTC4 S-deﬁcient and the CysLT1 and CysLT2 receptor–deﬁcient strains was reduced by more than one-half, thereby revealing a role for cys-LTs at least equal to that of the MC secretory granule amines in eliciting the plasma leakage at this site. The plasma leak in the LTC4 S-deﬁcient and CysLT1 receptor–deﬁcient strains, but not the CysLT2 receptor–deﬁcient strain, was also reduced by more than 50% after intraperitoneal injection of the microbial cell wall carbohydrate, zymosan (46–48). These studies reveal heterogeneity of the receptor expression of the microvasculature.

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In turning to more complex models, we began with bleomycin-induced pulmonary ﬁbrosis for which the C57B/6 strain is optimal. Twelve days after intratracheal administration of bleomycin, the extent of macrophage and ﬁbroblast accumulation with deposition of extracellular matrix proteins, including collagen, was much less in the LTC4 S-deﬁcient strain. By digital image analysis, the septal thickening of the lower lobes was reduced to one-half that observed in wild-type (WT) littermates. The cys-LTs were absent from the bronchoalveolar lavage (BAL) ﬂuid of the deﬁcient strain and abundant in the BAL ﬂuid of the WT littermates, while the quantities of LTB4 and PGE2 were similar. The CysLT2 receptor–deﬁcient strain was as protected against bleomycin-induced pulmonary ﬁbrosis as the LTC4 S-deﬁcient strain but accumulated the same cys-LT content in the BAL ﬂuid as the WT littermates (48). In contrast, the CysLT1 receptor–deﬁcient strain had greater septal thickening than sufﬁcient controls (49). That ﬁbrosis is prevented by disruption of the pathway at different points, biosynthesis and receptor-mediated function, encoded by genes on different chromosomes, is strong evidence for the role of cys-LTs in this form of chronic inﬂammation.

THE MAST CELL The MC, which is derived from a hematopoietic stem cell, circulates as an immature monocyte-like progenitor (MCp) lacking cytoplasmic granules, is distributed to tissues as an immature progenitor, and matures and differentiates in a tissue-determined fashion. The major phenotypes of tissue MCs in mouse, rat, and human are the constitutive connective tissue type that surrounds microvasculature, often in proximity to nerve endings, and the T cell–dependent mucosal type at intraepithelial locations such as intestine and airways. In the rat and mouse, these MC phenotypes are distinguished by differences in their secretory granule proteoglycans, their proﬁle of secretory granule pro-

teases, and their expression of eicosanoids with activation. These phenotypic distinctions are less perceptible in humans, although the T cell–dependence of the mucosal population is evident from their absence in patients with impaired T cell functions. In initial in vitro studies, we used MCs harvested from the rat peritoneal cavity and isolated on density gradients or human lung MCs obtained from surgical specimens. The MC numbers were limited and the preparation time was excessive. Thus, when knowledge of growth factors evolved through the studies of Ehud Razin and others, we changed to culturederived mouse bone marrow–derived MCs (BMMCs), which are abundant but immature, and human cord blood–derived MCs, which, although quite mature, are of limited number. We initially focused on the nature and number of the secretory granule neutral proteases and proteoglycans. With these markers for cell development, we moved in vivo to characterize the transendothelial migration of MCp to peripheral tissues, the phenotypic plasticity of tissue MCs with location and superimposed inﬂammation, and the tissue-based control of MC activation.

MCp: mast cell progenitor

Secretory Granule Proteases and Proteoglycans The secretory granule is best known as a source of histamine, a mediator of the nasal coryza and ocular/cutaneous pruritus in allergic disease. Both histamine and the neutral proteases bear positive charges, which interact with the anionic glycosaminoglycans polymerized onto a peptide core of the proteoglycan(s) in the secretory granule. An electron microscopic study of human lung MCs revealed that the secretory granules had a crystalline structure of deﬁned periodicity with a lattice- or scroll-like distribution. With IgEdependent activation, the granules swelled, their crystalline structure dissolved, and in association with contractile elements they moved to the membrane for fusion and extracellular release of contents (50). The ionic www.annualreviews.org • Doing What I Like

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interactions of the released complex were disrupted at physiologic pH, and many of the cationic moieties including histamine diffused away, leaving a “ghost” composed of the proteoglycans and highly cationic proteases (51). We were surprised that the dominant human MC protease, which we termed tryptase, was a tetramer with four active sites (52). Although some considered the tetramer presentation to be a consequence of our protein isolation, the ﬁndings were conﬁrmed by others with X-ray crystallography. Tryptase is the dominant neutral protease in both human MC phenotypes and can cleave human C3 to release the C3a anaphylatoxin (53). MC-carboxypeptidase A (MC-CPA) is a speciﬁc marker for connective tissue MCs in both mice and humans. MC-CPA is unique among carboxypeptidases in having a CPAlike substrate-binding pocket and enzymatic activity but an overall protein and gene structure more similar to carboxypeptidase B (54, 55). Recently, Rodewald and colleagues (56) reported that MC-CPA protected mice against snake venom sarafotoxin, identifying the speciﬁc protease responsible for a host defense MC function reported by others. On two occasions, transformed mouse cell lines developed in other laboratories profoundly advanced our program by solving a technical issue. The ﬁrst was the derivation of connective tissue–like MC lines (KiSV-MC) by coculture of mouse splenocytes with ﬁbroblasts that produce the Kirsten murine sarcoma virus. In contrast to earlier lines, some of the KiSV-MC lines exhibited maturation indistinguishable from that of mouse peritoneal MCs and provided enough secretory granule protein for isolation of the proteases (57). MC-CPA from KiSV-MCs and from peritoneal MCs were both 36 kDa and had identical N-terminal amino acid sequences; these cells, along with BMMCs, expressed the same transcript encoded by a single gene for MCCPA (58). The four other proteins obtained from KiSV-MCs, ranging from 28 to 32 kDa, were serine proteases based on [3 H]DFP binding (59). Their cDNAs indicated that

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mouse MC protease (mMCP)-2 (60), -4 (61), and -5 (62) were in the chymase gene family, whereas mMCP-6 was a tryptase (63). The tissue distribution of their mRNA indicated that mMCP-2 was prominent in mouse mucosal MCs, while mMCP-4, -5, and -6 and CPA characterized the connective tissue MC phenotype. We subsequently recognized a second secretory granule tryptase, mMCP-7, in the BALB/c but not the C57B/6 strain (64) and another chymase, mMCP-9, selectively expressed in mouse uterine MCs (65). The chymases are differently regulated in BMMCs, with mMCP-5 being induced by SCF (66) and the mucosal mMCP-1 and mMCP-2 by IL9 or IL-10 (67). By nuclear run-on analysis, all the beta chymases were being transcribed in the BMMCs, and the cytokines were simply stabilizing the transcripts to allow translation (68). Such a mechanism of transcript stabilization could account for the phenotypic diversity of the protease proﬁle in different tissue MCs and for the further changes with inﬂammation by Trichinella spiralis infection (69). These seminal studies involved postdoctoral fellows Bill Seraﬁn, Dale Reynolds, Patrick McNeil, and John Hunt and depended on a long-time colleague, Richard Stevens. The second transformed MC line from another laboratory that was essential to our progress was the v-abl immortalized MC line (V3-MC). Unlike the KiSV-MC, these cells did not shed virus and could be used to follow lineage maturation after adoptive transfer to a BALB/c recipient (70). The clonal line was immature and expressed protein for mMCP-5, mMCP-7, and mMC-CPA but not mMCP-2 or mMCP-6. After adoptive transfer and residence in the liver or spleen, the line expressed all proteases, as did the resident MCs. In contrast, the V3-MCs identiﬁed in the small intestine by the abl marker were agranular at day 6 after adaptive transfer and without detectable proteases, and by 14 days expressed only abundant mMCP-2, a marker for mucosal MCs. The distinct protease phenotypes of the V3-MCs in the two

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tissues reﬂecting additions and/or deletions elegantly demonstrated that protease expression was regulated by local factors. Because the proteases were bound in an active form in the secretory granule, we characterized the heparin moiety in rat peritoneal (71) and human lung MCs (72) after radiolabeling in vitro with [35 S]sulfate and [3 H]serine. This early work by Dean Metcalfe and Jerry Silbert showed that the rat proteoglycan was ten times larger than the human proteoglycan owing to the number and size of the glycosaminoglycans polymerized onto a glycine-/serine-rich small-core polypeptide (73). The proteoglycans in the BMMCs included heparin but were predominantly composed of chondroitin sulfate E glycosaminoglycans rich in Nacetylgalactosamine-4,6-disulfate sequences (74, 75). Irrespective of the size or nature of the glycosaminoglycans, the segment of the core polypeptide to which the glycosaminoglycans were attached was protease resistant. Cloning of the cDNA and gene that encode this peptide core in rat basophilic leukemia-1 cells (76), human promyelocytic HL-60 cells (77), and mouse BMMCs (78) revealed coding for a highly conserved N-terminal amino acid sequence and a stretch of alternating serine and glycine residues. Serglycin is the single peptide core for hematopoietic secretory granule proteoglycans, the selection of the types of glycosaminoglycan to be synthesized onto this peptide core is cell speciﬁc, and the protease resistance of serglycin allows storage association of active proteases to the glycosaminoglycans.

Development and Transendothelial Distribution of Mast Cell Progenitors (MCp) Investigations in the laboratories of Kitamura (79), Rodewald (80), and Schrader (81) indicated that MCp arise from bone marrow (BM) stem cells, circulate as agranular lineage progenitors, and settle into the small intestine at concentrations per million mononu-

clear cells (MNCs) that exceed the concentrations in other peripheral tissues. We assumed intestinal MCp to be the source of the Th2 cell–dependent MC hyperplasia that cleared adult worms (69), and Michael Gurish deﬁned the MCp integrins that sustained the intestinal reservoir. MNCs were harvested from the small intestine, BM, spleen, and lung, and the MCp per million MNCs was determined by limiting dilution and clonal expansion. The absence of β7 (CD49d) but not αE (CD103) or β2 (CD18) integrins resulted in a marked reduction of MCp in the small intestine but not in the lung, spleen, or BM of naive mice. To conﬁrm these ﬁndings, we eliminated MCp from WT mice by sublethal irradiation and introduced various monoclonal antibodies (mAbs) just after adoptive transfer of syngeneic BM. The administration of mAb to α4β7, α4, or β7 integrins and to their counterligands on the microvasculature, mucosal addressin cell adhesion molecule1 (MAdCAM-1) and vascular cell adhesion molecule-1 (VCAM-1), blocked reconstitution of the intestinal MCp, whereas mAb to αE or β1 integrins had no effect. The ﬁnding that blocking mAb to α4β7 was fully inhibitory even if administered four days after the syngeneic BM implied a priming phase for MC lineage development in recipient BM (82). This dynamic relationship between BM and the intestinal reservoir of MCp was conﬁrmed by showing that administration of blocking mAb every other day for one week to nonirradiated naive mice profoundly depleted the intestinal pool of MCp by blocking the regular inﬂux of new MCp (83). Furthermore, that the MCp reservoir was intact in mice null for the recombination activating (RAG)-2 and IL receptor common gamma chain genes indicated that this constitutive α4β7-dependent homing of MCp is independent of the Th2 cytokines needed to elicit mucosal MC hyperplasia (82). Finally, both mucosal and connective tissue MCs were deﬁcient in the β7 integrin-null mouse, indicating that a single lineage of MCp gives rise to both tissue phenotypes. www.annualreviews.org • Doing What I Like

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Mouse lung has minimal numbers of baseline MCp, and their recruitment with sensitization and inhalation antigen challenge depends partially on α4β7 but also involves α4β1. As lung lacks MAdCAM-1, adhesion with pulmonary inﬂammation is entirely through upregulated VCAM-1, likely accounting for the role of both α4-based integrins (84). The fact that blocking mAb to integrins of MCp prevents the accumulation of MCp in lung indicates that even with inﬂammation the initial host response is recruitment, not proliferation. The appreciation that β7-integrin expression is a requirement for transendothelial migration by MCp led to the discovery of a new bifunctional progenitor for both the MC and the basophil lineages. Candidate MCp progenitors in BM, spleen, and small intestine were depleted of irrelevant lineages, sorted by membrane phenotype, and characterized by growth in a cytokine mixture suitable for all hematopoietic lineages. In the C57B/6 strain, the spleen contained a population of bipotent β7+ /c-Kit+ /FcεR1+ basophil/MC progenitors (BaMCp) that gave rise in single-cell culture to colonies of either MCp or basophil progenitors (Bap). MCp but not Bap retained and increased β7 integrin expression. BaMCp administered intravenously to genetically MC-deﬁcient W/Wv mice reconstituted mature MCs in both spleen and stomach. The small intestine contained only MCp, suggesting that BaMCp do not leave the spleen. The granulocyte-related transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) played the major role in the fate decision of BaMCp, being retained by Bap and absent in MCp (85).

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Counterregulatory Signals Howard Katz, a new PhD in the laboratory of Steve Russell, had recognized that several mAbs obtained by immunizing Lewis rats with mononuclear phagocytes derived from BALB/c BM cultures in medium containing CSF-1 also bound BMMCs developed with 14

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IL-3. He entered our postdoctoral program for the purpose of unraveling the epitopes for these mAbs, of which one, gp49B1, has proven to be of particular interest as a constitutively expressed negative regulatory receptor. Immunoprecipitation of a membrane epitope on both BMMCs and peritoneal MCs by the IgM mAb/B23.1 revealed a heterogeneous protein with a mean molecular mass of 49 kDa (86). Puriﬁcation of gp49 from a KiSV-MC clone provided the N-terminal amino acid sequence used to prepare oligonucleotides for screening a KiSV-MC library. Two of the fulllength cDNAs obtained, gp49A and gp49B1, were 97% homologous and encoded members of the Ig superfamily with two type 2 Ig-like extracellular domains, whereas the third, gp49B2, was identical to gp49B1 except for the lack of the entire transmembrane domain encoded by exon 6 (87, 88). That the putative tandem immunoreceptor tyrosine-based inhibition motifs (ITIMs) recognized in the cytosolic domain of gp49B1 were functional was established in vitro (89). Mice lacking the gp49B gene but not the g49A gene were generated and assessed for MC function by PCA (90). The net ear swelling of the sensitized ear due to MC activation and plasma extravasation was twofold greater in the gp49B1-deﬁcient strain than in the sufﬁcient WT littermates, and the number of MCs degranulated per unit area was also twofold greater. When the sensitizing dose of mAb IgE was reduced by one log, the net swelling of the sensitized ear in the deﬁcient strain was equal to that obtained in the WT strain with the standard tenfold greater sensitizing dose. In active cutaneous anaphylaxis of mice systemically sensitized to OVA and showing similar levels of speciﬁc IgE, an intradermal dose of OVA that elicited a robust vascular leak in the gp49B1-deﬁcient strain caused no measurable ear swelling in the WT mice. These studies not only uncovered a control receptor of IgE-dependent MC activation but, most important, recognized that such inﬂammation was normally dampened in situ by

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an innate constitutive mechanism. That the activation of ear MCs by SCF also led to a signiﬁcantly greater vascular leak and MC degranulation in the gp49B1-deﬁcient mice as compared to the sufﬁcient littermates showed that regulation was not limited to adaptive immunity (91). There was yet a surprise. When lipopolysaccharide (LPS) was injected intradermally into the mouse ear to see if the gp49B1-null strain would have an exaggerated Toll-like receptor (TLR)-initiated MC-dependent swelling response, there was no early difference between the deﬁcient and sufﬁcient strains. Instead, by 24 h there was gross hemorrhage in the LPS-injected ear of the gp49B1-deﬁcient strain but none in the ear of the sufﬁcient littermates. The macroscopic bleeding visually apparent against the white fur of the gp49B1-deﬁcient BALB/c strain was reminiscent (to someone who had been working in immunology in the late 1950s) of a Schwartzman reaction. The microangiopathy of a Schwartzman reaction is due to a coagulopathy, not a vasculitis. On microscopic analysis, we saw venules occluded by thrombi composed of neutrophils, platelets, and ﬁbrin accompanied by a vascular margination and tissue inﬁltration by PMNs. The MCs were intact and not different in the deﬁcient strain. The lesion was prevented by administration of mAbs that depleted PMNs or blocked the interaction of their β2 integrin with the intercellular adhesion molecule 1 (ICAM-1) or by therapeutic inhibition of coagulation. Unexpectedly, the PMN expressed gp49B1, which is upregulated by LPS in WT mice (92). The “one-shot” Schwartzman reaction occurred in the deﬁcient strain owing to the absence of an innate control, gp49B1, of a TLR response on their PMNs. A member of the human leukocyte Ig-like receptor family (LIR-5) homologous to gp49B1 has been cloned (93) and renamed human LILRB4 to correspond to mouse LILRB4/gp49B1.

THE ALTERNATIVE COMPLEMENT PATHWAY In the 1950s, Pillemer and colleagues recognized that a plasma protein, designated properdin (P), was required for complementmediated bactericidal and hemolytic activity that was not antibody dependent (94). The properdin system/alternate complement pathway is composed of six proteins, of which four are speciﬁc to this pathway (P, factor B, factor D, and factor H) and two [C3 and C3b inactivator (C3bINA)] are shared with the classical antibody-dependent pathway. The most critical step in both activating pathways is the proteolytic cleavage of C3 to provide C3b for assembly of the ampliﬁcation C3 convertase, C3bBb. The same alternative pathway proteins are used for formation of a weak alternative pathway-initiating C3 convertase built with uncleaved C3. Unraveling the relationship of the initiation and ampliﬁcation phases of alternative pathway C3 cleavage followed the introduction of a quantitative, sensitive hemolytic assay for the assembly of C3bBb by my colleagues, Douglas Fearon and Shaun Ruddy (95). Sheep erythrocytes with afﬁxed C3b interacted with B in the presence of D to generate C3bBb sites, and the number of these sites could be calculated by lysis with additional C3 and C5-9. The cell-based assay had linear stoichiometry for generation of the C3bBb sites with incremental inputs of B and a ﬁxed dose of D, and a hemolytic efﬁciency comparable to cells bearing the classical C3 convertase. Our use of this assay included characterization of the proteins involved in the assembly, stabilization, and inactivation of C3bBb; recognition of the initiating alternative pathway C3 convertase, C3Bb; and demonstration of the integrated activation and ampliﬁcation of the alternative pathway on an activating particle or cell. Most important to the understanding of the alternative pathway was our discovery of the stabilizing function of P and of the destabilizing protein H for C3bBb. Properdin

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bound to C3b to form a trimolecular complex from which the decay of Bb was markedly retarded (96). Conversely, H terminated C3bBb function by dissociation-decay of Bb even in the presence of P (97, 98). This counterregulatory action allowed inactivation of C3b by C3bINA to prevent regeneration of the hemolytic site. With these insights into ampliﬁcation, we used the same proteins to seek the initiating alternative pathway C3 convertase, C3Bb, in the ﬂuid phase by comparing the dose-response effects of C3 and C3b for D-mediated cleavage inactivation of B. The time-dependent sigmoidal inactivation curve for B with C3 and D was delayed relative to that with C3b and was not inhibited by addition of C3bINA, indicating that C3Bb provided the C3b for subsequent formation of the ampliﬁcation convertase (99). Properdin dose-dependently augmented B inactivation with C3 and D, indicating stabilization of C3Bb. That D was a constitutively active serine protease (100) suggested a continuous low-grade cleavage of C3 in the presence of B and P and circumvention of the regulatory controls when C3b was deposited on an activating particle. To prove this mechanism, we used zymosan, the same insoluble polysaccharide derivative from yeast cell walls that Pillemer had used in recognizing the alternative complement-activating pathway (94). When zymosan was added to a mixture of C3, B, D, P, C3bINA, and H, an initial low-grade cleavage of C3 was followed by ampliﬁed cleavage that suggested a two-step process. Moreover, C3b bound to zymosan was relatively resistant to inactivation by C3bINA, and the hemolytic site, PC3bBb, assembled on zymosan was protected against H dissociation-decay, demonstrating that regulation of the ampliﬁcation site was retarded by the character of an activating particle (101). In a parallel study with the same six proteins, the addition of rabbit, but not sheep, erythrocytes initiated consumption of B and C3 in the ﬂuid phase and formation of lytic sites on the cell (102). Fearon then showed that enzymatic or chemical removal

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of membrane sialic acid groups from sheep erythrocytes converted these cells to activators of the alternative complement pathway while reducing the regulatory efﬁciency of C3bINA and H (103). Thus, the constitutive action of D to form C3Bb provides the C3b that, when afﬁxed to a protective nonself surface, forms a stabilized ampliﬁcation C3 convertase, PC3bBb, that provides diverse proinﬂammatory or host protective functions. The pattern recognition activation of the alternative complement pathway is a prime example of innate immunity. Our seminal insight that unraveled the activation and ampliﬁcation of the alternative pathway was that it might not begin with recruitment of a proenzyme but rather that nonself might be recognized by circumvention of surveillance mechanisms, H and C3bINA, that protect self.

TRANSLATIONAL INVESTIGATIONS The linkage of our bench research in our three focal areas to the clinic was most dynamic in the early years, when less was known, and continued at a reduced level until the early 1990s. Each clinical application of an insight gained at the bench involved a critical collaboration with either an astute full-time clinician or an established clinical investigator.

The cys-LTs After toiling with SRS-A for two decades, I was immensely curious about whether the cys-LTs provided by E.J. Corey would have the smooth muscle actions in humans that we had observed in animals. My equally curious colleagues, Bob Lewis and Nick Soter, joined me in an initial dose-response study in ourselves to intradermal LTD4 . The magnitude and persistence of the elicited wheal and ﬂare responses were so compelling that we initiated a second study with biopsies in which our cutaneous responses to the cys-LTs were compared with those to the other MCderived eicosanoids, LTB4 and PGD2 , and to

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buffer. Equimolar amounts of LTC4 , LTD4 , and LTE4 elicited a local erythema and wheal of 6 h and 2 h duration, respectively, whereas the similar response to a higher dose of PGD2 was less sustained. The tissue showed dermal edema with marked and uniform dilation of capillaries, superﬁcial and deep venules, and arterioles as well as activation-related changes in the endothelium. There was no cellular inﬁltration in the cys-LT-injected sites. In contrast, LTB4 elicited a transient wheal and ﬂare followed 3–4 h later by induration that was characterized by a dermal inﬁltration composed almost solely of neutrophils. The combination of LTB4 and PGD2 intensiﬁed the cellular inﬁltrate. The ﬁndings that nanomole amounts of leukotrienes were active in human skin and that each subclass gave the expected biology were immensely rewarding to us (104). The Human Studies Committee delayed our planned investigation of the airway smooth muscle actions of aerosolized cysLTs to be certain that the skin exposure had no adverse effects. The concentrations of aerosolized LTC4 that reduced by 30% the maximum expiratory ﬂow rate (measured at 30% of vital capacity above residual volume) were in ug/ml, whereas those for histamine were in mg/ml, revealing a more than 1000fold greater potency for LTC4 in normal volunteers (105). LTC4 induced a fall in airﬂow that was slow in onset, prolonged, and associated with audible wheezing without a cough, whereas histamine acted rapidly and elicited a cough but not a wheeze. LTD4 was also three logs more potent than histamine, and the more biologically stable LTE4 was one log more potent as a bronchoconstrictor in normal human subjects. In contrast, in patients with bronchial asthma, the respective potencies of LTD4 and LTE4 relative to histamine were less than a log (106, 107). Although this ﬁnding would still classify the cys-LTs as potent bronchoconstrictors in individuals with bronchial asthma, it also suggests that their chronic overproduction somehow ameliorates receptor responses.

The Mast Cell In studies initiated in the clinic, a distinct form of physical allergy, exercise-induced anaphylaxis (EIA), was identiﬁed. When Al Sheffer described a number of patients “allergic to exercise” to the point of vascular collapse with jogging/running, indoor/outdoor team sports, tennis, and dancing, but without occurrence with every repetition of the exercise, it seemed reasonable to further expand the series. When the number reached 16, it was obviously a syndrome. EIA was deﬁned by a premonitory feeling of body warmth and itching unrelated to ambient temperature and progression to cutaneous erythema, conﬂuent hives, laryngeal edema with stridor or hoarseness, gastrointestinal cramps/colic, and frequently culmination in vascular collapse (108). Of seven patients with EIA who exercised on a treadmill with a moving grade while wearing an occlusive suit, four developed erythema and conventional-sized hives and two of these manifested laryngeal hoarseness. The four with symptoms showed a rise in blood histamine concentration, implicating MC activation (109), whereas the blood histamine concentration did not change in the subjects with EIA who did not respond to this challenge. In a repeat study, cutaneous biopsies of the patients with EIA with symptoms and signs showed classical MC degranulation that was absent in the nonresponders (110). In contrast to patients with cholinergic urticaria, who experienced punctate hives, elevations in blood histamine, and pulmonary symptoms with signiﬁcantly altered pulmonary function with the exercise protocol (111), pulmonary mechanics were not altered in the challenged patients with EIA. Patients with EIA can abort an episode by stopping the exercise with the onset of premonitory symptoms and can ameliorate/prevent attacks by allowing a suitable interval after a meal and/or by using various MC-directed interventions before exercise. Studies from the bench to the clinic identiﬁed a new MC-derived mediator, PGD2 , that accounted for intractable hypotension in

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occasional patients with systemic mastocytosis. In collaboration with John Oates and Jack Roberts, we assessed the eicosanoid products of MC activation. Gas chromatography-mass spectroscopy (GC-MS) analysis of calcium ionophore-stimulated or anti-IgE-activated puriﬁed rat peritoneal MCs revealed net generation of PGD2 with only minimal quantities of other eicosanoids. Similarly, activation of dispersed human lung MCs by anti-IgE showed selective PGD2 generation relative to other prostanoids and a linear relationship to histamine release (112, 113). The failure of H1 and H2 antagonists to control the severe body ﬂushing and hypotension in two patients with systemic mastocytosis led to a GC-MS analysis of urine and the structural identiﬁcation of a metabolite of PGD2 that was not present in more than 200 urine samples from normal subjects or patients with other diseases (114). The hypotension in one of these patients was controlled by treatment with highdose aspirin in combination with H1 and H2 antagonists. The evidence that MCs in patients with systemic mastocytosis can spontaneously release more than preformed secretory granule mediators accounts for our use of cromolyn sodium, a MC-stabilizing agent, along with H1 and H2 antagonists and, when indicated by severe ﬂushing, aspirin in their management (115).

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The Complement Pathways Disordered regulation of the complement system became a clinical focus because of our preclinical studies and ability to develop sensitive stoichiometric hemolytic assays for proteins whose absence or sustained presence deﬁned the syndrome. Such studies included patients with inborn deﬁciency of C1INH and bouts of HAE and others with autoantibody-mediated C1INH deﬁciency; patients with autoantibody-mediated stabilization of the ampliﬁcation C3 convertase by nephritic factor (NEF) and membranoproliferative glomerulonephritis; and patients with acquired clonal loss of decay18

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accelerating factor (DAF) and paroxysmal nocturnal hemoglobinuria (PNH). Once Al Sheffer learned that Donaldson & Evans (116) had recognized a C1INH deﬁciency in patients with HAE by screening serum for lack of ability to inhibit C1 esterase activity, he appeared in my laboratory to request a suitable assay for this potentially fatal disease among his patients. Stoichiometric hemolytic assays for C2 and C4, substrates of C1, showed their concentrations in HAE to be two standard deviations below the mean for normal in kindreds lacking the protein or those with a nonfunctional protein and to be further reduced by consumption with a clinical episode (117, 118). The ﬁnding by Spaulding (119) that androgen treatment could prevent attacks of angioedema in patients with HAE led us to use attenuated androgens, which reduced but did not eliminate the risk of androgen-related side effects (120). The concentrations of C1INH in untreated HAE patients during asymptomatic periods were less than one quarter normal even though inheritance is autosomal dominant, suggesting consumption of the product of the good gene by the uninhibited C1. Reasonable prevention or amelioration of attacks was achieved with a dose of attenuated androgen that increased the mean functional concentrations of C1INH and C4 to less than one-half of the lower limit of normal, demonstrating a modest dose threshold for clinical beneﬁt (121). Acquired C1INH deﬁciency with clinical angioedema in patients with B cell proliferative disorders was recognized to be distinct from HAE because there was depletion of functional C1 and its binding subunit, C1q, as well as C4 and C2 (122). Analysis of three such patients showed circulating anti-idiotypes directed to the monoclonal Ig expressed on the surface of their lymph node B cells and on circulating B cells with ﬁxed C1q (123). Patients with PNH have erythrocytes with an acquired abnormal membrane sensitivity to complement-mediated lysis (PNH-E) and experience spontaneous episodes of intravascular hemolysis, often at night. Two membrane

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control proteins, C3b receptor (CR1) and DAF, extracted and puriﬁed from human erythrocytes, can act exogenously to destabilize both the ampliﬁcation (C3bBb) and classical (C4b2a) C3 convertases (124, 125). We found that speciﬁc immunoprecipitates of surfacelabeled PNH-E lacked a 70 MW DAF but expressed a 250 MW CR1. Further, PNH-E, unlike normal erythrocytes, failed to absorb the DAF-neutralizing activity of speciﬁc rabbit IgG (126). These ﬁndings provided the ﬁrst in situ evidence that an endogenous membrane protein can downregulate the potential pathobiologic effects of ampliﬁcation resulting from the low-grade turnover of C3 by identifying an acquired clonal deletion of DAF in PNH-E. Patients with chronic glomerulonephritis and a reduced serum C3 concentration have normal concentrations of C4 and reduced concentrations of B, implicating the alternative complement pathway, while patients with systemic lupus erythematosus and reduced C3 concentrations have reduced concentrations of C4 with or without reduced concentrations of B, implicating classical immune complex activation with or without ampliﬁcation (127). C3 nephritic factor (C3 NeF) in serum of patients with low C3 concentrations and membranoproliferative glomerulonephritis was known to augment C3 consumption. Mohamed Daha and Doug Fearon found that chromatographically puriﬁed C3 NeF stabilized the hemolytic function of C3bBb on erythrocytes and interacted with C3, B, and D in the ﬂuid phase to form a trimolecular 10S complex of C3bBb/C3NeF (128). The C3NeF recovered chromatographically after decay of the 10S convertase was highly puriﬁed, fully functional (129), and composed of heavy and light chains (130). Others had recognized that C3 NeF was an Ig by depletion of the activity from serum with particle-bound anti-IgG. We provided direct evidence that it was an autoantibody with speciﬁcity that stabilized the ampliﬁcation convertase without a role for P and with resistance to induced decay by H (97).

EPILOGUE As physicians have entered my laboratory after experiencing a daily sense of accomplishment as house ofﬁcers, it was critical that I help them endure the initial difﬁculties inherent to bench research. Most important, I have tried to convey that advice is not criticism and that failure is predictable if the projects chosen have merit. Over the years, postdoctoral fellows have been routinely designated as ﬁrst author on manuscripts when a project reached fruition and as last author when they became faculty with mentoring responsibilities. I edited and rewrote many drafts of each manuscript, making sure that the literature was fully recognized and sometimes laying out an urgent set of additional experiments that we had not identiﬁed in laboratory meetings. Since 1966, the “ﬁnal” text has been edited by Arlene Stolper Simon. She has edited every manuscript of which I am an author, the sections of every volume for which I served as an editor, and every grant application that I submitted. Colleagues often mention that they can recognize faculty who trained here by a certain consistency in their oral and written presentations. A few years ago, I titled my acceptance of the Kober medal “It Only Gets Better” (131) to convey that experience with focus provides insights and an inclination for measured risk that is productive for research in a setting with talented trainees and wise colleagues. Those individuals noted in this document have generally progressed to faculty level, thereby extending our period of collaboration. I have concentrated on areas with a longitudinal span and have not included descriptions for our studies of chemotaxis (Edward Goetzl, Stephen Wasserman), kinin generation (Allen Kaplan, Jocelyn Spragg), classical complement pathway (Irma Gigli, Michel Kazatchkine), eosinophils (Barry Kay, Marc Rothenberg), ﬁsh oil–derived leukotrienes (Tak Lee), and some of our early work on SRSA biology by Daniel Stechschulte and Martin Valentine, my ﬁrst postdoctoral fellows. By being an administrative minimalist with an www.annualreviews.org • Doing What I Like

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open door and much sharing of tasks, I have been able to concentrate on my triad of interests with results beyond my expectations. The respect for each other’s laboratories that grew between Bengt Samuelsson, E.J. Corey, Jeffrey Drazen, and me enhanced progress in the SRS-A/cys-LT ﬁeld and built long-term friendships. In the complement ﬁeld, there were times when the laboratories of Fred Rosen, Hans Muller-Eberhard, and my own were in competition, but we managed those

occasions so as to sustain our collaborations and friendships. My wife, Joycelyn Chapman Austen, has been especially generous in tolerating my avocation, in indulging the intensity of the ﬁveyear grant renewals, and in meeting every responsibility to our four wonderful children in a manner that reﬂected our joint interests. These children, and now our eight grandchildren, take my efforts to provide guidance with humor and tolerance.

DISCLOSURE STATEMENT The author is not aware of any biases that might be perceived as affecting the objectivity of this review.

LITERATURE CITED 1. Austen KF. 1978. Homeostasis of effector systems which can also be recruited for immunologic reactions. Pres. Address Am. Assoc. Immunol. J. Immunol. 121:793–805 2. Austen KF, Koch-Weser J, Field RA. 1956. Cardiorespiratory problems in severe poliomyelitis observed during the recent epidemic. N. Engl. J. Med. 254:790–93 3. Drazen JM. 2004. Presentation of the Kober Medal to K. Frank Austen. J. Clin. Invest. 114:1174–76 4. Livingstone JB, Austen KF, Kunz LJ. 1957. A study of intercurrent bacterial respiratory infections in bulbospinal poliomyelitis. N. Engl. J. Med. 257:861–66 5. Austen KF, Carmichael MW, Adams RD. 1957. Neurologic manifestations of chronic pulmonary insufﬁciency. N. Engl. J. Med. 257:579–90 6. Austen KF, Rubini ME, Meroney WH, Wolff J. 1958. Salicylates and thyroid function. I. Depression of thyroid function. J. Clin. Invest. 37:1131–43 7. Wolff J, Austen KF. 1958. Salicylates and thyroid function. II. The effect on the thyroidpituitary interrelation. J. Clin. Invest. 37:1144–52 8. Austen KF. 1960. The differentiation of the chloroform, peptone and antigen antibody inducible esterase activities of human serum from plasmin. Immunology 3:152–73 9. Austen KF, Brocklehurst WE. 1961. Anaphylaxis in chopped guinea pig lung. I. Effect of peptidase substrates and inhibitors. J. Exp. Med. 113:521–39 10. Austen KF, Brocklehurst WE. 1961. Anaphylaxis in chopped guinea pig lung. II. Enhancement of the anaphylactic release of histamine and slow reacting substance by certain dibasic aliphatic acids and inhibition by monobasic fatty acids. J. Exp. Med. 113:541–57 11. Austen KF, Brocklehurst WE. 1961. Anaphylaxis in chopped guinea pig lung. III. Effect of carbon monoxide, cyanide, salicylaldoxine, and ionic strength. J. Exp. Med. 114:29– 42 12. Humphrey JH, Austen KF, Rapp HJ. 1963. In vitro studies of reversed anaphylaxis with rat cells. Immunology 6:226–45 13. Stroud RM, Austen KF, Mayer MM. 1965. Catalysis of C’2 ﬁxation by C’1a: reaction kinetics, competitive inhibition by TAMe, and transferase hypothesis of the enzymatic action of C’1a on C’2, one of its natural substrates. Immunochemistry 2:219–34 20

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14. Murphy RC, Hammarstrom S, Samuelsson B. 1979. Leukotriene C: a slow-reacting substance from murine mastocytoma cells. Proc. Natl. Acad. Sci. USA 76:4275– 79 15. Stechschulte DJ, Austen KF, Bloch KJ. 1967. Antibodies involved in antigen-induced release of slow reacting substance of anaphylaxis (SRS-A) in the guinea pig and rat. J. Exp. Med. 125:127–47 16. Orange RP, Stechschulte DJ, Austen KF. 1970. Immunochemical and biologic properties of rat IgE. II. Capacity to mediate the immunologic release of histamine and slow reacting substance of anaphylaxis (SRS-A). J. Immunol. 105:1087–95 17. Ishizaka T, Ishizaka K, Orange RP, Austen KF. 1970. The capacity of human immunoglobulin E to mediate the release of histamine and slow reacting substance of anaphylaxis (SRS-A) from monkey lung. J. Immunol. 104:335–43 18. Morse HC, Bloch KJ, Austen KF. 1968. Biologic properties of rat antibodies. II. Timecourse of appearance of antibodies involved in antigen-induced release of slow reacting substance of anaphylaxis (SRS-arat ): association of this activity with rat IgGa. J. Immunol. 101:658–63 19. Orange RP, Murphy RC, Karnovsky ML, Austen KF. 1973. The physicochemical characteristics and puriﬁcation of slow reacting substance of anaphylaxis. J. Immunol. 110:760– 70 20. Orange RP, Murphy RC, Austen KF. 1974. Inactivation of slow reacting substance of anaphylaxis (SRS-A) by arylsulfatases. J. Immunol. 113:316–22 21. Weller PF, Corey EJ, Austen KF, Lewis RA. 1986. Inhibition of homogeneous human eosinophil arylsulfatase B by sulﬁdopeptide leukotrienes. J. Biol. Chem. 261:1737–44 22. Jakschik BA, Falkenhein S, Parker CW. 1977. Precursor role of arachidonic acid in release of slow reacting substance from rat basophilic leukemia cells. Proc. Natl. Acad. Sci. USA 74:4577–81 23. Lewis RA, Wasserman SI, Goetzl EJ, Austen KF. 1974. Formation of slow-reacting substance of anaphylaxis in human lung tissue and cells before release. J. Exp. Med. 140:1133–46 24. Lam BK, Owen WF Jr, Austen KF, Soberman RJ. 1989. The identiﬁcation of a distinct export step following the biosynthesis of leukotriene C4 by human eosinophils. J. Biol. Chem. 264:12885–89 25. Drazen JM, Austen KF. 1974. Effects of intravenous administration of slow reacting substance of anaphylaxis, histamine, bradykinin, and prostaglandin F2 on pulmonary mechanics in the guinea pig. J. Clin. Invest. 53:1679–85 26. Marfat A, Corey EJ. 1985. Synthesis and structure elucidation of leukotrienes. In Advances in Prostaglandin, Thromboxane, and Leukotriene Research, ed. JE Pike, DR Morton Jr, 14:155–228. New York: Raven 27. Lewis RA, Austen KF, Drazen JM, Clark DA, Marfat A, Corey EJ. 1980. Slow reacting substances of anaphylaxis: identiﬁcation of leukotrienes C-1 and D from human and rat sources. Proc. Natl. Acad. Sci. USA 77:3710–14 28. Morris HR, Taylor GW, Piper PJ, Samhoun MN, Tippins JR. 1980. Slow reacting substances (SRSs); the structure identiﬁcation of SRSs from rat basophil leukemia (RBL-1) cells. Prostaglandins 19:185–201 29. Lewis RA, Drazen JM, Austen KF, Clark DA, Corey EJ. 1980. Identiﬁcation of the C(6)S-conjugate of leukotriene A with cysteine as a naturally occurring slow reacting substance of anaphylaxis (SRS-A). Importance of the 11-cis-geometry for biological activity. Biochem. Biophys. Res. Commun. 96:271–77 www.annualreviews.org • Doing What I Like

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30. Lee TH, Austen KF, Corey EJ, Drazen JM. 1984. Leukotriene E4 -induced airway hyperresponsiveness of guinea pig tracheal smooth muscle to histamine and evidence for three separate sulﬁdopeptide leukotriene receptors. Proc. Natl. Acad. Sci. USA 81:4922– 25 31. Drazen JM, Austen KF, Lewis RA, Clark DA, Goto G, et al. 1980. Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro. Proc. Natl. Acad. Sci. USA 77:4354–58 32. Lee CW, Lewis RA, Tauber AI, Mehrotra M, Corey EJ, Austen KF. 1983. The myeloperoxidase-dependent metabolism of leukotrienes C4 , D4 and E4 to 6-transleukotriene B4 diastereoisomers and the subclass-speciﬁc S-diastereoisomeric sulfoxides. J. Biol. Chem. 258:15004–10 33. Yoshimoto T, Soberman RJ, Lewis RA, Austen KF. 1985. Isolation and characterization of leukotriene C4 synthetase of rat basophilic leukemia cells. Proc. Natl. Acad. Sci. USA 82:8399–403 34. Yoshimoto T, Soberman RJ, Spur B, Austen KF. 1988. Properties of highly puriﬁed leukotriene C4 synthase of guinea pig lung. J. Clin. Invest. 81:866–71 35. Penrose JF, Gagnon L, Goppelt-Struebe M, Myers P, Lam BK, et al. 1992. Puriﬁcation of human leukotriene C4 synthase. Proc. Natl. Acad. Sci. USA 89:11603–6 36. Lam BK, Penrose JF, Freeman GJ, Austen KF. 1994. Expression cloning of a cDNA for human leukotriene C4 synthase, a novel integral membrane protein conjugating reduced glutathione to leukotriene A4 . Proc. Natl. Acad. Sci. USA 91:7663–67 37. Penrose JF, Spector J, Baldasaro M, Xu K, Boyce J, et al. 1996. Molecular cloning of the gene for human leukotriene C4 synthase: organization, nucleotide sequence, and chromosomal localization to 5q35. J. Biol. Chem. 271:11356–61 38. Lam BK, Penrose JF, Rokach J, Xu K, Baldasaro MH, Austen KF. 1996. Molecular cloning, expression, and characterization of mouse leukotriene C4 synthase. Eur. J. Biochem. 238:606–12 39. Penrose JF, Baldasaro MH, Webster M, Xu K, Austen KF, Lam BK. 1997. Molecular cloning of the gene for mouse leukotriene C4 synthase. Eur. J. Biochem. 248:807– 13 40. Hsieh FH, Lam BK, Penrose JF, Austen KF, Boyce JA. 2001. T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood–derived mast cells: profound induction of leukotriene C4 synthase expression by interleukin 4. J. Exp. Med. 193:123–33 41. Mellor EA, Frank N, Soler D, Hodge MR, Lora JM, et al. 2003. Expression of the type 2 receptor for cysteinyl leukotrienes (CysLT2 R) by human mast cells: functional distinction from CysLT1 R. Proc. Natl. Acad. Sci. USA 100:11589–93 42. Lam BK, Penrose JF, Xu K, Baldasaro MH, Austen KF. 1997. Site-directed mutagenesis of human leukotriene C4 synthase. J. Biol. Chem. 272:13923–28 43. Schmidt-Krey I, Kanaoka Y, Mills DJ, Irikura D, Haase W, et al. 2004. Human leukotriene C4 synthase at 4.5 A˚ resolution in projection. Structure 12:2009– 14 44. Ago H, Kanaoka Y, Irikura D, Lam BK, Shimamura T, et al. 2007. Crystal structure of LTC4 synthase, the membrane protein for cysteinyl leukotriene biosynthesis. Nature 448:609–12 45. Kanaoka Y, Maekawa A, Penrose JF, Austen KF, Lam BK. 2001. Attenuated zymosaninduced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase. J. Biol. Chem. 276:22608–13

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46. Maekawa A, Kanaoka Y, Lam BK, Austen KF. 2001. Identiﬁcation in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing. Proc. Natl. Acad. Sci. USA 98:2256–61 47. Maekawa A, Austen KF, Kanaoka Y. 2002. Targeted gene disruption reveals the role of cysteinyl leukotriene 1 receptor in the enhanced vascular permeability of mice undergoing acute inﬂammatory responses. J. Biol. Chem. 277:20820–24 48. Beller TC, Maekawa A, Friend DS, Austen KF, Kanaoka Y. 2004. Targeted gene disruption reveals the role of the cysteinyl leukotriene 2 receptor in increased vascular permeability and in bleomycin-induced pulmonary ﬁbrosis in mice. J. Biol. Chem. 44:46129– 34 49. Beller TC, Friend DS, Maekawa A, Lam BK, Austen KF, Kanaoka Y. 2004. Cysteinyl leukotriene 1 receptor controls the severity of chronic pulmonary inﬂammation and ﬁbrosis. Proc. Natl. Acad. Sci. USA 101:3047–52 50. Caulﬁeld JP, Lewis RA, Hein A, Austen KF. 1980. Secretion in dissociated human pulmonary mast cells: evidence for solubilization of granule contents before discharge. J. Cell Biol. 85:299–312 51. Schwartz LB, Riedel C, Caulﬁeld JP, Wasserman SI, Austen KF. 1981. Cell association of complexes of chymase, heparin proteoglycan, and protein after degranulation by rat mast cells. J. Immunol. 126:2071–78 52. Schwartz LB, Lewis RA, Austen KF. 1981. Tryptase from human pulmonary mast cells. Puriﬁcation and characterization. J. Biol. Chem. 256:11939–43 53. Schwartz LB, Kawahara MS, Hugli TE, Vik D, Fearon DT, Austen KF. 1983. Generation of C3a anaphylatoxin from human C3 by human mast cell tryptase. J. Immunol. 130:1891– 95 54. Reynolds DS, Gurley DS, Stevens RL, Sugarbaker DJ, Austen KF, Seraﬁn WE. 1989. Cloning of cDNAs that encode human mast cell carboxypeptidase A, and comparison of the protein with mouse mast cell carboxypeptidase A and rat pancreatic carboxypeptidases. Proc. Natl. Acad. Sci. USA 86:9480–84 55. Reynolds DS, Gurley DS, Austen KF. 1992. Cloning and characterization of the novel gene for mast cell carboxypeptidase A. J. Clin. Invest. 89:273–82 56. Schneider LA, Schlenner SM, Feyerabend TB, Wunderlin M, Rodewald H-R. 2007. Molecular mechanism of mast cell-mediated defense against endothelial and snake venom sarafotoxin. J. Exp. Med. 204:2629–39 57. Reynolds DS, Seraﬁn WE, Faller DV, Wall DA, Abbas AK, et al. 1988. Immortalization of murine connective tissue-type mast cells at multiple stages of their differentiation by coculture of splenocytes with ﬁbroblasts that produce Kirsten sarcoma virus. J. Biol. Chem. 263:12783–91 58. Reynolds DS, Stevens RL, Gurley DS, Lane WS, Austen KF, Seraﬁn WE. 1989. Isolation and molecular cloning of mast cell carboxypeptidase A. J. Biol. Chem. 264:20094– 99 59. Reynolds DS, Stevens RL, Lane WS, Carr MH, Austen KF, Seraﬁn WE. 1990. Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases. Proc. Natl. Acad. Sci. USA 87:3230–34 60. Seraﬁn WE, Reynolds DS, Rogelj S, Lane WS, Conder GA, et al. 1990. Identiﬁcation and molecular cloning of a novel mouse mucosal mast cell serine protease. J. Biol. Chem. 265:423–29 61. Seraﬁn WE, Sullivan TP, Conder GA, Ebrahimi A, Marcham P, et al. 1991. Cloning of the cDNA and gene for mouse mast cell protease 4: demonstration of its late transcription www.annualreviews.org • Doing What I Like

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in mast cell subclasses and analysis of its homology to subclass-speciﬁc neutral proteases of the mouse and rat. J. Biol. Chem. 266:1934–41 McNeil HP, Austen KF, Somerville LL, Gurish MF, Stevens RL. 1991. Molecular cloning of the mouse mast cell protease-5 gene: a novel secretory granule protease expressed early in the differentiation of serosal mast cells. J. Biol. Chem. 266:20316–22 Reynolds DS, Gurley DS, Austen KF, Seraﬁn WE. 1991. Cloning of the cDNA and gene of mouse mast cell protease-6: transcription by progenitor mast cells and mast cells of the connective tissue subclass. J. Biol. Chem. 266:3847–53 Hunt JE, Stevens RL, Austen KF, Zhang J, Xia Z, Ghildyal N. 1996. Natural disruption of the mouse mast cell protease 7 gene in the C57BL/6 mouse. J. Biol. Chem. 271:2851–55 Hunt JE, Friend DS, Gurish MF, Feyfant E, Sali A, et al. 1997. Mouse mast cell protease 9, a novel member of the chromosome 14 family of serine proteases that is selectively expressed in uterine mast cells. J. Biol. Chem. 272:29158–66 Gurish MF, Ghildyal N, McNeil HP, Austen KF, Gillis S, Stevens RL. 1992. Differential expression of secretory granule proteases in mouse mast cells exposed to interleukin 3 and c-kit ligand. J. Exp. Med. 175:1003–12 Ghildyal N, Friend DS, Nicodemus CF, Austen KF, Stevens RL. 1993. Reversible expression of mouse mast cell protease 2 mRNA and protein in cultured mast cells exposed to interleukin-10. J. Immunol. 151:3206–14 Xia Z, Ghildyal N, Austen KF, Stevens RL. 1996. Post-transcriptional regulation of chymase expression in mast cells: a cytokine-dependent mechanism for controlling the expression of granule neutral proteases of hematopoietic cells. J. Biol. Chem. 271:8747– 53 Friend DS, Ghildyal N, Austen KF, Gurish MF, Matsumoto R, Stevens RL. 1996. Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype. J. Cell Biol. 135:279–90 Gurish MF, Pear WS, Stevens RL, Scott ML, Sokol K, et al. 1995. Tissue-regulated differentiation and maturation of a v-abl-immortalized mast cell-committed progenitor. Immunity 3:175–86 Yurt RW, Leid RW Jr, Austen KF, Silbert JE. 1977. Native heparin from rat peritoneal mast cells. J. Biol. Chem. 252:518 Metcalfe DD, Lewis RA, Silbert JE, Rosenberg RD, Wasserman SI, Austen KF. 1979. Isolation and characterization of heparin from human lung. J. Clin. Invest. 64:1537–43 Metcalfe DD, Smith JA, Austen KF, Silbert JA. 1980. Polydispersity of rat mast cell heparin. Implications for proteoglycan assembly. J. Biol. Chem. 255:11753–58 Razin E, Stevens RL, Akiyama F, Schmid K, Austen KF. 1982. Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate. J. Biol. Chem. 257:7229–36 Seraﬁn WE, Katz HR, Austen KF, Stevens RL. 1986. Complexes of heparin proteoglycans, chondroitin sulfate E proteoglycans, and [3 H]diisopropyl ﬂuorophosphate-binding proteins are exocytosed from activated mouse bone marrow-derived mast cells. J. Biol. Chem. 261:15017–21 Avraham S, Stevens RL, Gartner MC, Austen KF, Lalley PA, Weis JH. 1988. Isolation of a cDNA that encodes the peptide core of the secretory granule proteoglycan of rat basophilic leukemia-1 cells and assessment of its homology to the human analogue. J. Biol. Chem. 263:7292–96

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77. Stevens RL, Avraham S, Gartner MC, Bruns GAP, Austen KF, Weis JH. 1988. Isolation and characterization of a cDNA that encodes the peptide core of the secretory granule proteoglycan of human promyelocytic leukemia HL-60 cells. J. Biol. Chem. 263:7287– 91 78. Avraham S, Austen KF, Nicodemus CF, Gartner MC, Stevens RL. 1989. Cloning and characterization of the mouse gene that encodes the peptide core of secretory granule proteoglycans and expression of this gene in transfected rat-1 ﬁbroblasts. J. Biol. Chem. 264:16719–26 79. Kitamura Y, Go S, Hatanaka K. 1978. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood 52:447–52 80. Rodewald HR, Dessing M, Dvorak AM, Galli SJ. 1996. Identiﬁcation of a committed precursor for the mast cell lineage. Science 271:818–22 81. Crapper RM, Schrader JW. 1983. Frequency of mast cell precursors in normal tissues determined by an in vitro assay: antigen induces parallel increases in the frequency of P cell precursors and mast cells. J. Immunol. 131:923–28 82. Gurish MF, Tao H, Abonia JP, Arya A, Friend DS, et al. 2001. Intestinal mast cell progenitors require CD49dβ7 (α4β7 integrin) for tissue-speciﬁc homing. J. Exp. Med. 194:1243–52 83. Abonia JP, Austen KF, Rollins BJ, Joshi SK, Flavell RA, et al. 2005. Constitutive homing of mast cell progenitors to the intestine depends on autologous expression of the chemokine receptor CXCR2. Blood 105:4308–13 84. Abonia JP, Hallgren J, Jones T, Shi R, Xu Y, et al. 2006. Alpha-4 integrins and VCAM-1, but not MAdCAM-1, are essential for recruitment of mast cell progenitors to the inﬂamed lung. Blood 108:1588–94 85. Arinobu Y, Iwasaki H, Gurish MF, Mizuno S, Shigematsu H, et al. 2005. Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis. Proc. Natl. Acad. Sci. USA 102:18105–10 86. Katz HR, Benson AC, Austen KF. 1989. Activation and phorbol ester-stimulated phosphorylation of a plasma membrane glycoprotein antigen expressed on mouse IL-3dependent mast cells and serosal mast cells. J. Immunol. 142:919–26 87. Arm JP, Gurish MF, Reynolds DS, Scott HC, Gartner CS, et al. 1991. Molecular cloning of gp49, a cell surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily. J. Biol. Chem. 266:15966– 73 88. Castells MC, Wu X, Arm JP, Austen KF, Katz HR. 1994. Cloning of the gp49B gene of the immunoglobulin superfamily and demonstration that one of its two products is an early-expressed mast cell surface protein originally described as gp49. J. Biol. Chem. 269:8393–401 89. Katz HR, Vivier E, Castells MC, McCormick MJ, Chambers JM, Austen KF. 1996. Mouse mast cell gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs and suppresses mast cell activation when coligated with the high afﬁnity Fc receptor for IgE. Proc. Natl. Acad. Sci. USA 93:10809–14 90. Daheshia M, Friend DS, Grusby MJ, Austen KF, Katz HR. 2001. Increased severity of local and systemic anaphylactic reactions in gp49B1-deﬁcient mice. J. Exp. Med. 194:227– 33 91. Feldweg AM, Friend DS, Zhou JS, Kanaoka Y, Daheshia M, et al. 2003. gp49B1 suppresses stem cell factor-induced mast cell activation-secretion and attendant inﬂammation in vivo. Eur. J. Immunol. 33:2262–68 www.annualreviews.org • Doing What I Like

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92. Zhou JS, Friend DS, Feldweg AM, Daheshia M, Li L, et al. 2003. Prevention of lipopolysaccharide-induced microangiopathy by gp49B1: evidence for an important role for gp49B1 expression on neutrophils. J. Exp. Med. 198:1243–51 93. Arm JP, Nwankwo C, Austen KF. 1997. Molecular identiﬁcation of a novel family of human Ig superfamily members that possess immunoreceptor tyrosine-based inhibition motifs and homology to the mouse gp49B1 inhibitory receptor. J. Immunol. 159:2342–49 94. Pillemer L, Blum L, Lepow IH, Ross OA, Todd EW, Wardlaw AC. 1954. The properdin system and immunity. I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science 120:279–85 95. Fearon DT, Austen KF, Ruddy S. 1973. Formation of a hemolytically active cellular intermediate by the interaction between properdin factors B and D and the activated third component of complement. J. Exp. Med. 138:1305–13 96. Fearon DT, Austen KF. 1975. Properdin: binding to C3b and stabilization of the C3bdependent convertase. J. Exp. Med. 142:856–63 97. Weiler JM, Daha MR, Austen KF, Fearon DT. 1976. Control of the ampliﬁcation convertase of complement by the plasma protein β1H. Proc. Natl. Acad. Sci. USA 73:3268–72 98. Whaley K, Ruddy S. 1976. Modulation of the alternative complement pathways by beta 1H globulin. J. Exp. Med. 144:1147–63 99. Fearon DT, Austen KF. 1975. Initiation of C3 cleavage in the alternative complement pathway. J. Immunol. 115:1357–61 100. Fearon DT, Austen KF, Ruddy S. 1974. Properdin factor D: characterization of its active site and isolation of the precursor form. J. Exp. Med. 139:355–66 101. Fearon DT, Austen KF. 1977. Activation of the alternative complement pathway due to resistance of zymosan-bound ampliﬁcation convertase to endogenous regulatory mechanisms. Proc. Natl. Acad. Sci. USA 74:1683–87 102. Fearon DT, Austen KF. 1977. Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins. J. Exp. Med. l46:22–33 103. Fearon DT. 1978. Regulation by membrane sialic acid of beta 1H-dependent decaydissociation of ampliﬁcation C3 convertase of the alternative complement pathway. Proc. Natl. Acad. Sci. USA 75:1971–75 104. Soter NA, Lewis RA, Corey EJ, Austen KF. 1983. Local effects of synthetic leukotrienes (LTC4 , LTD4 , LTE4 and LTB4 ) in human skin. J. Invest. Dermatol. 80:115– 19 105. Weiss JW, Drazen JM, Coles N, McFadden ER Jr, Weller PF, et al. 1982. Bronchoconstrictor effects of leukotriene C in humans. Science 216:196–98 106. Grifﬁn M, Weiss JW, Leitch AG, McFadden ER Jr, Corey EJ, et al. 1983. Effects of leukotriene D on the airways in asthma. N. Engl. J. Med. 308:436–39 107. Davidson AB, Lee TH, Scanlon PD, Solway J, McFadden ER Jr, et al. 1987. Bronchoconstrictor effects of leukotriene E4 in normal and asthmatic individuals. Am. Rev. Respir. Dis. 135:333–37 108. Sheffer AL, Austen KF. 1980. Exercise-induced anaphylaxis. J. Allergy Clin. Immunol. 66:106–11 109. Sheffer AL, Soter NA, McFadden ER Jr, Austen KF. 1983. Exercise-induced anaphylaxis: a distinct form of physical allergy. J. Allergy Clin. Immunol. 71:311–l6 110. Sheffer AL, Tong AKF, Murphy GF, Lewis RA, McFadden ER Jr, Austen KF. 1985. Exercise-induced anaphylaxis: a serious form of physical allergy associated with mast cell degranulation. J. Allergy Clin. Immunol. 75:479–84

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111. Soter NA, Wasserman SI, Austen KF, McFadden ER Jr. 1980. Release of mast cell mediators and alterations in lung function in patients with cholinergic urticaria. N. Engl. J. Med. 302:604–8 112. Roberts LJ II, Lewis RA, Oates JA, Austen KF. 1979. Prostaglandin, thromboxane, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid production by ionophore-stimulated rat serosal mast cells. Biochim. Biophys. Acta 575:185–92 113. Lewis RA, Soter NA, Diamond PT, Austen KF, Oates JA, Roberts LJ II. 1982. Prostaglandin D2 generation after activation of rat and human mast cells with anti-IgE. J. Immunol. 129:1627–31 114. Roberts LJ II, Sweetman BJ, Lewis RA, Austen KF, Oates JA. 1980. Increased production of prostaglandin D2 in patients with systemic mastocytosis. N. Engl. J. Med. 303:1400–4 115. Soter NA, Austen KF, Wasserman SI. 1979. Oral disodium cromoglycate in the treatment of systemic mastocytosis. N. Engl. J. Med. 301:465–69 116. Donaldson VH, Evans RR. 1963. A biochemical abnormality in hereditary angioneurotic edema: absence of serum inhibitor of C’1-esterase. Am. J. Med. 35:37–44 117. Austen KF, Sheffer AL. 1965. Detection of hereditary angioneurotic edema by demonstration of a profound reduction in the second component of human complement. N. Engl. J. Med. 272:649–56 118. Ruddy S, Austen KF. 1967. A stoichiometric assay for the fourth component of complement in whole human serum using EAC’1agp and functionally pure human second component. J. Immunol. 99:1162–72 119. Spaulding WB. 1955. Hereditary angioneurotic oedema in two families. Can. Med. Assoc. J. 73:181–87 120. Sheffer AL, Fearon DT, Austen KF. 1979. Clinical and biochemical effects of impeded androgen (oxymethalone) therapy of hereditary angioedema (HAE). J. Allergy Clin. Immunol. 64:275–80 121. Sheffer AL, Fearon DT, Austen KF. 1981. Clinical and biochemical effects of stanozolol therapy for hereditary angioedema. J. Allergy Clin. Immunol. 68:181–87 122. Caldwell JR, Ruddy S, Schur PH, Austen KF. 1972. Acquired C1 inhibitor deﬁciency in lymphosarcoma. Clin. Immunol. Immunopathol. 1:39 123. Geha RS, Quinti I, Austen KF, Cicardi M, Sheffer A, Rosen FS. 1985. Acquired C1inhibitor deﬁciency associated with antiidiotypic antibody to monoclonal immunoglobulins. N. Engl. J. Med. 312:534–40 124. Fearon DT. 1980. Identiﬁcation of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J. Exp. Med. 152:20–30 125. Nicholson-Weller A, Burge J, Fearon DT, Weller PF, Austen KF. 1982. Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system. J. Immunol. 129:184–89 126. Nicholson-Weller A, March JP, Rosenfeld SI, Austen KF. 1983. Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deﬁcient in the complement regulatory protein, decay accelerating factor. Proc. Natl. Acad. Sci. USA 80:5066– 70 127. Hunsicker LG, Ruddy S, Carpenter CB, Schur PH, Merrill JP, et al. 1972. Metabolism of third complement component (C3) in nephritis: role of the classical and alternate (properdin) pathways for complement activation. N. Engl. J. Med. 287:835–40 128. Daha MR, Fearon DT, Austen KF. 1976. C3 nephritic factor (C3NeF): stabilization of ﬂuid phase and cell-bound alternative pathway convertase. J. Immunol. 116:1–7 www.annualreviews.org • Doing What I Like

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129. Daha MR, Austen KF, Fearon DT. 1977. The incorporation of C3 nephritic factor (C3NeF) into a stabilized C3 convertase, C3b, Bb (C3NeF) and its release after decay of convertase function. J. Immunol. 19:812–17 130. Daha MR, Austen KF, Fearon DT. 1978. Heterogeneity, polypeptide composition and antigenic reactivity of C3NeF. J. Immunol. 120:1389–94 131. Austen KF. 2004. Acceptance of the Kober Medal: It only gets better. J. Clin. Invest. 114:1177

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Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin Burnham Institute for Medical Research, La Jolla, California 92037; email: [email protected], [email protected], [email protected], [email protected], [email protected], [email protected]

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Key Words

First published online as a Review in Advance on October 11, 2007

LYP, PTPN22, CD45

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090418 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0029$20.00

Abstract Protein tyrosine phosphatases (PTPs) are important regulators of many cellular functions and a growing number of PTPs have been implicated in human disease conditions, such as developmental defects, neoplastic disorders, and immunodeﬁciency. Here, we review the involvement of PTPs in human autoimmunity. The leading examples include the allelic variant of the lymphoid tyrosine phosphatase (PTPN22), which is associated with multiple autoimmune diseases, and mutations that affect the exon-intron splicing of CD45 (PTPRC). We also ﬁnd it likely that additional PTPs are involved in susceptibility to autoimmune and inﬂammatory diseases. Finally, we discuss the possibility that PTPs regulating the immune system may serve as therapeutic targets.

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INTRODUCTION

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T regulatory (Treg) cells: specialized T cells (CD4+ CD25+ Foxp3+ ) that suppress activation of the adaptive immune system, contributing to self-tolerance and immune system homeostasis Tolerance: a state of unresponsiveness of cells of the adaptive immune system to self-antigens TCR: T cell antigen receptor

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Over 100 years ago Paul Ehrlich ﬁrst proposed the concept of horror autotoxicus, the unthinkable possibility that an organism’s immune system would mount a response against itself. Today, we know that breakdown of self-tolerance is not uncommon and can result in serious immune-mediated damage and destruction of an individual’s own cells and tissues. There are currently over 80 diseases classiﬁed as autoimmune, affecting approximately 5% of the human population. Moreover, autoimmune disorders are among the top 10 leading causes of death and affect at least 10 million individuals in the United States alone. Autoimmunity develops through a combination of environmental and genetic factors resulting in a broad spectrum of human illnesses. Many genes identiﬁed as causing or predisposing to autoimmunity encode proteins that are involved in lymphocyte, macrophage, or dendritic cell signal transduction (1–7). Studies conducted with patient samples and with numerous animal models of autoimmune disease have demonstrated that subtle imbalances in the regulation of the activation and/or function of immune cells can precipitate the development of autoimmunity. Although several autoimmune diseases are characterized by the presence of autoantibodies produced by autoreactive B cells, genetic evidence [e.g., major histocompatibility complex (MHC) association] and animal models point to a central role of autoreactive T cells as the primary mediators of autoimmune disease. More recently, T cell subsets, such as CD4+ CD25+ Foxp3+ T regulatory (Treg) cells and T helper 17 (Th17) cells, have been implicated in the maintenance of peripheral tolerance (discussed below) and in promoting inﬂammation and tissue destruction (8, 9), respectively. Even though the etiology of each autoimmune disease is different (and often poorly understood), it appears that a common theme is a failure of immunologic tolerance, presumably by one or more lymphocyte subsets. Central tolerance is generated through mechanisms of Vang et al.

thymic negative selection wherein developing T cells with medium to high afﬁnity for tissuespeciﬁc self-antigens are deleted, thereby eliminating putative autoreactive T cells from the peripheral T cell repertoire. Alternatively, thymocytes with T cell antigen receptors (TCRs) recognizing self-antigens can develop into Treg cells, which then function in the periphery to control self-reactive T cells that have escaped clonal deletion (10). Autoimmunity may develop if TCR signal strength is altered, for example by mutations in signaling proteins, leading to decreased effectiveness of negative selection allowing for the “escape” of autoreactive T cells and/or altering the development of Treg cells in the thymus. Analogous mechanisms are operative in B cells where high-afﬁnity/avidity self-antigens induce rapid elimination (clonal deletion) of newly formed B cells in the bone marrow. Low-afﬁnity/avidity interactions, by contrast, induce an anergic state where B cells are short lived and will succumb to apoptosis in the sustained presence of self-antigen (11, 12). Peripheral tolerance encompasses a myriad of mechanisms that include clonal anergy via activation by immature dendritic cells, clonal ignorance (13), clonal exhaustion, immune privilege, and regulation by Treg cells (14–16). Of note, Treg cells generated in the periphery, so-called adaptive Tregs, through the concerted actions of autoantigen and transforming growth factor-β (TGF-β), also function to suppress T cell activation. B cells can acquire self-reactivity as a consequence of V gene hypermutation during T cell–dependent differentiation in the germinal center. In such a case, altered antigen receptor speciﬁcity could lead to the efﬁcient uptake and presentation of autoantigens as well as the generation of plasma cells producing autoantibodies. Together, the mechanisms of peripheral tolerance are aimed at preventing activation of autoreactive T cells, thereby avoiding immune responses against self and, ultimately, tissue damage. Because of the increasing prevalence of autoimmune diseases, particularly in the

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industrialized world, a better understanding of the molecular mechanisms of autoimmunity would be helpful for the development of more effective and speciﬁc treatments. Signaling molecules in immune cells are promising targets for such treatments. Protein tyrosine phosphatases (PTPs) constitute a group of signaling mediators playing important regulatory roles in immune cells and may prove useful as targets for medical treatment of autoimmune disorders. This review focuses on the known roles of PTPs in autoimmunity.

PROTEIN TYROSINE PHOSPHATASES The human genome contains a total of 107 genes encoding either experimentally veriﬁed PTPs or proteins with a domain homologous to the catalytic domain of these PTPs, i.e., probable PTPs (17). The corresponding gene products of 81 of these genes are predicted to be active protein phosphatases; 13 dephosphorylate inositol phospholipids, 2 dephosphorylate mRNA, and 11 are catalytically inactive. On the basis of the primary structure of the catalytic domains, PTPs can be divided into four evolutionarily distinct classes. Class I PTPs constitute the largest group with 99 members, including 38 classical PTPs and 61 VH1-like (dual-speciﬁc) phosphatases (DSPs). The classical PTPs can further be divided into receptor and nonreceptor PTPs. There are seven different subgroups of DSPs [MAPK phosphatases (MKPs), atypical DSPs, slingshots, PRLs, CDC14s, phosphatase and tensin homologs (PTENs), and myotubularins]. Class II contains only one member [low-molecular-weight phosphotyrosine phosphatase (LMPTP)], whereas class III has three members (CDC25 A, B, and C). All the members of these three classes of PTPs use a cysteine-based catalytic mechanism. In contrast, class IV PTPs are aspartate based and are currently represented by the four Eya genes. The expression patterns of individual PTPs vary from ubiquitous to strictly tissue

speciﬁc. Most cells express 30% to 60% of the entire complement of PTPs. Neuronal and hematopoietic cells tend to express a relatively high number of PTPs. T cells, for instance, contain between 60 and 70 different PTPs, and a similar set is found in B cells (18). Several PTPs are restricted to hematopoietic cells, for example LYP (lymphoid tyrosine phosphatase, also known as PTPN22); the mouse ortholog is called PEP [proline-, glutamicacid-, serine-, and threonine-rich (PEST)domain-enriched PTP)], SHP1 (Src homology 2-domain-containing PTP 1), CD45, and HePTP (hematopoietic PTP). Of the 60–70 PTPs expressed in T cells, approximately 20 regulate signaling events between the TCR and transactivation of the interleukin-2 (IL-2) gene (18). Most of these PTPs affect TCR signaling in an inhibitory manner, but a few (such as CD45, LMPTP, and SHP2) have a positive regulatory role. The importance of PTPs in immune cell signaling has recently been extensively reviewed (18, 19), and here we focus only on the involvement of PTPs in autoimmune disease (Table 1).

PTP: protein tyrosine phosphatase LYP: lymphoid tyrosine phosphatase Proline-rich motif/domain: a motif/domain that contains the proline-rich sequence PxxP and is found in numerous signaling proteins

LYMPHOID TYROSINE PHOSPHATASE (LYP; PTPN22) AND AUTOIMMUNITY Structure and Function of LYP The human PTPN22 gene is located on chromosome 1p13.3–13.1. It encodes an 807amino acid residue protein, LYP (20), which belongs to the PEST group of nonreceptor classical class I PTPs. The two other members of this family are PTP-PEST (PTP with PEST domain, genomic designation PTPN12) and PTP-HSCF (PTP hematopoietic stem-cell fraction, genomic designation PTPN18). LYP contains an N-terminal PTP domain, a central region of unknown function, and a C-terminal portion of approximately 200 amino acids containing four proline-rich motifs termed P1-P4. P4 is part of the so-called www.annualreviews.org • Phosphatases and Autoimmunity

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List of PTPs involved in autoimmunity, their mechanisms and phenotypesa

Common name

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Genomic designation

Synonyms

Molecular mechanism(s) for disease development

Cellular mechanism(s) for disease development

Phenotype(s)b

Gain-of-function mutation, reduced TCR signaling

Reduced negative selection in thymus? Reduced activity of Treg cells? Altered activity of other hematopoietic cells?

Associated with T1D, RA, JIA, SLE, Graves’ disease, myasthenia gravis, generalized vitiligo, Wegener’s granulomatosis

LYP (PEP in mouse)

PTPN22

PTP-PEST

PTPN12

PTPG1, PTP-P19

Mutations in CD2BP1 cause loss of PTP-PEST binding. Altered spatial regulation of PTP-PEST?

Exaggerated proliferation and inﬁltration of neutrophils. Altered activities of B or T cells?

Indirectly associated with the autoinﬂammatory condition FRA/PAPA syndrome

CD45

PTPRC

B220, Gp180, LCA, Ly5, T220

Aberrant CD45 activity due to abnormal splicing, the produced CD45RA isoforms are less likely to dimerize and hence are less likely to be autoinhibited

Altered B and/or T cell function? In mice with aberrant CD45 activity, B cells are hyperresponsive and hyperproliferative

Associated with autoimmune hepatitis and systemic sclerosis, and perhaps MS. In mice with aberrant CD45 activity, SLE-like disease and lymphoproliferative disorder are observed

IA-2

PTPRN

Islet cell antigen 512

The normal protein serves as autoantigen

Serves as autoantigen in pancreatic β-cells in T1D

T1D

Ia-2β

PTPRN2

Phogrin, PTPRP, RPTPπ

The normal protein serves as autoantigen

Serves as autoantigen in pancreatic β-cells in T1D

T1D

SHP1

PTPN6

HCP, Hcph, PTP1C, SH-PTP1

In mice, loss of expression or loss of function

In mice, inﬂammation caused by aberrant myeloid cells, B cell abnormalities

In mice, motheaten phenotype; B cells alone cause SLE-like disease

a The list includes the most relevant PTPs with regard to autoimmunity. The PTPs are listed in the order they appear in the text. Unless otherwise stated, the information given applies for humans. b Abbreviations: T1D, type 1 diabetes; RA, rheumatoid arthritis; JIA, juvenile idiopathic arthritis; SLE, systemic lupus erythematosus; FRA/PAPA, familial recurrent arthritis/pyogenic sterile arthritis, pyoderma gangrenosum, and acne syndrome; MS, multiple sclerosis.

C-terminal homology (CTH) domain, which is found in all members of the PEST group of PTPs (21, 22). LYP may also exist as an alternatively spliced form called LYP2, which has a shorter C terminus, resulting in the absence of P2, P3, and P4 (CTH domain) (20), 32

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although this form has not been detected by others. The mouse ortholog of LYP is called PEP (23), and it was given the genomic designation PTPN8 because initially it was not clear that LYP and PEP were species orthologs. Both LYP and PEP are expressed exclusively

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in hematopoietic cells and share 89% identity between their PTP domains and 61% identity for their noncatalytic portions. Much of the current knowledge regarding LYP/PEP is derived from studies of PEP, and it should be stressed that data obtained with PEP may not necessarily apply completely to LYP, as these two PTPs show subtle biochemical differences in some assays (T. Vang & T. Mustelin, unpublished observations). The subcellular localization of LYP is mainly cytoplasmic, but some PEP can also be found in the nucleus (20, 24). Factors determining the localization of LYP/PEP in cells are poorly understood, but several binding partners have been identiﬁed. The best characterized is C-terminal Src kinase (Csk), which through its Src homology 3 (SH3) domain binds to the P1 region in LYP/PEP (25–27). This interaction is of relatively high stoichiometry: In mouse T cells, approximately 5% of total cellular Csk associates with 25% to 50% of total cellular PEP (25); the corresponding numbers for LYP in human cells are not known. Like other PEST group PTPs, both LYP and PEP contain a CTH domain at their C termini to which the coiled-coil domain of the cytoskeletalassociated protein proline-serine-threonine PTP-associated protein (PSTPIP) can bind (T. Vang, A.V. Miletic & T. Mustelin, unpublished observations). The human ortholog of PSTPIP is called CD2-binding protein 1 (CD2BP1). There are also reports that LYP can interact with the adaptor molecule Grb2 (28) as well as the adaptor molecule and the E3 ligase c-Cbl (20). Both PEP and LYP inhibit TCR signaling by acting immediately downstream of the TCR. Speciﬁcally, PEP has been implicated in the dephosphorylation of the positive regulatory tyrosine residue in the activation loop of the Src family kinases (SFKs) FynT (Y417) and Lck (Y394), as well as ZAP-70 (29, 30). PEP also negatively impacts TCR-induced phosphorylation of the tyrosines within the immunoreceptor tyrosinebased activation motifs (ITAMs) in CD3/ζ-

chains, but these effects may be indirect because SFKs (particularly Lck) are responsible for phosphorylation of ITAMs (29). A recent study using a substrate-trapping mutant version of LYP combined with mass spectrometry identiﬁed the following substrates: Lck (Y394), the ITAMs of CD3/ζ-chains, ZAP-70 (Y493), Vav, and valosin-containing protein (31). The ability of LYP/PEP to bind Csk may be important. Although PEP dephosphorylates the positive regulatory tyrosine in the activation loop of Lck (Y394) and FynT (Y417), Csk phosphorylates the C-terminal negative regulatory tyrosine in Lck (Y505) and FynT (Y528) (29, 30). However, recent data contradict the notion that Csk association promotes LYP/PEP function in TCR signaling. First, PEP cannot bind Csk when the latter interacts with the membrane protein Cbp/PAG (Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched membrane domains), which exclusively partitions into lipid rafts and contributes to raft targeting of Csk (32). Second, PEP is in fact not found in lipid rafts (32). Therefore, at present it is unclear how PEP targets lipid raft–resident proteins such as Lck and FynT. In comparison, in human T cells, a small fraction of LYP partitions into lipid rafts, but this partitioning is independent of Csk binding, highlighting interesting differences between PEP and LYP (T. Vang, A.V. Miletic & T. Mustelin, unpublished observations). Mice deﬁcient in PEP have normal resting T cell numbers and subpopulations but exhibit enhanced memory T cell responses (33). Furthermore, restimulation of T cells from these animals is associated with elevated and sustained TCR-induced phosphorylation of both Lck-Y394 and of ZAP-70, as well as augmented proliferation. The lack of a noticeable phenotype in naive T cells from PEP−/− mice may be due to redundancy with PTP-PEST (Y. Arimura & T. Mustelin, unpublished observations). A recent study found that microRNA-181a targets and downregulates PEP expression, resulting in elevated www.annualreviews.org • Phosphatases and Autoimmunity

C-terminal homology (CTH) domain: a proline-rich sequence found at the C terminus of PEST group PTPs Csk: C-terminal Src kinase Src homology 3 (SH3) domain: a protein domain of approximately 60 amino acids capable of binding proline-rich motifs SFK: Src family kinase

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Single-nucleotide polymorphism (SNP): one nucleotide in a gene differs between members of a species or between paired chromosomes in an individual causing a DNA sequence variation T1D: type 1 diabetes RA: rheumatoid arthritis

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Lck-Y394 phosphorylation in resting T cells and rendering the cells hyperresponsive to TCR stimulation (34). In addition to PEP, microRNA-181a also targets DUSP6, a MAP kinase-speciﬁc phosphatase, which may contribute to the downstream response (34). In accordance with these observations for PEP, acute elimination of LYP in Jurkat T cells using RNA interference resulted in increased TCR-mediated activation of nuclear factor κB (35). Although most of our knowledge about LYP/PEP is derived from studies on T cells, LYP/PEP is also expressed in all other leukocyte lineages. In PEP-deﬁcient mice, the germinal center B cell population and IgG production are elevated, but it remains to be determined whether these effects are B cell intrinsic or secondary to defects in the T cell compartment (33).

Association of the C1858T Polymorphism in PTPN22 with Autoimmune Diseases In 2004, we reported that the C1858T single-nucleotide polymorphism (SNP) in the PTPN22 gene was associated with increased risk of type 1 diabetes (T1D) in two independent populations (6). The following year, association of T1D with the T1858 allele was conﬁrmed in additional large population samples (36–38). Furthermore, the same association was found for rheumatoid arthritis (RA) (35, 39–42), juvenile idiopathic arthritis ( JIA) (42, 43), systemic lupus erythematosus (SLE) (39, 41, 44), Graves’ disease (36, 45), myasthenia gravis (46), generalized vitiligo (47), and Wegener’s granulomatosis (48). Interestingly, some diseases were demonstrated not to be associated with the T1858 allele, including multiple sclerosis (MS) (41, 43, 49), inﬂammatory bowel diseases such as Crohn’s disease (50– 52) and ulcerative colitis (52, 53), celiac disease (42, 54), primary sclerosing cholangitis (42), primary biliary cirrhosis (55), psoriasis (43), and psoriatic arthritis (43). In addition, several other disorders (such as Hashimoto’s 34

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thyroiditis, primary Sjogren’s syndrome, and systemic sclerosis) have been investigated with regard to involvement of the C1858T SNP, but these data are somewhat conﬂicting, and more studies are required. A recent metaanalysis conﬁrmed the association between the T1858 allele and the diseases T1D, RA, JIA, SLE, and Graves’ disease, while there was no association with MS, inﬂammatory bowel disease, psoriasis, or Addison’s disease (56). In general, PTPN22 appears to be coupled to the collection of autoimmune diseases that typically are characterized by circulating autoantibodies. On the basis of many genetic studies conducted with different populations, there is a clear geographic gradient with regard to the frequency of the disease-associated T1858 allele in Europe. Even though this allele is relatively rare in southern European populations (2% in Italy, 6% in Spain), the frequency increases northward through Europe (8% in the United Kingdom, 12% in Sweden, 15.5% in Finland). Interestingly, in African American and Asian populations, the T1858 allele is virtually absent, suggesting a northern European origin and/or selective advantage for the T1858 allele in this region. Nevertheless, an adequate number of studies have now been conducted to demonstrate that the autoimmune disease association with the T1858 allele is population independent. In addition to the C1858T SNP (rs2476601), there are numerous other SNPs in the human PTPN22 gene. An initial investigation of the role of these other SNPs in RA patients conﬁrmed that C1858T is the major disease-associated SNP in PTPN22, but also suggested minor involvement of at least one other SNP (rs3789604) (57). Subsequent studies have supported the notion that C1858T is the only SNP in PTPN22 associated with RA (58–60). Similarly, T1D patients have also been investigated with regard to other SNPs in PTPN22. Whereas one report suggested that a promoter SNP in PTPN22 (-G1123C, rs2488457) confers increased risk

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of T1D (61), a later study could not conﬁrm this connection (62), again supporting a role for C1858T as the major T1D-associated SNP in PTPN22 (63). However, it is still possible that additional polymorphisms may be discovered for PTPN22 and that these are independently related to different and/or overlapping groups of autoimmune disorders. For instance, one recent study suggested that the C1858T SNP was not associated with psoriasis but that there was evidence for a susceptibility locus for this disease somewhere else in PTPN22 or in its vicinity (64). The involvement of PTPN22 in multiple human autoimmune disorders places this gene in company of general autoimmunity genes (such as MHC and CTLA4) that increase the risk for numerous autoimmune diseases. The MHC locus (HLA variants) has the greatest impact among these genes, followed by PTPN22 and CTLA4 susceptibility variants. For T1D, HLA contributes approximately 40% to the familial clustering, while the corresponding number for PTPN22 is about 2% (65). Even so, the odds ratio is typically ∼1.5 for the T1858 allele in T1D, and that is substantially greater than the 1.1 value calculated for the CTLA4 variant predisposing for T1D (66). Obviously, the presence of many susceptibility loci and other factors, although of little impact on their own, can add up and contribute a signiﬁcant portion to the genetic predisposition for T1D. To better predict the risk for autoimmune disorders, MHC haplotype could perhaps be combined with PTPN22 genotype and an additional set of nongenetic factors. Studies published so far have not revealed ﬁrm genetic interactions between PTPN22 and MHC in RA or T1D, but two recent reports demonstrated a strong connection between the disease-associated T1858 allele and the occurrence of anticyclic citrullinated peptide antibodies in RA (67, 68). When combined, these two factors led to an up to 350-fold increased risk of developing RA (67, 68). The concurrent presence of these two factors also predicted

the later development of RA with extremely high accuracy (68). Similarly, the T1858 allele confers increased risk for development of insulin autoantibodies (as well as of other autoantibodies) and progression from insulitis to clinical T1D (69). For other autoimmune diseases, additional factors may likewise enhance the predictive power of PTPN22 C1858T. The C1858T SNP may also potentially serve as a prognostic factor. A link between the T1858 allele and the course of disease or other variables of autoimmune disorders has been reported. For instance, the T allele is preferentially associated with rheumatoid factor (RF)-positive RA and probably not with RFnegative disease (35, 40, 70), although there are some conﬂicting results (43, 50). There also seems to be a dose-dependent effect of the T allele because two copies of this allele give a much higher risk for RF-positive RA compared with one copy alone (40). Furthermore, a recent study demonstrated that RA disease progression rate (evaluated as tissue damage) was increased in patients carrying the T allele (71). The C1858T SNP is also associated with an earlier age at RA onset (67). A similar pattern is observed for other diseases. For example, Graves’ disease will debut at a younger age in T1858/T1858 homozygous patients compared with patients with C1858/C1858 genotype, whereas heterozygous patients develop the disease at an intermediate age (72). In contrast, no correlation between the T allele and severity of Graves’ disease has been demonstrated (72). Likewise, the age at onset for T1D is lower for patients with the T1858/T1858 genotype compared with the C1858/C1858 genotype, but no difference has been seen with regard to autoantibodies (38, 73). Clearly, more work is required to clarify whether and how the C1858T SNP is associated with the clinical course and severity of autoimmune diseases. Such studies will also answer the question of whether determination of a patient’s PTPN22 genotype will be useful for evaluation of disease prognosis and treatment. www.annualreviews.org • Phosphatases and Autoimmunity

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Effects of the Disease-Associated LYP∗ W620 Allele at the Molecular Level

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The C1858T SNP changes amino acid 620 in LYP from an arginine to a tryptophan (6). We refer to the two forms as LYP∗ R620 (encoded by the C1858 allele) and LYP∗ W620 (encoded by the T1858 allele). Residue 620 is located within the P1 region, which interacts with the SH3 domain of Csk. The structure of the Csk SH3 domain with a bound peptide, including the P1 region from PEP (this peptide sequence differs only by one amino acid residue from the corresponding human sequence), was solved by NMR (27). Interestingly, the Csk SH3 domain contains two peptide recognition surfaces: the usual polyproline helix-binding surface of SH3 domains as well as a hydrophobic patch that interacts with hydrophobic residues C-terminal to the polyproline helix of the PEP P1 region. In the P1 sequence PPLPERTPESFIV, the underlined residues are responsible for the binding to the polyproline helix-binding surface, and the two C-terminal residues (I and V) mediate the interaction with the hydrophobic patch. Both of these sets of interactions contribute to the binding afﬁnity. The P1 region in the human LYP∗ R620 (PPLPVRTPESFIV, R620 in bold) differs from the mouse sequence only in residue V619, which corresponds to a glutamic acid in the mouse. However, this residue is not involved in SH3 binding, and because the amino acid sequences of the Csk SH3 domain in humans and mice are identical, it is likely that the PEP-P1/Csk-SH3 and LYP∗ R620P1/Csk-SH3 interactions are virtually identical. These structures also strongly predict that the R620W change in LYP will severely impair the formation of the LYP-Csk complex. This model was conﬁrmed experimentally, both with recombinant and endogenous proteins. Thus, although LYP∗ R620 bound strongly to the SH3 domain of Csk, the corresponding interaction between LYP∗ W620 and Csk was undetectable (5, 6, 35).

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Because of earlier reports that PEP and Csk in complex downmodulate TCR signaling in a cooperative manner (29, 30), we and others expected that the inability of LYP∗ W620 to bind Csk would result in less efﬁcient inhibition of TCR signaling by LYP∗ W620 compared with LYP∗ R620. However, all experimental data clearly demonstrated that the opposite was the case. When expressed in either Jurkat T cells or in primary human T cells, both LYP∗ R620 and LYP∗ W620 reduced TCR signaling in a dosedependent manner, but at equivalent expression levels LYP∗ W620 was consistently more potent (5). This was observed for all TCRsignaling parameters tested, including LckY394 phosphorylation, ζ-chain phosphorylation, LAT phosphorylation, intracellular calcium ﬂux, Erk1/2 phosphorylation, activation of the proximal IL-2 promoter (containing NFAT and AP1 sites), as well as IL-2 secretion. Furthermore, T cells from the peripheral blood of T1D patients with LYP∗ W620 (heterozygous T1858/C1858 genotype) secreted less IL-2 in response to TCR/CD28 costimulation than T cells from patients homozygous for LYP∗ R620. All T cell subsets were similar between the two groups, excluding the possibility that the reduced IL-2 secretion levels were due to a skewing in T cell subsets. Importantly, when T cells from the two patient groups were stimulated with a combination of phorbol ester and ionomycin to bypass proximal TCR signaling, IL-2 secretion levels were indistinguishable, demonstrating that the only differences between the two groups were present in signaling events immediately downstream of the TCR. These ﬁndings raise the question: What is/are the mechanism(s) by which LYP∗ W620 is a stronger inhibitor of TCR signaling? There are several possibilities. Although residue 620 is more than 300 amino acid residues C-terminal to the PTP domain, LYP∗ W620 is approximately 1.5-fold more active a PTP than LYP∗ R620 (5). Although this increase in activity may not seem signiﬁcant, it is consistent with our observation that

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even a modest overexpression of LYP in T cells has a profound inhibitory effect on TCR signaling. It remains unclear, however, how a mutation so far away from the PTP domain can affect the PTP activity. In the absence of crystal structures of the two LYP variants, one can only speculate that the noncatalytic portion of LYP may play a regulatory role in the function of the catalytic domain. In support of this notion, we found that deletion of the C-terminal half of PEP resulted in a ﬁvefold increase in its PTP activity (30), suggesting an interaction between the N and C termini of PEP. Thus, it is possible that binding of ligands to the C-terminal part of LYP may affect the catalytic activity of LYP/PEP, perhaps in a manner reminiscent of the way SH2 domain ligands activate SHP1 or SHP2. Even though Csk coimmunoprecipitated with LYP∗ R620 in the above-mentioned PTP assays, no coimmunoprecipitation was seen between LYP∗ W620 and Csk (5). Theoretically, the absence of Csk binding per se could enhance the catalytic activity of LYP (in what would be the opposite type of regulation compared with SHP1). However, such a model is less likely because the two LYP variants expressed and puriﬁed from a Csk-deﬁcient system did not exhibit changes in PTP activities when increasing amounts of Csk were added (T. Vang & T. Mustelin, unpublished observations). Another possibility is that the R620W mutant confers upon the P1 region of LYP the capacity to interact more strongly with other proteins and that these proteins can induce a more catalytically preferable conformation of the LYP PTP domain. However, in the absence of any experimental evidence, all of these possibilities remain completely speculative. It is reasonable to assume that there is an equilibrium between free LYP and LYP bound to known (Csk, Grb-2, c-Cbl, PSTPIP) or unknown proteins. Any skewing of this equilibrium may have profound cellular effects. In murine T cells, 25% to 50% of total cellular PEP are bound to Csk (25) in what appears to be a high-afﬁnity interaction with a Kd =

0.8 μM (27). This is probably also true for human LYP∗ R620 and Csk. In comparison, most SH3 domain-mediated interactions have a Kd ≥ 10 μM. Thus, a disruption of the LYPCsk interaction, as is the case for LYP∗ W620, may result in a more than 1.5-fold increase in the pool of free LYP (i.e., not bound to Csk), which may bind other proteins. Such a mechanism, combined with altered ligand properties for the P1 region of LYP∗ W620, may contribute to make LYP∗ W620 a stronger inhibitor of TCR signaling compared with LYP∗ R620. We currently have some experimental evidence in support for this model (T. Vang & T. Mustelin, unpublished data). Another explanation for the augmented inhibitory effect of LYP∗ W620 on TCR signaling is altered subcellular localization, which may be tightly connected with a new set of interacting partners for LYP∗ W620 compared with LYP∗ R620. There appear to be subtle differences between the two LYP variants as evaluated by subcellular localization analyses and biochemical fractionation studies (T. Vang, A.V. Miletic & T. Mustelin, unpublished data), but more studies are required to clarify these issues. A ﬁnal explanation for the increased inhibitory potential of LYP∗ W620 is an alteration in the substrate speciﬁcities and/or substrate afﬁnities compared with LYP∗ R620, although this has yet to be experimentally tested.

Src homology 2 (SH2) domain: a protein domain of approximately 100 amino acids capable of binding tyrosine phosphorylated proteins

Effects of the Disease-Associated LYP∗ W620 at the Cellular and Systemic Level A simplistic model of autoimmunity would predict that T cells with abnormalities augmenting TCR signaling would be likely to cause disease. However, there is emerging evidence that the picture is much more complex. For instance, peripheral T cells from T1D patients are hyporesponsive rather than hyperresponsive to TCR stimulation in vitro (74). Furthermore, thymocytes from nonobese diabetic mice are also hyporesponsive with regard to TCR-mediated activation www.annualreviews.org • Phosphatases and Autoimmunity

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and proliferation (75–77). Indirect evidence also suggests that thymocyte hyporesponsiveness because of abnormalities in early TCR signaling can play a causative role in autoimmune disorders (2). Together, these ﬁndings may explain why LYP∗ W620 as a more potent inhibitor of TCR signaling can still enhance the risk for a variety of autoimmune diseases. This also explains why patients with the T1858/T1858 genotype are at a higher risk for autoimmune diseases than heterozygotes (T1858/C1858), because there is a dosedependent effect of LYP∗ W620. Another question is how LYP∗ W620 affects T cells so that autoimmunity develops. At present, there is a lack of experimental data covering this ﬁeld. However, one can envision two possibilities, and they are not mutually exclusive. First, the reduced signal transduction ability downstream of the TCR, caused by LYP∗ W620, may result in a skewing of the TCR repertoire toward generation of more autoreactive TCRs in the thymus. The resulting failure in deleting these autoreactive thymocytes by negative selection gives rise to a peripheral T cell population with enhanced ability for self-recognition. Second, the presence of LYP∗ W620 in Treg cells may inhibit signaling in these cells so that they become insufﬁcient in their ability to control the activity of autoreactive T cells in peripheral blood and tissues. Interestingly, a recent report demonstrated that PTPN22 is a direct Foxp3 target gene and that, although stimulation of Foxp3− hybridoma cells resulted in upregulation of PTPN22, this increase was not observed in Foxp3+ hybridoma cells or in ex vivo Treg cells (78). There are still several unanswered questions. All our knowledge about the cellular effects of LYP∗ W620 comes from studies on Jurkat T cells or on primary T cells (mainly CD4+ T helper cells). It is possible that LYP∗ W620 affects the development of autoimmunity through more complex mechanisms involving other immune cell lineages. The potential role for Treg cells has already been mentioned. Furthermore, some of the

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autoimmune disorders associated with the T1858 allele of PTPN22 are not regarded as primarily T cell mediated. A possible role in B cell biology has been suggested by the association of the T allele with RF-positive RA but not with RF-negative RA (35, 40, 70). In contrast, in T1D, there is no signiﬁcant correlation between the T allele and the appearance or levels of autoantibodies. Also, the role of LYP in the autoantibody response may not necessarily be intrinsic to B cells but rather a consequence of altered T cell help. The uncertainty regarding which cells are phenotypically affected by LYP∗ W620 in different disease settings is further exempliﬁed by a recent study demonstrating that carriers of the T1858 allele are more prone to certain infectious diseases, such as invasive pneumococcal infections and Gram-positive empyema (79). These ﬁndings may reﬂect reduced antibody responses owing to the presence of LYP∗ W620 in B cells, or they may even be caused by the effects of LYP∗ W620 in neutrophils. To make the picture even more complex, T cells have also been shown to play an important protective role in the early phase of the immune response toward pneumococcal lung infection (80). LYP∗ W620 may also simultaneously exert effects in all these cell lineages. Moreover, because LYP is found in all leukocytes, it may play a role in the function of dendritic cells, macrophages, and natural killer cells. Certainly, more studies are required to clarify which hematopoietic cells are responsible for the different effects of LYP∗ W620. On the basis of current knowledge, we favor a model wherein LYP∗ W620 functions primarily in T cell–mediated autoimmunity by reducing TCR signaling in the thymus and/or periphery, although we are aware that effects in other immune cell lineages are likely. Our model is supported by the role T cells play during the initiation phase of the autoimmune diseases associated with the C1858T SNP. In addition, altered TCR signaling has been reported in both T1D and RA as well as in animal models of these diseases, for

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instance the nonobese diabetic mouse model in which peripheral T cells exhibit hyporesponsiveness to TCR engagement in the early phase of the autoimmune disease (75, 77). Interestingly, diminished TCR signaling in developing thymocytes owing to a mutation in ZAP-70 (which serves as a substrate for LYP) also results in autoimmune disease in mice (2). Finally, the two other shared autoimmunity genes, MHC and CTLA4, play a role in antigen recognition by T cells and in negative regulation of TCR signaling, respectively.

Why Has Evolution Kept the C1858T SNP? A fascinating question is why evolution has kept the C1858T SNP. From a historical perspective, the relatively high prevalence of autoimmune disorders currently seen is a characteristic trait of modern society. Although it is evident that the T1858 allele is tightly associated with several autoimmune disorders, some of which have a relatively poor prognosis, the negative effects of the C1858T SNP on the immune system of the modern human being may have been beneﬁcial in ancient times. Interestingly, a recent report demonstrated that the T allele is associated with increased risk of atherosclerosis in men as evaluated by intima-media thickness of the carotid artery (81). A corresponding association was not found in women, but here there was an association between the T1858 allele and several risk factors for atherosclerosis, including body mass index (BMI), waist circumference, waist-to-hip ratio, and elevated serum levels of triglycerides and C-reactive protein. Like autoimmunity, atherosclerosis is a disease that has reached epidemic levels in the modern world. However, individuals with typical syndrome X characteristics (high BMI, elevated hip-to-waist ratio, big waist circumference, and elevated serum lipids) probably had a survival advantage in ancient times because they could easily gain weight when food supplies were adequate and survive for longer periods of times when food supplies were low. It was

also recently reported that carriers of the T allele are less susceptible to developing clinically signiﬁcant tuberculosis (82). Given the worldwide epidemic of tuberculosis in historical times, individuals with protection against this disease may have had a survival advantage. However, carriers of the T allele have not always had an advantage in ﬁghting infections, as carriers of the T allele are at higher risk for certain infectious diseases, such as invasive pneumococcal infections and Gram-positive empyema (79). Taken together, the data suggest that the T1858 allele has been a selection factor. Because the T allele is found in Europeans but is virtually absent in Africans and Asians, there may have been a regional advantage of the T allele. Alternatively, the T allele may have originated in northern Europe, but did not provide sufﬁcient advantage to spread worldwide.

INVOLVEMENT OF OTHER PEST GROUP PTPs IN AUTOIMMUNITY In addition to LYP, there are two other PEST group PTPs in humans, namely PTPPEST (PTPN12) and PTP-HSCF (PTPN18) (19). These PTPs share certain structural features, including an N-terminal PTP domain and a noncatalytic C-terminal portion containing different sequence motifs. All PEST group PTPs also contain a proline-rich CTH domain. However, the sequence motifs within the noncatalytic parts of the three family members differ to a certain degree, thereby providing a basis for shared as well as unique interaction partners. PTP-PEST is ubiquitously expressed, whereas PTP-HSCF is primarily found in brain, stem cells, and hematopoietic cells where expression tends to be higher in primitive cells compared with mature cells. In immune cells, there may be a certain degree of redundancy between the different PEST group PTPs ( Y. Arimura & T. Mustelin, unpublished observations), and this can explain the seemingly mild phenotype observed in PEP-deﬁcient mice (33). That www.annualreviews.org • Phosphatases and Autoimmunity

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LYP/PEP act primarily on SFKs and their substrates (CD3/ζ-chain ITAMs and ZAP70) suggests that PTP-PEST and/or PTPHSCF may target the same proteins. However, these two PTPs also seem to have unique functions. For instance, PTP-PEST dephosphorylates several cytoskeletal and focal adhesion proteins (83, 84), and PTP-HSCF may play a role in regulation of the Tec family protein tyrosine kinases (PTKs) (85). To date, there are no reports of a direct involvement of PTP-PEST or PTP-HSCF in autoimmune disease. Because LYP∗ W620 is a gain-of-function variant and there are likely redundant functions between LYP and at least PTP-PEST, one could envision that activating mutations in the PTPN12 gene could also be associated with autoimmunity. Indeed, there are several SNPs in PTPN12, but no disease association with one or more of these SNPs has been published. It is also worth mentioning that the PTPN12 gene is located on chromosome 7q in a region that exhibits evidence of linkage to inﬂammatory bowel disease (86). PTP-PEST, LYP, and PTP-HSCF may be indirectly involved in an autoimmunityrelated group of disorders called autoinﬂammatory diseases, which are conditions involving inﬂammation due to breakdown in self-tolerance but where detection of antigenspeciﬁc T cells and/or autoantibodies fails (87). The autoinﬂammatory condition familial recurrent arthritis (FRA)/PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne) is caused by mutations in the coiled-coil domain of a cytoskeletalassociated protein called CD2BP1 (in humans) or PSTPIP (in mice) (88). All PEST group PTPs can normally through their CTH domain bind the coiled-coil domain of CD2BP1/PSTPIP (19) (T. Vang, A.V. Miletic & T. Mustelin, unpublished data), but the disease-associated mutated form of CD2BP1 exhibits more than 90% reduction in its ability to interact with PTP-PEST (88). Most likely, the binding between disease-associated CD2BP1 and LYP/PTP-HSCF is affected

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in a similar way. C-terminal to the coiledcoil domain, CD2BP1 contains an SH3 domain that interacts with CD2, Abl, and WASP (Wiskott-Aldrich syndrome protein). Notably, the CD2/CD2BP1/PTP-PEST complex negatively affects T cell responses (89). Similarly, the CD2BP1/PTP-PEST complex also exists in other hematopoietic cells, and the disruption of this complex in neutrophils from FRA/PAPA syndrome patients is most likely the underlying cause for the accumulation of dysfunctional neutrophils in the affected organs in this disease (88). Furthermore, CD2BP1 can also bind to pyrin, which is primarily expressed in myeloid cells and is mutated in familial Mediterranean fever, another autoinﬂammatory disease (90).

CD45 AND AUTOIMMUNITY CD45 (PTPRC ) is expressed in all nucleated hematopoietic cells and consists of a highly glycosylated extracellular part, a single transmembrane domain, and a cytoplasmic portion containing two consecutive PTP domains (called D1 and D2) (91). The membrane distal D2 is catalytically inactive but necessary to support the PTP activity of the membrane proximal D1. Different isoforms of CD45 are expressed owing to alternative splicing of exons 4 (gives rise to CD45RA), 5 (gives CD45RB), and 6 (gives CD45RC); CD45RABC includes all three exons. The isoform CD45RO is a result of exons 4–6 spliced out. Different CD45 isoforms are expressed during the life span of immune cells. For instance, naive peripheral T cells primarily express CD45RB, and memory T cells predominantly express CD45RO. CD45 is extremely abundant in T cells. In fact, it constitutes 10% of all surface protein. With regard to TCR signaling, CD45 has a nonredundant positive regulatory role, which involves activation of the SFKs Lck and FynT through dephosphorylation of the C-terminal tyrosine residues Y505 and Y528, respectively (18). Consistent with this function, patients lacking CD45 suffer from severe combined immunodeﬁciency

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(92). In the peripheral blood of these patients, B cell numbers are normal, but the T cell population is greatly diminished, and these few T cells are unresponsive to mitogenic stimulation (92). CD45-deﬁcient mice exhibit similar B and T cell phenotypes to that which is observed in patients lacking CD45 (93). Interestingly, although B cells are generated in CD45−/− mice, BCR-mediated positive and negative selection events are impaired (94). With respect to autoimmunity, autoreactive B cells are generated in CD45−/− mice as a consequence of altered threshold signaling by self-antigen, but autoantibodies are not generated because of impairments in late B cell differentiation (95). In T cells, CD45 activity is regulated at several levels, including spatiotemporal mechanisms, phosphorylation, and autoinhibition by dimerization (18) (Figure 1). Notably, CD45 splice variants with a smaller extracellular portion (CD45RO) homodimerize more easily compared with those with a larger extracellular domain (CD45RA, CD45RAB, and CD45RABC) (96). Because naive and memory T cells express different repertoires of CD45 isoforms, such a mechanism may allow differential regulation of CD45 between subsets of T cells. The molecular basis for autoinhibition by dimerization was proposed to involve a wedge-like structure in CD45, blocking the catalytic site in the other CD45 molecule of the dimer (97). This model was supported by the ﬁnding that disruption of the function of the putative wedge by mutating a key residue (from glutamic acid to arginine at position 624) abolished dimerizationinduced inhibition of CD45 (97). Moreover, knockin mice with a corresponding mutation (from glutamic acid to arginine at position 613, hereafter called CD45-E613R) in CD45 developed a phenotype characterized by a lymphoproliferative syndrome as well as autoimmune disease (such as lupus nephritis), suggesting aberrant CD45 activity in these animals (98). However, the authors found no evidence for activation of Lck in CD45E613R knockin T cells, and these cells exhib-

ited the same proliferative responses in vitro compared with wild-type T cells, indicating that the wedge model cannot explain important features of CD45 regulation in T cell biology (98). Furthermore, the crystal structure of CD45 is incompatible with the wedge model for several reasons (99). First, even when highly concentrated, the CD45 cytoplasmic region does not dimerize. Second, the intramolecular D1-D2 interaction appeared very tight and may not allow the deformation needed for an intermolecular D1-D1 interaction. And ﬁnally, the wedge region is not involved in any intermolecular interactions. At present, there is some debate regarding the role of the CD45 wedge. On the basis of the published coordinates for the CD45 structure, the researchers behind the CD45E613R knockin mice conducted a re-analysis of the CD45 structure (100). They conﬁrmed the existence of the wedge, but its involvement in CD45 regulation is still an open question. The latter study also encompassed a more thorough investigation of the CD45E613R knockin mice, demonstrating that the observed phenotype in these mice (lymphoproliferative disorder) is due to hyperresponsive B cells, whereas T cells do not play any role (100). CD45 has also been linked to several autoimmune disorders in humans. The C77G SNP in PTPRC (the gene encoding CD45) was initially reported to associate with MS (101). This mutation disrupts an exonic splicing silencer for exon 4 in the PTPRC gene, with the consequence that high levels of exon 4–encoded CD45 isoforms are produced (i.e., CD45RA) in all cell lineages (102). As already mentioned, CD45RA isoforms do not dimerize as easily as CD45RO. Therefore, according to the model for autoinhibition by dimerization, patients with the C77G mutation should have aberrant CD45 activity in cells that normally only would express CD45RO, thereby suggesting a mechanism for disease development. However, subsequent studies have not been able to conﬁrm the association between the C77G SNP and MS (103, 104). www.annualreviews.org • Phosphatases and Autoimmunity

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a Lipid raft

FN SH3

D1 SH2

Lck

D2 Phosphorylated Y505

b

CD45RABC

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CD45R0

More active

Less active

More active

CD45RABC

c

Less active

d

CD45R0

CD45AP

Fodrin

CK2, PKC, Csk

Figure 1 CD45 activity in T cells is regulated at several levels. (a) While Lck preferentially partitions into lipid rafts, approximately 95% of all CD45 molecules are excluded from rafts. Therefore, it remains controversial whether CD45-mediated activation of Lck (through dephosphorylation of Y505 in Lck) occurs within lipid rafts or outside. (b) CD45 activity is inhibited by homodimerization. Because CD45 isoforms with smaller extracellular portions (such as CD45RO) tend to homodimerize more easily compared with isoforms with larger extracellular parts (such as CD45RABC), the PTP activity of the former will be lower. (c) CD45 isoform–speciﬁc interactions with other transmembrane proteins may affect the juxtapositioning of CD45 with its substrates (such as Lck). (d ) CD45 may be regulated by numerous other proteins, for instance by binding CD45-associated protein (CD45AP) or fodrin (a cytoskeletal protein). CD45 can also be phosphorylated by casein kinase 2 (CK2), protein kinase C (PKC), and Csk. FN, ﬁbronectin-like; SH, Src homology.

Nevertheless, there seems to be a connection between the C77G mutation and increased risk for systemic sclerosis (105) and autoimmune hepatitis (106), but not with T1D or 42

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Graves’ disease (107). Another mutation in PTPRC, C59A, has also been reported to interfere with alternative splicing in several members of a MS multiplex family (i.e., a large

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family in which several members have MS), leading to CD45RA expression on memory T cells (108).

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INVOLVEMENT OF OTHER TRANSMEMBRANE PTPs IN AUTOIMMUNITY In addition to CD45, there are 20 other transmembrane (or receptor-like) PTPs in humans. A large number of these are found in leukocytes, and there is currently limited evidence for an involvement of these PTPs in autoimmune diseases. Because LYP most likely is associated with autoimmunity through regulation of SFKs, we can speculate that transmembrane PTPs acting on SFKs are connected as well; in addition to CD45, those would include PTPα and PTPε. Interestingly, two other transmembrane PTPs have been implicated as major autoantigens in pancreatic β-cells in connection with T1D, namely PTPRN (IA-2) and PTPRN2 (IA-2β, phogrin) (109, 110).

SHP1 AND SHP2 IN AUTOIMMUNITY SHP1 (PTPN6 ) consists of two N-terminal SH2 domains, a PTP domain, and a Cterminal tail. It is expressed in hematopoietic cells and is a well-characterized PTP that negatively regulates immune cell activation through recruitment to immunoreceptor tyrosine-based inhibition motifs (ITIMs) in signaling molecules. SHP1 catalyzes dephosphorylation of ITAMs, the Syk family kinases Syk and ZAP-70, SLP-76, PI3 kinase, and Vav, to name a few. The association of SHP1 with autoimmunity was ﬁrst revealed in motheaten and viable motheaten mice in which mutations cause abnormal splicing of SHP1 transcripts. As a result, motheaten mice lack SHP1 protein, while viable motheaten mice express catalytically defective protein (111, 112). Motheaten and viable motheaten mice (hereafter referred to as motheaten mice) display aggressive hyperinﬂammation characterized by hyperactive T

and B cells, as well as myeloid and phagocytic cells. Moreover, motheathen mice express autoantibodies and display glomerulonephritis (113). Notably, SHP1-deﬁcient mice show a two- to threefold increase in the percentage of Treg cells, potentially explaining the minimal contribution of T cells compared with myeloid cells in the inﬂammation displayed by motheaten mice (114). Importantly, conditional deletion of SHP1 in B cells causes autoantibody production and glomerulonephritis, demonstrating that these hallmarks of the motheaten phenotype are B cell intrinsic and not secondary to dysregulated macrophage activity (19). Polymorphisms in the gene encoding SHP1 (PTPN6 ) have been found in humans, but unlike PTPN22, no association with autoimmune disease has been found. Of note, it has been reported that some SLE patients express lower levels of SHP1 as well as of CD45 (115). It is also worth mentioning that SHP1 mutations have been demonstrated in numerous hematological diseases, including myelodysplastic syndrome (116), and in certain types of lymphoma (117). SHP2 (PTPN11) is widely expressed and, similar to SHP1, contains two SH2 domains, a PTP domain, and a C-terminal tail. SHP2 is generally a positive regulator of signaling through receptor tyrosine kinases and cytokine receptors as well as of integrin signaling. TCR signaling is also positively regulated by SHP2 (118). Our preliminary ﬁndings from the analysis of mice lacking SHP2 in B cells indicates that it has a minimal role in BCR signaling but negatively regulates germinal center formation (R.V. Kolla, F. Princen, D. Ostertag, G.S. Feng & R.C. Rickert, in preparation). Although there are no reports of involvement of SHP2 in autoimmune diseases, mutations in the gene encoding SHP2 (PTPN11) are associated with Noonan syndrome (NS), a disorder characterized by multiple developmental defects, and LEOPARD (multiple lentigines, electrocardiographic-conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, www.annualreviews.org • Phosphatases and Autoimmunity

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and sensorineural deafness) syndrome, a rare, autosomal dominant disorder that shares many of the clinical features of NS. Interestingly, mutations in NS code for gainof-function proteins, whereas LEOPARD syndrome is characterized by a dominantnegative, loss-of-function SHP2 protein (119). Mutations in PTPN11 have also been associated with Helicobacter pylori-induced gastric atrophy/cancer (120) and in numerous myeloid leukemias and lymphomas (121, 122). Annu. Rev. Immunol. 2008.26:29-55. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

OTHER NONRECEPTOR PTPs IN AUTOIMMUNITY Although no PTPs aside from the aforementioned PTPs have been linked to autoimmunity, it is conceivable that mutations altering the function of other PTPs, perhaps combined with genetic or environmental factors, may precipitate autoimmune disease. Gene knockout studies are beginning to reveal the roles of many nonreceptor PTPs in lymphocyte development and activation. Mice lacking expression of TCPTP (PTPN2) show defects in T and B cell development and activation (123), which may be caused by its function in regulation of STAT1 signaling in the nucleus (124). In contrast, PTPH1 (PTPN3)deﬁcient mice displayed no discernible defects in TCR signaling despite a proposed function of PTPH1 in the dephosphorylation of ITAMs (125). Like PTPH1, PTPMEG1 (PTPN4) and HePTP (PTPN7) have been implicated in negative regulation of TCR signaling (126). Although HePTP expression is induced by IL-2, suggesting that it may be involved in contraction of T cell responses following activation (127–129), and HePTP functions to regulate Erk and p38, the knockout mice displayed no noticeable phenotype aside from a two- to ﬁvefold increase in Erk1/2 and p38 activation. PTPD1 (PTPN21) reportedly associates with Tec kinases and functions to activate Tec and Itk (130). The precise function of PTP-BAS (FAP-1, PTPN13) in T lymphocytes is unknown; however, it may function to regulate 44

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apoptosis of developing or activated T cells as it associates with, and inhibits, cell surface expression of FAS (131). Future studies, including SNP analysis, may reveal roles for these and other PTPs in human autoimmunity.

DUSPs IN AUTOIMMUNITY—FOCUS ON MAPK PHOSPHATASES MAPK (mitogen-activated protein kinase) phosphatases (MKPs, a subgroup of the DUSPs) are important for regulation of MAPKs, which can function either to amplify or to attenuate innate immune responses and are essential for lymphocyte development, activation, and homeostasis (132). In this regard, at least 10 MKPs are expressed in developing thymocytes, and MKP expression levels vary at different stages of T cell development and can be modulated by TCR signaling (133). Together, these data suggest that MKPs potentially play an important role in thymic selection and/or Treg cell development. Gene-targeting studies have revealed the speciﬁcity, redundancy, and cross talk between MKPs in both innate and adaptive immunity (134). MKP-1 (DUSP1) is a phosphatase that primarily regulates activation of p38 and JNK1/2, with little effect on Erk1/2 activation (135). Mice lacking MKP-1 revealed a critical function for MKP-1 in regulation of innate immune responses to microbial components via a negative-feedback loop that includes p38 and JNK. Moreover, compared with wild-type controls, MKP-1-deﬁcient mice showed increased severity of collagen-induced arthritis resulting from increased levels of systemic TNF-α and IL-6, as well as high levels of antitype II collagen antibodies, suggesting that in addition to an important function in innate immune cells, MKP-1 may also be critical in regulation of T and/or B lymphocytes. Although a MKP-1 SNP has been identiﬁed in ovarian cancer to date, no associations have been made between MKP-1 and autoimmune disease (136).

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PAC-1 (DUSP2) expression is induced in T cells in response to antigen receptor stimulation and functions to negatively regulate JNK activation but augments both p38 and ERK1/2 activation. Unlike MKP-1−/− mice, PAC-1-deﬁcient mice show decreased inﬂammatory cytokine production by myeloid cells following stimulation with Toll-like receptor ligands (137). PAC-1−/− CD4+ T cells have diminished proliferation and cytokine production, indicating that PAC-1 is a positive regulator of both innate and adaptive immune cell signaling. In addition, PAC-1 is a direct transcriptional target of p53 in response to cell stress, suggesting that PAC-1 may be important in cellular apoptosis and growth suppression, both of which are critical in contraction of immune responses and are aberrant in many autoimmune disorders (138). MKP-5 (DUSP10) is constitutively expressed in naive CD4+ T cells and is downregulated following TCR activation (139). Following mitogenic stimulation, MKP-5deﬁcient T cells display diminished proliferation but increased cytokine production by both Th1 and Th2 cells, as well as increased IFN-γ and TNF-α production by cytolytic CD8+ T cells in vitro. Notably, although primary responses to lymphocytic choriomeningitis virus infection were similar between wild-type and MKP-5−/− mice, upon secondary infection, MKP-5−/− CD4+ and CD8+ T cells had increased cytokine production. Thus, MKP5 functions to diminish T cell responses, thereby preventing excessive, and potentially harmful, T cell activation during an immune response. Several other MKPs have been implicated in regulation of T cell activation and homeostasis; however, the precise function and association with autoimmunity of these MKPs are not yet established. For example, MKP-2 (DUSP4), MKP-3 (DUSP6 ), hVH3 (DUSP5), MKP-7 (DUSP16 ), and LMWDSP20 (DUSP18) are all implicated in regulation of ERK1/2 and/or JNK1/2 activation (140–143). VHR (DUSP3) is a negative regulator of TCR signaling whose activation is,

in part, mediated by ZAP-70 phosphorylation (144), and expression of VHR controls cell cycle, differentiation, or senescence through regulation of ERK1/2 and JNK1/2 MAPKs (145). VHX (DUSP22), like VHR, is implicated in negative regulation of TCR signaling (146), whereas MKP-6 (DUSP14) associates with CD28 and negatively regulates CD28 signals (147). Interestingly, MGC1136 (DUSP24) is upregulated in tolerant T cells and thus could function in induction or maintenance of lymphocyte tolerance (148).

POTENTIAL FOR TREATMENT OF AUTOIMMUNE DISORDERS USING PTP INHIBITORS? PTPs have recently been implicated in an increasing number of human diseases. That, in turn, has begun to elicit a growing interest in PTPs as drug targets and the development of potent and selective PTP inhibitors (149). The spark that truly ignited the quest for PTP inhibitors with great market potential was the paper reporting the PTP1B knockout mouse, which indicated that PTP1B acts as a negative regulator of insulin signaling (123). Inhibition of PTP1B would conceivably alleviate insulin resistance in type 2 diabetes and would improve the effects of insulin on both glucose balance and fatty acid metabolism. Although the enthusiasm for PTP inhibitors was initially dampened by the notions that PTPs were less speciﬁc than, for example, PTKs and that the structure of the active site of PTPs did not allow for the generation of selective inhibitors, it has now become increasingly clear that PTPs indeed have unique, nonredundant, important functions and a great deal of speciﬁcity in vivo. The question of how selective small-molecule inhibitors can be developed is perhaps not quite resolved, but many promising examples have been published. The crystallization of many PTPs has revealed that the surface topology surrounding the catalytic pocket of each PTP has numerous unique features that can be utilized for the rational structure-based design of highly selective www.annualreviews.org • Phosphatases and Autoimmunity

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compounds (149). The highest afﬁnity and the most selective inhibitors reported so far have been those that interact with several such distinct surface features. These features together with new techniques in PTP inhibitor design (150) provide great opportunities for the development of sufﬁciently selective and efﬁcient PTP inhibitors. In our view, speciﬁc criteria have to be fulﬁlled for selecting a particular PTP as a drug target for the treatment of autoimmune disease. First, the target enzyme should have higher activity in autoimmunity than in healthy subjects. Second, the protein should be expressed mostly in immune cells. Third, the biological function of the PTP should be reasonably well known. At this point, only LYP fulﬁlls these criteria, although the biology of this PTP is only partially understood. Most importantly, the activity of the disease-associated LYP∗ W620 enzyme is

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about 50% higher than the nonmutated form (LYP∗ R620) (5), and the protein is found only in hematopoietic cells (20). Thus, in theory, a selective small-molecule LYP inhibitor could eliminate the effects of LYP∗ W620 on TCR signaling. Such an inhibitor would need to reduce the activity of LYP∗ W620 only to a certain extent. Under these conditions, TCR signaling should be corrected to normal levels, and the autoimmunity-inducing process precipitated by LYP∗ W620 would be neutralized. However, because of the complexity and plasticity of the immune system and the many different roles that signaling molecules can have, the timing and strategy of such a treatment would need to be carefully evaluated. Once suitable small-molecule PTP inhibitors with viable pharmacological properties have been developed, their potential for treatment of autoimmune disorders can be experimentally evaluated.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS This work was supported by grants from the U.S. National Institutes of Health (AI53585 to T.M.), the Oxnard Foundation (to T.M.), and the Norwegian Cancer Society (to T.V.). We apologize to all colleagues whose papers we could not cite owing to space constraints.

NOTE ADDED IN PROOF A recent study demonstrated no association between Graves’ disease and individual SNPs in PTPN12, but some of these SNPs were associated with ophthalmopathy and/or interacted with a previously associated thyrotropin hormone receptor SNP (151). Another study has shown that mice with speciﬁc deletion of SHP1 in B cells develop an SLE-like condition, suggesting that SHP1 deﬁciency in B cells alone is sufﬁcient to cause autoimmune disease (152).

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145. Cerignoli F, Rahmouni S, Ronai Z, Mustelin T. 2006. Regulation of MAP kinases by the VHR dual-speciﬁc phosphatase: implications for cell growth and differentiation. Cell Cycle 5:2210–15 146. Alonso A, Merlo JJ, Na S, Kholod N, Jaroszewski L, et al. 2002. Inhibition of T cell antigen receptor signaling by VHR-related MKPX (VHX), a new dual speciﬁcity phosphatase related to VH1 related (VHR). J. Biol. Chem. 277:5524–28 147. Marti F, Krause A, Post NH, Lyddane C, Dupont B, et al. 2001. Negative-feedback regulation of CD28 costimulation by a novel mitogen-activated protein kinase phosphatase, MKP6. J. Immunol. 166:197–206 148. Perco P, Blaha P, Kainz A, Mayer B, Hauser P, et al. 2006. Molecular signature of mice T lymphocytes following tolerance induction by allogeneic BMT and CD40-CD40L costimulation blockade. Transpl. Int. 19:146–57 149. Tautz L, Pellecchia M, Mustelin T. 2006. Targeting the PTPome in human disease. Expert Opin. Ther. Targets 10:157–77 150. Pellecchia M, Becattini B, Crowell KJ, Fattorusso R, Forino M, et al. 2004. NMR-based techniques in the hit identiﬁcation and optimisation processes. Expert Opin. Ther. Targets 8:597–611 151. Syed AA, Simmonds MJ, Brand OJ, Franklyn JA, Gough SC, Heward JM. 2007. Preliminary evidence for interaction of PTPN12 polymorphism with TSHR genotype and association with Graves’ ophthalmopathy. Clin. Endocrinol. In press 152. Pao LI, Lam KP, Henderson JM, Kutok JL, Alimzhanov M, et al. 2007. B cell-speciﬁc deletion of protein-tyrosine phosphatase Shp1 promotes B-1a cell development and causes systemic autoimmunity. Immunity 27:35–48

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:29-55. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity∗ Rosanne Spolski and Warren J. Leonard Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892-1674; email: [email protected], [email protected]

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Key Words

First published online as a Review in Advance on November 8, 2007

cytokine, Blimp-1, plasma cell differentiation, Stat3, antitumor, adaptive immunity, innate immunity

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090316 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0057$20.00 ∗ The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

Abstract Interleukin-21 (IL-21), a potent immunomodulatory four-α-helicalbundle type I cytokine, is produced by NKT and CD4+ T cells and has pleiotropic effects on both innate and adaptive immune responses. These actions include positive effects such as enhanced proliferation of lymphoid cells, increased cytotoxicity of CD8+ T cells and natural killer (NK) cells, and differentiation of B cells into plasma cells. Conversely, IL-21 also has direct inhibitory effects on the antigen-presenting function of dendritic cells and can be proapoptotic for B cells and NK cells. IL-21 is also produced by Th17 cells and is a critical regulator of Th17 development. The regulatory activity of IL-21 is modulated by the differentiation state of its target cells as well as by other cytokines or costimulatory molecules. IL-21 has potent antitumor activity but is also associated with the development of autoimmune disease. IL-21 transcription is dependent on a calcium signal and NFAT sites, and IL-21 requires Stat3 for its signaling. The key to harnessing the power of IL-21 will depend on better understanding its range of biological actions, its mechanism of action, and the molecular basis of regulation of expression of IL-21 and its receptor.

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INTRODUCTION

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Immune responses to foreign antigens have evolved to comprise the two interacting arms of cellular and humoral responses: the innate and adaptive immune systems. The cellular components of the innate immune system, including dendritic cells (DCs), natural killer (NK) cells, macrophages, and granulocytes, possess pattern recognition receptors capable of detecting conserved structural components of pathogens whose recognition then initiates immediate responses. One of the key responses of the innate immune system is the production of cytokines that then regulate the antigen-driven differentiation of the adaptive immune system, composed of naive lymphoid B and T cells, which then results in the development of antigen-speciﬁc effector responses. Cytokines produced by the innate immune system as well as cytokines produced by activated T cells drive the expansion and effector functions of the adaptive immune response as well as the downregulation of responses once the offending agent has been eradicated. Much attention has been paid over the past several decades to the array of cytokines that are produced by these immune cells and play such an important role in the ampliﬁcation and control of responses to pathogens. Understanding the mechanisms involved in the production and function of these cytokines is key to predicting and employing clinical strategies for controlling these responses. One of the most important families of cytokines includes the type I four-α-helicalbundle cytokines, which comprise many of the interleukins and colony-stimulating factors as well as a range of other molecules such as erythropoietin, growth hormone, and prolactin. In one subfamily of this set of cytokines, the receptors share the common cytokine receptor γ chain, γc , which is mutated in humans with X-linked severe combined immunodeﬁciency (XSCID) (1), a disease characterized by the absence of T and NK cells but the presence of nonfunctional B cells. This set of cytokines includes IL-2, IL-4, IL-7, IL-9, IL-

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15, and IL-21, the most recently discovered member (2, 3) (see Figure 1). The IL-21 receptor was discovered in 2000 as an orphan receptor, ﬁrst denoted as NILR for novel interleukin receptor and now as IL21R (4, 5). IL-21 was cloned as the ligand for this novel receptor (5) and was ﬁrst observed to be produced by CD4+ T cells and to modulate the proliferation and effector function of other lymphoid cells. Subsequently, however, IL-21 was observed to act on multiple nonlymphoid lineages as well and to be produced by innate immune natural killer T (NKT) cells and the more recently identiﬁed Th17 lineage. This has expanded our understanding of the broad potential roles for this cytokine in the development and control of immune responses. Moreover, IL-21 has been shown to have strong antitumor action via its effects on both NK and CD8+ T cells and also has been identiﬁed as a key component in the development of autoimmune disease. The dissection of these beneﬁcial and pathogenic effects of IL-21 has begun to offer a new appreciation of the complexity of the interaction between the innate and adaptive immune responses.

IL-21 Receptor and Ligand Structure The IL-21 receptor (IL-21R) was ﬁrst discovered by genomic and cDNA sequencing projects as an open reading frame that putatively encoded a type I cytokine receptor (4, 5). Its predicted amino acid sequence was most related to the IL-2 receptor β chain, and like IL-2Rβ, IL-21R appeared to be lymphohematopoietic restricted. Moreover, IL-21R was located immediately downstream of IL4Rα on human chromosome 16 (4). Thus, IL-21R was clearly related to the γc family of cytokines. Indeed, when identiﬁed, the ligand for this novel type I receptor was most similar to IL-2, IL-4, and IL-15 (5). The functional receptor for IL-21 is IL-21R + γc (6, 7). IL-21R was observed initially to be expressed on T, B, and NK cells (4, 8).

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IL-2Rα α

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• Ig class switch • T cell proliferation • Antigen-induced cell death • Boosting of cytolitic activity of NK cells • Development of Treg cells

• NK cell development • CD8+ memory T cell homeostasis

• T cell development in humans and mice

• Memory T cell development

• Mucus production • Mast cell proliferation

• Comitogen for T cell proliferation • Cooperative expansion of CD8+ T cells • Antitumor agent • Plasma cell differentiation • Implicated in autoimmunity (see Fig. 4)

Figure 1 Cytokine receptors containing the common cytokine receptor γ chain (γc ). The IL-21 receptor is a member of a family of receptors that share γc . In addition to γc , each of these receptors has one or more distinctive receptor components. Mutations in γc result in X-linked severe combined immunodeﬁciency (XSCID); the severity of this disease results from defective signaling through all these receptors.

Expression on B cells was the highest, even on resting cells, with constitutive expression in a number of cell lines (4, 8). Low-level IL-21R expression on T cells was also observed but was signiﬁcantly increased following T cell receptor (TCR) stimulation (4, 8). Interestingly, like that of IL-2Rβ, expression of IL21R was also augmented in cells transformed with HTLV-I (4). Recently, the three-dimensional structure of human IL-21 has been solved by heteronuclear NMR spectroscopy (9). As anticipated, it is a typical up-up-down-down four-α-helicalbundle cytokine. A segment of the molecule involving helix C that is important for receptor binding is relatively unstable, and stabilization of this region in a human IL-21 analog results in a tenfold increase in biological potency (9).

The Molecular Basis for IL-21 Signaling Like other type I cytokines, IL-21 signals via the Jak-STAT pathway (see Figure 2). Analogous to IL-2, IL-4, IL-7, IL-9, and IL-15, Jak1 and Jak3 are the Janus family tyrosine kinases that are activated by IL-21 (4, 6, 7). IL21 can activate Stat1, Stat3, and both Stat5a and Stat5b (10, 11). However, the activation of Stat5a and Stat5b is relatively weak and transient, whereas the activation of Stat3 is the most sustained. Stat3 appears to be the most important STAT protein for IL-21 signaling. Indeed, there is defective signaling to IL-21 in T cells that lack expression of Stat3 (11). The IL-21R cytoplasmic domain contains six tyrosine residues. One of these, Tyr 510, is phosphorylated and serves as a critical docking site for both Stat1 and Stat3. On the basis

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MEK

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IL-21 target genes Granzyme A, Bcl-3, Jak3, granzyme B

Figure 2 Signaling pathways for the IL-21 receptor (IL-21R). Upon IL-21 binding, Jak1 and Jak3, which interact with IL-21R and γc , respectively, are activated and then phosphorylate Stat3 and Stat1 and weakly phosphorylate Stat5 proteins. This leads to STAT dimerization and translocation to the nucleus, with subsequent binding to target gene regulatory elements. A critical tyrosine in the IL-21R cytoplasmic domain (Y510) is primarily responsible for the docking of Stat1 and Stat3. Five other cytoplasmic tyrosines are not shown. Additionally, ligand binding to the IL-21R can lead to activation of the MAP kinase (MAPK) and the PI 3-kinase (PI 3-K) pathways. Target genes activated by IL-21 have been identiﬁed, but the involvement of each of these signaling pathways in the regulation of these genes remains to be determined. Co, co-activator; POL, RNA polymerase; TF, transcription factor.

of analysis using the chemical inhibitors wortmannin and PD98059, we can conclude that PI 3-kinase and MAP kinase pathways also contribute to IL-21 signaling. These different signaling pathways may function in distinct phases of lineage development and function.

IL-21 Ligand and Receptor Regulation IL-21 is produced by CD4+ T cells (5) as well as by NKT cells (13). Although the genes encoding IL-21 and IL-2 are adjacent to each other, the regulation of these genes is signiﬁ60

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cantly distinct. Both cytokines can be induced in CD4+ T cells by signaling via the TCR, but IL-21 mRNA can be induced by a calcium signal alone in preactivated T cells, whereas IL-2 mRNA induction requires both a calcium signal and protein kinase C (14). Nuclear factor of activated T cells (NFAT) binding sites in the IL-21 promoter region contribute to the regulation of IL-21 transcription (14, 15). Interestingly, NFATC2 binds in vivo, but mice lacking NFATC2 still express IL-21, indicating functional redundancy with other NFAT family proteins (14). Mycobacterial antigens

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(BCG) upregulate levels of IL-21 in NKT cells in both mouse and human systems (16), revealing that innate immune signals can also induce IL-21 production. IL-21 receptor expression has been detected on CD4+ T cells, CD8+ T cells, B cells, NK cells, DCs, macrophages, and keratinocytes (4, 5, 8, 17–19), suggesting that IL21 has a broad range of actions. Although IL-21 is not required for hematopoiesis, as demonstrated by analysis of IL-21R knockout (KO) mice (20), IL-21R mRNA has also been detected on a population of bone marrow progenitors, and IL-21 can expand hematopoietic progenitor cells both in vivo and in vitro (20a). Within the T cell lineage, IL-21R expression is induced as cells differentiate from double negative (CD4− CD8− ) thymocytes to double positive (CD4+ CD8+ ) thymocytes (8), but this expression may not be absolutely essential for thymocyte differentiation because there is normal thymic development in IL-21R KO mice (20). Low but detectable levels of IL21R are found on mature CD4+ and CD8+ T cells (8), and these levels are upregulated in response to either TCR or IL-21 (8, 21). TCRmediated IL-21R expression is regulated in part by the induction and dephosphorylation of the transcription factor Sp1 (21). Within the B cell lineage, IL-21R is expressed at a low level at the pre–B cell stage of development; this level persists through the ﬁrst transitional (T1) stage but then increases at the second transitional (T2) stage (22). Mature follicular B cells express higher basal levels of IL-21R than are found on mature T cells (8), and these levels are further increased by signals either through the B cell receptor (BCR) or through CD40 (23). Marginal zone B cells respond to IL-21 but have lower IL-21R expression than do follicular B cells (22). Plasma cells have no detectable surface IL-21R (23), in keeping with their terminally differentiated and nonproliferative state. Interestingly, however, myeloma plasmacytoma cells do express surface IL-21R (24), which may provide a distinct survival advantage for these cells in vivo.

IL-21 AND B CELL FUNCTION IL-21 Plays a Critical Role in Immunoglobulin Production The role of IL-21 in B cell function has been investigated in both in vitro studies and in vivo systems employing IL-21R KO and IL-21 transgenic mice. IL-21 is not essential for B cell development; no defects in B cell subsets within bone marrow or periphery have been observed in IL-21R KO mice (20). B cells from IL-21R KO mice normally proliferate in response to lipopolysaccharide (LPS), anti-CD40, or the combination of IL4 plus anti-IgM (20). The most striking defect in naive IL-21R KO mice is a reduced level of serum IgG1, yet an increased level of IgE (20). Upon immunization with T cell– dependent antigens, IL-21R KO mice have strikingly impaired production of antigenspeciﬁc IgG1 and signiﬁcantly higher levels of antigen-speciﬁc IgE (20), an unexpected result given that IgG1 and IgE are usually coordinately regulated. These elevated levels of IgE in IL-21R KO mice were consistent with experiments demonstrating that IL-21 administered to wild-type (WT) mice at the time of immunization can lead to reduced IgE responses, as well as with in vitro experiments showing that IL-21 can reduce levels of germline Cε transcripts, leading to reduced IgEspeciﬁc switching (25). Interestingly, in vitro experiments using human peripheral blood B cells revealed that IL-21 can both positively and negatively regulate IgE production, depending on the context. For example, IL-21 in combination with PHA and IL-4 inhibited IgE, whereas IL-21 in combination with anti-CD40 plus IL-4 led to increased IgE levels (26). The precise molecular mechanism(s) by which IL-21 regulates IgE production remains to be fully delineated, particularly because IL-21 can also be proapoptotic for B cells (see below). IL-4 is required for IgE production, and as expected, IL-4/IL-21R double-knockout (DKO) mice could not produce IgE,

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conﬁrming that IL-4 is indeed necessary for the enhanced levels of IgE seen in the IL-21R KO mice. But surprisingly, the DKO mice exhibited a pan-hypogammaglobulinemia, with essentially absent levels of IgG1, IgG2a, IgG2b, and IgG3 and greatly reduced levels of IgM (20). Thus, IL-21 and IL-4 cooperatively regulate immunoglobulin (Ig) production. These observations may also explain the B cell phenotype in humans with XSCID (2, 20). In this severe immunodeﬁciency, B cells develop normally but are nonfunctional; patients exhibit a severe pan-hypogammaglobulinemia. In the mouse, elimination of γc results in a loss not only of T cells but also of B cells, given a critical role in the mouse, but not human, for IL-7 signaling in B cell development (27). By keeping IL-7 signaling intact and thus allowing B cells to develop but by eliminating signaling by IL-4 and IL-21, we apparently have mimicked in mice the human XSCID B cell phenotype (20).

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IL-21 Induces B Cell Apoptosis in a Context-Dependent Manner One of the most puzzling aspects of IL-21 biology is that, in contrast to other members of the γc -dependent cytokines, IL-21 can be potently proapoptotic for B cells. Initially, IL21 was found to augment anti-CD40-induced human B cell proliferation but inhibit proliferation to anti-IgM and IL-4 (5). The inhibition of LPS-induced proliferation by IL-21 results at least in part from a strong proapoptotic signal from IL-21 (8, 28, 29). The degree of IL-21-induced apoptosis is dependent on the context of B cell activation: Apoptosis dominates when B cells are activated with Toll-like receptor (TLR) signals such as LPS or CpG but augments proliferation when B cells are activated with BCR signals (antiIgM) plus T cell–derived costimulatory signals such as those provided by anti-CD40 (8, 28, 29). The apoptotic signal is caspase dependent because it can be inhibited with caspase inhibitors (28). Although apoptosis can

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be prevented by the overexpression of the antiapoptotic Bcl-2 protein (28, 29), IL-21 has no effect on Bcl-2 protein levels in B cells (29). Analysis of genes involved in apoptosis revealed that IL-21 increases mRNA and protein levels of the proapoptotic mitochondrial protein Bim and decreases levels of Bcl-xL (8). IL-21-induced apoptosis was eliminated in B cells from Bim KO mice, conﬁrming that Bim-1 plays a role in the IL-21-mediated death of B cells. Other antiapoptotic proteins may be involved in the rescue from IL-21mediated apoptosis. For example, IL-4 can rescue B cells from LPS + IL-21-induced cell death through upregulation of Bcl-xL, and this rescue is dependent on the presence of Bcl-6 (30). IL-21-mediated induction of apoptosis may in part also account for the inhibitory effects of IL-21 on IgE production. Vaccination with Mycobacterium bovis bacillus (BCG) activated Vα14 NKT cells to express high levels of IL-21, which in turn preferentially induced apoptosis of IgE-expressing B cells but not apoptosis of other Ig-isotypeexpressing B cells (16). The mechanism for this speciﬁc apoptosis seems to involve IL21-induced formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2modifying factor (Bmf), which is speciﬁcally expressed in the IgE-expressing population of B cells. Bmf thereby inhibits the usual antiapoptotic activity of Bcl-2, leading to speciﬁc apoptosis of IgE-producing B cells and the subsequent loss of IgE production. The above results indicate that IL-21 can differentially inﬂuence the outcome of an antibody response, depending on the costimulatory signals present at the time that B cells encounter antigen (see Figure 3). B cells receiving a polyclonal, nonspeciﬁc signal such as those mediated by the TLRs would thus potentially produce nonantigen-speciﬁc Igs, including autoreactive Igs. Expansion of this potentially deleterious population would be prevented by the presence of IL-21 at the time of encounter. In contrast, a B cell that interacts speciﬁcally via its BCR and receives speciﬁc

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Without BCR signal or T cell interaction

With BCR signal and/or T cell interaction IL-21

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IL-4

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IL-21

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Pathology and immune disorders

Figure 3 Effects of IL-21 on B cell differentiation and survival. Signaling through the IL-21 receptor (IL-21R) has two potential outcomes for naive B cells. (Left) In the absence of a B cell receptor (BCR) or T cell interaction signal or in the presence of a Toll-like receptor (TLR)-dependent signal, IL-21 induces apoptosis of a naive B cell. (Right) In the presence of a BCR signal and/or costimulatory interactions with T cells, IL-21 induces proliferation, isotype class switching, and differentiation to either memory B cells or terminally differentiated plasma cells. Memory B cells maintain IL-21R and respond to IL-21 plus antigen signals to differentiate into plasma cells, with subsequent downregulation of IL-21R expression.

T cell help would receive a positive costimulatory signal from IL-21.

IL-21 Drives Terminal Differentiation of B Cells to Plasma Cells The analysis of IL-21R KO mice had indicated a critical role for IL-21 in Ig production. The role of IL-21 in antibody responses was further investigated through the use of IL-21 transgenic mice as well as through hydrodynamic transfection of mice with an IL21 expression plasmid (29). In both systems, IL-21-induced apoptosis could be detected by annexin V staining of naive B cells ex vivo. Surprisingly, in both mouse models, there

were increased numbers of total splenic B cells rather than the expected decrease resulting from IL-21-induced apoptosis. Most of the increase in B cell numbers resulted from increases in the number of immature B cells and in the number of postswitch B cells and plasma cells. However, there was no change in the number of mature B cells. This was consistent with the increased concentrations of serum IgM and IgG1. In vitro experiments using murine splenic B cells showed that IL-21 in combination with anti-IgM could directly induce the differentiation and accumulation of Syndecan-1+ plasma cells in these cultures (29). The ability of IL-21 to promote differentiation to plasma cells was explained by its potent induction of B lymphocyte–induced www.annualreviews.org • Interleukin-21

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maturation protein-1 (Blimp-1) (29), a transcription factor that acts as a master switch for the program of transcriptional changes involved in terminal differentiation of B cells to plasma cells (31). Surprisingly, in both primary splenic B cells as well as in a B cell line, IL-21 also induced the production of the transcription factor Bcl-6 (29). Blimp-1 and Bcl-6 function as mutually exclusive transcription factors that negatively regulate the expression of each other and correlate with the plasma cell and the memory cell phenotype, respectively (31, 32). It is not yet clear whether both transcription factors are induced by IL21 within the same individual cells; if so, their coinduction by IL-21 may be limited to a speciﬁc stage of B cell development prior to the commitment to either memory cell or plasma cell lineages. Corresponding to its effects on mouse B cells, IL-21 also plays a major role in stimulating the differentiation of human B cells. Naive cord blood B cells as well as postswitch memory B cells can be driven to differentiate to plasma cells by IL-21 in combination with either BCR and/or CD40 signals (33). IL-21 costimulation of human B cells also induced high levels of Blimp-1 as well as activation-induced cytidine deaminase (AID), but surprisingly this did not induce somatic hypermutation (33). Although IL-21 acted as a switch factor for production of both IgG1 and IgG3 by human peripheral B cells (34), costimulation of naive cord blood B cells with IL-21 plus anti-CD40 induced predominantly the IgG3 isotype (33), suggesting that molecular differences in the responding populations can account for switch preferences. Interestingly, although IL-21 and IL-4 cooperate in the production of Ig, as seen in the absence of Ig responses in IL-4/IL-21R DKO mice (20), these two cytokines appear antagonistic in their effects in both murine and human B cell differentiation into plasma cells: IL-4 inhibited IL-21-mediated plasma cell differentiation by B cells stimulated with either anti-IgM or anti-IgM plus anti-CD40 but did not inhibit B cells stimulated with anti-

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CD40 alone (29, 33). The functional interaction between IL-4 and IL-21 is thus complex and dependent on the nature of costimulatory signals as well as the developmental stage of the target B cell. Although IL-21R levels are higher on naive than on memory B cells (23), IL-21 can also induce rapid plasma cell differentiation in a population of human marginal zone memory B cells (35). This differentiation occurs in response to the combination of IL21 plus BAFF/BLyS, a TNF family cytokine produced by the local DC population (36). The combination of IL-21 with BAFF leads to the synergistic induction of both Blimp-1 and AID, providing a possible mechanism for rapid upregulation of IgG-secreting plasma cells in an antigen-independent manner (35).

IL-21 EFFECTS ON CD4+ T CELL FUNCTION IL-21 Is Produced by Multiple T Helper Populations The functional capacity of CD4+ T cell populations is highly dependent on the cytokines that are available in the environment at the time of TCR priming. Th1 cells arise in response to DC-derived IL-12, produce IFNγ and TNF-α, and are involved in mediating strong inﬂammatory responses to intracellular pathogens. IL-4-mediated Th2 cell differentiation results in cells that produce cytokines, including IL-4, IL-5, and IL-13, which mediate antibody responses to extracellular pathogens. Th17 cells, the most recently identiﬁed CD4+ T cell subset, differentiate in response to TGF-β and IL-6 signals and produce IL-17, which mediates neutrophil differentiation and inﬁltration during various infections (37). Each of these CD4+ T cell populations can produce IL-21, although to different extents, and can respond to IL-21 with distinct differentiative responses. Nevertheless, the subset(s) of cells responsible for IL-21 production at speciﬁc phases of various in vivo immune responses is not yet fully clear.

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Several laboratories originally examined the Th subset expression of IL-21 and obtained ostensibly inconsistent results. One study reported that IL-21 mRNA and protein are produced by Th2-polarized cells but not by Th1polarized cells (38), but another used a DNA array analysis of expression in human Th1and Th2-polarized CD4 T cells and showed that IL-21 mRNA was expressed predominantly in Th1 cells (39). Furthermore, IL-21 mRNA expression was highest in a population of follicular Th cells that could augment B cell antibody responses to antigen (39). Recent work has identiﬁed Th17 cells as producing signiﬁcantly higher levels of IL-21 mRNA and protein than either Th1 or Th2 cells do (40, 41). IL-21 itself and IL-6 were identiﬁed as essential for the upregulation of IL-21 in this lineage (40–42); Stat3 signaling was essential (41, 42). In the committed Th17 cell, TCR signaling can further upregulate IL21 production. The functional signiﬁcance of Th17 expression of IL-21 is further discussed below.

Regulation of Th1 versus Th2 Differentiation In Vitro by IL-21 Although both Th1 and Th2 cells produce IL-21, the effects of IL-21 on differentiation within these subsets are only beginning to be delineated. Analogous to effects of IL-21 on B cell proliferation and function, the end result may depend on other cytokines or immune populations as well as on the developmental stage of the target cell. The initial observation that IL-21 can function as a Th2 cytokine was based in part on in vitro experiments showing that IL-21 could inhibit IFN-γ expression only when IL-21 was present at the time of naive CD4+ T cell priming under Th1 conditions (38). There was not, however, a general downregulation of the Th1 program in that IL-21 had no effect on T-bet or IL-12Rβ2 expression, both of which are induced in Th1 cells even when Th1 cells are primed in the presence of IL-21. The speciﬁc decrease of IFN-γ by IL-21 was mediated by the direct

repression of Eomesodermin, a T-box transcription factor important for IFN-γ induction (43). Moreover, in populations of human peripheral blood T cells that were preactivated with TCR, IL-21 could induce the expression of a panel of Th1 genes, including those encoding IFN-γ, T-bet, and IL-12Rβ2, suggesting that the effects of IL-21 on already activated cells may be distinct from those on naive T cells (44).

Regulation of Th1 versus Th2 Responses In Vivo by IL-21 The role that IL-21 plays in the in vivo regulation of Th1 versus Th2 polarization has been studied via the use of IL-21R KO mice in a number of immunization and infection models. IL-21R KO mice exhibit normal development of CD4+ T cells both in the thymus and in the periphery (20). Additionally, in vitro stimulation of naive CD4+ T cells under Th1- or Th2-polarized conditions showed no differences in WT versus IL-21R KO levels of IFN-γ or IL-4, suggesting that IL-21 is not essential for the normal differentiation of these two subsets (20). When IL21R KO mice were examined in a delayedtype hypersensitivity model in which footpad swelling was measured after an antigenic challenge, these KO mice had higher inﬂammatory responses than did WT mice (38). Ex vivo antigen-speciﬁc challenge of CD4+ T cells in these challenged mice revealed higher production of IFN-γ by KO than by WT cells. Another group examined the expression of IL-21 during the time course of infection with Schistosoma mansoni, a parasite that induces a Th2-dependent granuloma formation (45). IL-21 levels were measured in strains of mice that developed massive Th1 (IL-4/IL-10 DKO) or Th2 (IL-12/IL-10 DKO) responses to the parasite. Although infection of these mice with schistosome eggs induced highly polarized responses in the lungs, as evaluated by the production of IL-13 and IFN-γ, IL21 was produced during the infection in both strains of mice, indicating that IL-21 does not www.annualreviews.org • Interleukin-21

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behave as a classical Th1 or Th2 cytokine (45). When IL-21R KO mice were infected with S. mansoni, there was not a corresponding increase in Th1 cytokine production, despite a reduced Th2 response (i.e., decreased production of IL-4, IL-10, and IL-13) to the parasite in the granulomatous tissues of the lungs. Consistent with the decreased Th2 response in the IL-21R KO mice, there was more rapid resolution of the lung granulomas in these mice, indicating that IL-21 plays a role in the initiation and maintenance of a granulomatous inﬂammatory response. In spite of the reduced Th2 responses in the S. mansoni– infected mice, there was no difference in the cytokine proﬁle of CD4+ T cells stimulated ex vivo with antigen, suggesting that in vivo IL21 deﬁciency may not alter the Th priming of CD4+ T cells per se but rather may lead to a depressed Th2 response through mechanisms that are not yet clear. In a separate study, IL21R KO mice that were infected with Heligmosomoides polygyrus intestinal parasites developed fewer and smaller granulomas and had a reduced eosinophilia in the blood, suggesting a defective Th2 type response (46). However, analysis of ex vivo cytokine production from infected mice revealed no differences in levels of IFN-γ or IL-4 (46).

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IL-21 Critically Regulates Th17 Development IL-17-producing Th17 cells have distinctive developmental and functional properties that differ from those ascribed to Th1 and Th2 CD4+ effector T cells (47, 48). TGF-β, an immunosuppressive cytokine with a role in the generation of T regulatory (Treg) cells (49), also plays a key role in the induction of the Th17 differentiation pathway (50). However, Treg and Th17 cells are induced by TGF-β in a mutually exclusive manner; IL-6 shifts the balance in favor of Th17 cells and decreases the development of Treg cells (51). An analysis of TCR-stimulated CD4+ T cells revealed that the IL-21 gene was one of the genes most highly induced by IL-6 (42). 66

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Indeed, IL-21 mRNA and protein are very highly expressed in Th17 cells, at levels approximately ﬁvefold higher than in Th1- and Th2-polarized cells (40, 41). The induction of IL-21 leads to a further autocrine upregulation of IL-21 (42). Interestingly, IL-21 induced IL-23R expression on these CD4+ T cells (41, 42). IL-23R forms dimers with the IL-12Rβ1 subunit, which is shared by both IL-12R and IL-23R (52). Although IL-23 is an important factor in inﬂammatory disease in both mice and humans, its receptor is not present on naive T cells, and IL-23 may play a role in the expansion of already differentiated Th17 cells (53). The induction of IL-23R by IL-21 in naive CD4+ T cells is therefore a critical step in the differentiation and possibly in the expansion of Th17 cells in vivo. IL-21 and IL-23 both upregulated expression of the orphan nuclear receptor RORγt, which is essential for Th17 differentiation (54) and which leads to further upregulation of IL-21 (42). IL-17 production was signiﬁcantly lower in CD4+ T cells from IL-21R KO mice that were induced in vitro with TGF-β and IL-6, demonstrating that the induction of IL-21 by IL-6 leads to an ampliﬁcation of this differentiation pathway (41, 42). In fact, no IL-23R was induced by TGF-β + IL-6 in CD4+ T cells from IL-21 KO or IL-21R KO mice, indicating a critical role for IL-21 in controlling IL-23R expression (41, 42). The initial induction of IL-21 in Th17 cells therefore is critical for the establishment of an autocrine ampliﬁcation pathway for maximal IL-17 production.

IL-21 and Treg Induction IL-21 also plays an indirect role in the regulation of Treg differentiation. Interestingly, IL6 is critical in the inhibition of Treg differentiation by TGF-β and in the induction of Th17 differentiation (51). Indeed, IL-6 KO mice do not produce Th17 cells but produce a dominant FoxP3+ Treg population (40). However, in mice that are deﬁcient in both IL-6 and FoxP3+ Treg cells, Th17 cells are once again

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present, and these cells are produced in response to IL-21 in combination with TGFβ, demonstrating the presence of an IL-6independent pathway for Th17 production. The negative effects of IL-21 on Treg production are further demonstrated by the presence of a three- to fourfold increased FoxP3+ CD4+ T cell population in IL-21 KO mice (41).

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IL-21 Regulates Proliferation and Effector Function of CD8+ T Cells IL-21 is produced by CD4+ T cells and, as indicated above, inﬂuences the development of speciﬁc functional subsets within this lineage; however, the CD8+ T cell lineage is perhaps the primary target of the proliferative effects of IL-21. Nevertheless, CD8+ T cell development appears normal in the IL21R KO mice (20), indicating compensatory redundancy of these proliferative effects. In vitro experiments demonstrate that IL-21 by itself has very little if any effect on naive or memory phenotype CD8+ T cell proliferation and expansion but that it has a profound synergistic effect on proliferation in combination with either IL-7 or IL-15 (55), cytokines previously identiﬁed as playing major roles in homeostatic expansion of naive or memory CD8+ T cells (56, 57). This synergistic effect is especially evident when CD8+ T cells are stimulated in the absence of TCR signals (55), suggesting that IL-21 may play a role in antigen-independent expansion of this lineage in vivo. Gene expression analysis by DNA microarrays reveals that subsets of mRNAs are regulated individually by IL-21 or IL-15, but an additional set of mRNAs are cooperatively or distinctively regulated by the combination of IL-21 and IL-15, including granzyme B, which is important in cytolytic function of CD8+ T cells, as well as c-jun, which plays a role in the control of proliferative responses (55). The molecular mechanism of this synergistic transcriptional activity by these two cytokines remains to be determined. It is interesting that IL-21 activates Stat1 and Stat3

whereas IL-15 activates Stat5; however, differential STAT protein activation alone cannot be the full explanation given that the cooperative effect of IL-15 with IL-21 cannot be mimicked by IL-2, even though, like IL15, IL-2 is an activator of Stat5 proteins. Despite the ability of IL-21 to synergistically upregulate proliferation in combination with either IL-7 or IL-15, IL-21 has distinct effects on the differentiation of CD8+ T cells. IL-15 treatment of naive CD8+ T cells induces an effector phenotype in CD8+ T cells characterized by reduced CD28 and CD62L surface proteins, but IL-21 acts to prevent the downregulation of these proteins and potentially serves to maintain the important costimulatory function mediated by them (58). Although IL-21 alone can lead to the downregulation of CD44 expression on CD8+ T cells, the combination of IL-21 with IL-15 enhanced the accumulation of CD44high CD8+ T cells (55). With regard to cytokine production by naive CD8+ T cells, IL-21 alone induced no accumulation of IFN-γ-producing cells, and IL-15 alone could induce these cells, but the combination led to a further increase in the number of IFN-γ-producing cells (55). The effects of IL-21 on antigen-speciﬁc CD8+ T cell proliferation and effector function have been examined in several experimental systems. Primary immunization of mice with vaccinia virus expressing HIV gp160 antigen induced signiﬁcantly lower expansion and cytolytic activity in IL-21R KO mice than in WT mice (55), indicating a role for IL-21 in antigen-speciﬁc expansion and functional differentiation of naive CD8+ T cells in vivo. When naive human CD8+ T cells were stimulated in vitro with mature DCs presenting a tumor-associated peptide, there was a greatly augmented proliferation when IL-21 was added, leading to the accumulation of a population of cytotoxic cells characterized by a CD28high surface phenotype and a tenfold higher afﬁnity for antigen and a signiﬁcantly increased production of IL-2, as compared with cells stimulated in the absence of IL21 (59). In contrast to the ability of IL-21 to www.annualreviews.org • Interleukin-21

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augment the antigen-independent proliferation of both naive and memory CD8+ T cells, IL-21 augmented proliferative and differentiative effects of antigen-dependent stimulation with naive but not with memory CD8+ T cells. The basis for this difference is not yet known. A study of the effects of IL-21 on CD8+ effector T cells in HIV-infected patients revealed that IL-21 could upregulate perforin production in the absence of cell activation or proliferation, whereas IL15-mediated upregulation of perforin was less substantial and occurred only in the presence of proliferation (60). This induction of perforin by IL-21 in memory T cells was greater in cells from HIV patients than from normal controls. Thus, the proliferative and functional effects of IL-21 differ for naive and memory CD8+ T cells.

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Actions of IL-21 on Natural Killer Cells NK cell development depends on the function of γc cytokines; γc KO mice are devoid of mature NK cells (61, 62). IL-21R KO mice have normal numbers of fully functional NK cells (20, 63), indicating that IL21 is not required for NK cell development, but it has become clear that IL-21 plays a role in NK cell maturation and functional development and that the actions of IL-21 on this lineage are stage speciﬁc. The original observation of an effect of IL-21 on NK cells was that IL-21 enhanced in vitro generation of NK cells from bone marrow precursors (5). Although γc KO mice lack mature NK cells, bone marrow NK cell precursors (CD122+ NK1.1− CD49b− ) develop even in the absence of γc -mediated signals, and a small subset of these precursors expresses IL21R (64). Increases in IL-21R levels on these precursors depended on IL-15, suggesting that IL-15 may regulate the ability of NK precursors to respond to IL-21 (64). Although IL-21 has not been found to affect NK cell generation, experiments using human cord blood NK cell precursors showed that these 68

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cells progressed further along the maturation pathway if IL-21 was added to the combination of IL-15, Flt3, stem cell factor, and IL-7, a set of cytokines required for in vitro generation of NK cells (65). Once committed immature NK cells are generated, IL-21 can also enhance their proliferative response to suboptimal IL-2 or IL-15 concentrations while having no effect on proliferation by itself (66). Interestingly, IL-21 had a biphasic effect on the growth of immature NK cells: Low doses of IL-21 enhance proliferation and high doses inhibit proliferation, even in the presence of IL-2 or IL-15 (66). IL-21 also has effects on mature NK cells, including effects on both proliferation and survival as well as on NK cell–speciﬁc cell surface receptors. IL-21 had a negative effect on the proliferation of NK cells that had been activated by IL-15, either inhibiting proliferation or increasing their apoptosis (67). In spite of the reduced proliferative and survival effects of IL-21 on NK cells, these cells exhibited enhanced cytolytic function, increased IFN-γ production, and conversion to a large granular phenotype, all indicative of enhanced effector function of these cells (63, 67). In addition, IL-21 inclusion in the in vitro–generated NK cell cultures resulted in changes in the expression of several NK cell inhibitory and activating receptors. For example, IL-2 and IL-15 could induce inhibitory Ly49 receptors on mature NK cells, but the inclusion of IL-21 downregulated these receptors (68). The NKG2D receptor was similarly downregulated by IL21 in cultures of human NK cells (69). This decreased expression was mediated by transcriptional repression of the DAP10 adaptor through which NKG2D signals. Consistent with these changes in NK receptor expression, IL-21 modestly inhibited NK cell lysis of NKG2D-sensitive targets (69). Although IL21 repressed NKG2D expression on mature peripheral blood NK cells, IL-21 enhanced NKG2D expression on murine bone marrow– derived NK cells, which represent a less mature population of NK cells. This underscores

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the stage-speciﬁc effects of IL-21 on NK cells (67). Thus, although IL-21 is not required for NK cell development, it inﬂuences the proliferation and functional activity of this lineage.

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IL-21 Effects on Natural Killer T Cells NKT cells are a population of T cells expressing a restricted TCR repertoire; they recognize glycolipids presented by CD1d as well as inhibitory and activating NK receptors (70). NKT cells have immunoregulatory activity on other subsets that is related to their secretion of cytokines and their potent cytotoxic activity. Similar to its effect on NK cells, IL-21 can increase the proliferation of NKT cells in response to in vitro stimulation with antiCD3 but only when combined with either IL2 or IL-15 (13). IL-21 can also stimulate the in vitro release of increased levels of IL-4 and IL13 by NKT cells. In addition, IL-21 upregulated effector function in NKT cells through the induction of granzyme B and conversion to a large granular cell morphology similar to what was found for NK cells (13). Interestingly, NKT cells are also potent producers of IL-21 when stimulated in vitro with anti-CD3 or in vivo with α-GalCer, a stimulatory glycolipid speciﬁc for NKT cells (13). Levels of IL-21 protein secreted by NKT were signiﬁcantly higher than those produced by splenic CD4+ T cells in response to anti-CD3 stimulation. The ability of NKT cells to produce large amounts of IL-21 in response to microbial stimuli opens the possibility that these innate immune cells can regulate the initial steps in the formation of an adaptive immune response by B and T cells.

IL-21 Inhibits Dendritic Cell Maturation and Function DCs are peripheral myeloid cells that have the capacity to recognize microbial components via surface receptors, endocytose these microbes, and then undergo maturation in response to some of these microbial compo-

nents. Subsequent to this maturation, DCs migrate to lymphoid organs, where they function as antigen-presenting cells for T cells. The initial evidence that IL-21 can affect the proliferation or differentiation of myeloid cells came from the observation that injection of an IL-21-encoding plasmid into WT mice led to increases in the numbers of both CD11b+ and Gr1+ cells in the periphery (71). Although most of the effects of IL-21 on lymphoid cells are stimulatory, involving enhanced proliferation or effector function, effects of IL-21 on DCs are largely inhibitory. DCs can be generated and expanded in vitro by culturing bone marrow precursor cells with GM-CSF. When DC cultures were expanded in this manner in the presence of either IL21 or IL-15, differences in their phenotype and function were evident (17). Although IL15-treated DCs behaved as mature DCs and could present antigen in both in vivo and in vitro assays, IL-21-treated DCs maintained an immature phenotype that was characterized by low MHC class II expression accompanied by increased uptake of antigen and lowlevel expression of CC-chemokine receptor 7 (CCR7) (17). When IL-21-primed DCs were stimulated with LPS, there was no upregulation of MHC class II, CD86, or CD80 costimulatory proteins, in contrast to the upregulation that is seen with IL-15-primed DCs, and the IL-21-primed DCs had inhibitory effects on T cell responses. Even when DCs were treated for only 2 h in vitro with IL-21 plus ovalbumin antigen and then adoptively transferred in vivo, they could inhibit T cell– mediated contact hypersensitivity responses (17). IL-21 can also exert proinﬂammatory effects on immune responses through the induction of the neutrophil chemoattractant CXCchemokine ligand 8 (CXCL8) in macrophages (72). Neutrophils apparently lack IL-21R but can be recruited to sites of inﬂammation indirectly through IL-21-mediated CXCL8 induction on macrophages. Additionally, the critical role played by IL-21 in the differentiation and expansion of Th17 cells leads to the www.annualreviews.org • Interleukin-21

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production of IL-17 family cytokines that affect neutrophil recruitment and function (40– 42). Thus, IL-21 can either dampen immune responses or exacerbate them, depending on the myeloid population that is targeted and the timing of exposure to IL-21 during the course of an immune response.

IL-21 Mediates Potent Antitumor Responses

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The ability of IL-21 to regulate both T cell– mediated and NK cell–mediated immune responses suggested possible antitumor effects. Indeed, IL-21 has been examined in a number of in vivo tumor models, and these studies have conﬁrmed the potent effects of IL21 as an antitumor agent in animal models. The success in these models has led to the use of IL-21 in several phase I clinical trials in advanced-stage melanoma patients. The mechanisms of the antitumor action of IL-21 involve augmented NK cell and CD8+ T cell cytotoxicity. Systemic expression of IL-21 by plasmidmediated in vivo delivery led to an inhibition of the growth of large preestablished melanomas and ﬁbrosarcomas (71). These effects were mediated predominantly by NK cells; ablation of this population reduced the antitumor effect, with only minimal effects seen by ablation of the CD8+ T cell population. Signiﬁcantly, there were no major in vivo toxic effects, even at high doses of IL-21 (71), unlike the severe toxicity observed with similar doses of IL-2 or IFN-α. In another study that used a different approach to achieve high systemic levels of cytokine, IL-21 and IL23 were constitutively expressed in pancreatic carcinomas, leading to retarded tumor growth in nude mice, an effect that was again predominantly mediated by NK cells (73). IL-21 overexpression in mammary adenocarcinoma cells also led to the prevention of tumor initiation, although in this system there was no role for NK cells, and the tumor prevention was completely dependent on CD8+ T cells (74). 70

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NK cell–mediated killing of tumors after treatment with IL-21 appears to depend on the presence of NKG2D ligands on the tumor target because there was no IL-21-mediated enhancement of rejection of tumors that did not express these ligands and killing could be blocked by antibodies to NKG2D (75). IL-21 could enhance killing of ligand-positive tumors even in Rag2 KO mice, demonstrating that this is an NK cell–mediated event not requiring an adaptive immune response. These experiments suggest that the action of IL-21 on NK cells may be limited to tumors involving the NKG2D recognition system. Although this study found no effects of IL-21 on NK cell expression of this receptor, other studies did ﬁnd either positive or negative effects of IL-21 on NKG2D expression (67, 69). Other tumor systems have allowed the delineation of IL-21-induced CD8+ T cell– mediated killing mechanisms. One study compared the antitumor activity of intraperitoneally delivered IL-2, IL-15, and IL-21 with syngeneic E.G7 thymomas. This study found that, although all three cytokines could induce greater survival than PBS, IL-21 was the most potent, and the administration of IL21 resulted in a doubling of the 50% survival time, with 20–30% of the mice surviving for more than four months after IL-21 was administered (76). When these long-term survivors were rechallenged with thymoma, all the mice survived for more than 100 days, and this survival was dependent on the presence of a persistent CD8+ T cell memory population that was less susceptible to apoptosis than were CD8+ T cells induced by treatment with IL-2 (76). In another study, mice with large, established melanomas were treated by adoptive transfer of in vitro expanded tumor-speciﬁc CD8+ T cells plus peptide vaccine, followed by intraperitoneal administration of either IL2, IL-15, or IL-21 or the combination of IL-15 plus IL-21 (55). Treatment with either IL-15 or IL-21 led to partial tumor regression, but consistent with the synergistic effects of these two cytokines on CD8+ T cell

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proliferation, the combination of IL-15 and IL-21 led to complete regression of a subset of these melanomas and long-term survival of the majority of the treated mice (55). Although treatment with IL-2 alone was less effective therapy than treatment with either IL-15 or IL-21, IL-21 acted synergistically with low-dose IL-2 after the adoptive transfer of naive tumor-speciﬁc CD8+ T cells into mice with preestablished melanomas (77). Nearly half of these treated mice survived for more than 150 days. When these long-term survivors were rechallenged with melanoma, all were protected, indicating the induction of long-term immunity as a result of the combination of IL-2 with IL-21. Although IL-21 has shown great potential as a cancer chemotherapeutic agent either alone or in combination with other cytokines, it also has potential for being used with other forms of therapy. The TRAIL/DR5 ligand/receptor pair controls apoptosis and has been the target for monoclonal antibody therapy in some tumors (78). AntiDR5 mAb can inhibit tumor growth in an FcR-dependent fashion mediated by NK cell antibody-dependent cellular cytotoxicity (79). IL-21 can enhance the ability of NK cells to lyse antibody-coated cancer cells (80). After anti-DR5 treatment of tumor-bearing mice, some tumor cells die, and these apoptotic tumor cells prime CTL to respond to tumorspeciﬁc antigens. When tumor-bearing mice were treated with anti-DR5, followed by IL21, there was enhanced suppression of metastasis of small, preestablished tumors as well as an enhanced CD8+ memory T cell response to secondary tumor challenge. In contrast, similar treatment did not eradicate large, established tumors (81). Another method for enhancing the innate immune response involves the use of the CD1d-reactive glycolipid α-GalCer. In vivo treatment of mice with α-GalCer potently activates NKT cells that then stimulate the activation and proliferation of other lymphoid populations (82). The combination of α-GalCer treatment and IL-21 administra-

tion resulted in synergistically enhanced prevention of tumor metastasis (83). Transfer of DCs pulsed with α-GalCer, followed by IL21, reduced already established metastatic tumors (83). Overall, these results suggest that the ability of IL-21 to enhance NK, NKT, and CD8+ T cell function can potentially be used in combination with numerous chemotherapeutic protocols to lead to further advances in the eradication of tumors. These preclinical studies have shown that IL-21 has signiﬁcant antitumor activity against a variety of tumors that is mediated by multiple mechanisms involving both the innate and adaptive immune systems. IL-21 has entered human clinical trials, and phase I results in patients with metastatic melanoma have been reported (84). Consistent with the animal models, IL-21 was well tolerated, and there were few adverse effects, unlike the capillary leak syndrome or neurotoxicity resulting from IL-2 and IFN-α therapy. IL-21 potently upregulated perforin and granzyme B mRNA in patients, at all except the lowest dose tested. One patient in the phase I trial achieved complete remission, and 9 of 29 had stable disease at the end of the study.

Role of IL-21 in Autoimmune Disease In light of the pleiotropic effects of IL-21 on the function of different components of the innate and adaptive immune systems, it was difﬁcult to predict the role that IL-21 would play in the various autoimmune diseases. The initial observation that suggested that IL-21 might play a role in the progression of B cell–mediated autoimmune disease was that, in the BXSB.B6-Yaa+ mouse model of systemic lupus erythematosus (SLE), the development of disease correlated with an increased serum expression of IL-21 (29). This was consistent with the increased serum Igs in these mice and the role for IL-21 in plasma cell differentiation. Another autoimmune mouse strain, the sanroque mutant, has a defect in the function www.annualreviews.org • Interleukin-21

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of a protein, Roquin, which is a negative regulator of the production of a unique population of follicular T helper (TFH) cells (85). These TFH cells produce high levels of IL-21, and sanroque mutant mice have increased levels of these TFH cells as well as increased production of IL-21 accompanied by augmented levels of antinuclear antibodies, glomerulonephritis, and peripheral lymphadenopathy (85). The hypothesis is that the increased levels of IL-21 lead to increased formation of high-afﬁnity autoreactive antibodies by follicular B cells. That these autoimmune phenotypes were accompanied by high levels of IL-21 suggested that blocking the IL-21 signal might ameliorate autoimmune symptoms. Such a study was performed in the lupus-prone MRL-Fas/lpr mouse model through the use of IL-21R-Fc fusion proteins as blocking agents (86). Analogous to the Yaa mice results, lpr CD4+ T cells produced higher levels of IL-21. IL-17 IL-21

Th17

Th2 IFN-γ

Cytotoxicity, proliferation, antitumor activity

NKT

Th1

Cytotoxicity, proliferation, antitumor activity

IL-21 DC

NK

APC function

CD8

B Proliferation/apoptosis, plasma cell differentiation, Ig production

Cytotoxicity, proliferation/survival, antitumor activity

Figure 4 IL-21 has pleiotropic effects on multiple target cells. IL-21 is produced by multiple subpopulations of CD4+ T cells and by natural killer T (NKT) cells (indicated by the red arrows), although the amounts secreted by T helper (Th) 17 and NKT cells are signiﬁcantly higher than those secreted by Th1 and Th2 cells. IL-21 can then function as an autocrine factor for these populations, with the indicated effects, or can then exert varied positive or negative effects on lineages that do not themselves produce IL-21. 72

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Treatment of these mice with the IL-21Rblocking agent led to a partial reduction of lymphadenopathy, morphological changes in kidney glomeruli, and slightly reduced levels of IgG1 and IgG2a. In a mouse model of collagen-induced arthritis, the IL-21Rblocking agent also slightly reduced inﬂammation (87), suggesting that interruption of the IL-21 signaling pathway may be beneﬁcial in several autoimmune diseases. One of the genetic loci that are associated with the autoimmune diabetic phenotype in the nonobese diabetic mouse (NOD) is the insulin-dependent diabetes susceptibility 3 locus (Idd3). This locus contains the genes encoding both IL-21 and IL-2 (88). Because these cytokines are known to play roles in the proliferation and function of CD8+ T cells and Treg cells, attempts have been made to identify mutations within this region that associate with diabetes prevalence in the population. One study in the NOD mouse found increased levels of IL-21 mRNA in T cells and suggested that high levels of IL-21 protein may promote homeostatic proliferation of the autoreactive CD8+ T cells that mediate destruction of the pancreatic islet β cells (89). However, a recent study has ruled out the possibility that IL-21 is the genetic determinant of Idd3 that predisposes one to the development of diabetes (90). IL-21 also has disease-promoting effects in experimental allergic encephalitis (EAE) (91), an experimental model of human multiple sclerosis, which is induced by immunization of mice with myelin antigen in the presence of adjuvants. When IL-21 is administered to mice before induction of disease, there is increased severity of disease characterized by increased numbers of inﬂammatory cells in the central nervous system. However, if IL-21 is administered after disease has been initiated, there is no effect on the disease severity. The ability of IL-21 to exacerbate disease is totally dependent on the presence of NK cells because depletion of these cells before disease induction abrogates the effect of IL-21 (91). Although these effects were

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attributed to the ability of IL-21 to activate NK cell–mediated inﬂammatory responses, recent studies have pointed to the role that IL-21 plays in the induction and expansion of the Th17 population in this EAE model (40, 41). IL-21 or IL-21R KO mice have a tenfold reduction in the number of IL-17-producing cells and greatly reduced EAE disease progression (40, 41), as do mice deﬁcient in IL17 (92). Interestingly, IL-21 KO mice have increased numbers of Treg cells (41). The enhanced autoimmune symptoms in the mice injected with IL-21 before the initiation of EAE may be the result of increased numbers of Th17 cells and reduced numbers of Treg cells (40).

CONCLUDING REMARKS Since the discovery of IL-21 and IL-21R in 2000, this γc family cytokine system has been demonstrated to have effects on an extremely broad set of target cells, including T cells, B cells, NK cells, NKT cells, and DCs (see Figure 4). The actions of IL-21 on each of these target cells can be either stimulatory

or suppressive, and the ultimate outcome depends on the manner by which the IL-21 signal is integrated with other signals received by a target cell. Although IL-21 was initially thought to be produced solely by antigenstimulated CD4+ T cells, the discovery that IL-21 is produced by NKT cells implies that it is a key player in early innate immune responses as well. The recent discovery that IL21 is produced by and plays a major role in the differentiation of the Th17 lineage has expanded our understanding of the ways that IL21 may contribute to inﬂammatory responses. Moreover, studies of cancer and autoimmune models suggest that administering IL-21 or blocking the action of IL-21 holds promise in a number of disease settings. IL-21 is thus an exciting cytokine with pleiotropic actions on multiple lineages whose modulation has clear therapeutic beneﬁts in animal models. Understanding how and when it exerts various effects in vivo are some of the major basic science challenges, with the goal that future studies will both advance our scientiﬁc knowledge and contribute to moving IL-21 into the therapeutic setting.

FUTURE ISSUES 1. To be able to speciﬁcally amplify or neutralize the effects of IL-21 in pathological situations, investigators will require an understanding of the stage-speciﬁc and context-speciﬁc signaling events involved in the response to IL-21. 2. The in vivo sites where IL-21-producing cells are found are not yet deﬁned. This awaits the construction of reporter mice, with the hope that mRNA expression will correlate with protein expression. The availability of these mice will allow an understanding of the physiological expression of IL-21 as well as mechanisms for controlling levels of IL-21. 3. An in-depth understanding of the molecular basis of IL-21 and IL-21R gene expression will be important to better understand and develop ways of controlling the expression of this cytokine and its receptor. Analogously, a more detailed understanding of IL-21-induced signaling pathways is also important. 4. A major clinical issue will be balancing the immunostimulatory effects of IL-21 on lymphoid lineages with the largely immunosuppressive actions on DCs and the apoptotic effects on inappropriately stimulated B cells.

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5. The effects of IL-21 on CD8+ T cell phenotype and function suggest that IL-21 will have an impact on immunological memory, with potential ramiﬁcations for vaccination strategies.

DISCLOSURE STATEMENT The authors have issued patents and/or patent applications related to IL-21.

ACKNOWLEDGMENT Annu. Rev. Immunol. 2008.26:57-79. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

This work was supported by the Intramural Research Program, National Heart, Lung, and Blood Institute, NIH.

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84. Davis ID, Skrumsager BK, Cebon J, Nicholaou T, Barlow JW, et al. 2007. An open-label, two-arm, phase I trial of recombinant human interleukin-21 in patients with metastatic melanoma. Clin. Cancer Res. 13:3630–36 85. Vinuesa CG, Cook MC, Angelucci C, Athanasopoulos V, Rui L, et al. 2005. A RINGtype ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 435:452–58 86. Herber D, Brown TP, Liang S, Young DA, Collins M, Dunussi-Joannopoulos K. 2007. IL-21 has a pathogenic role in a lupus-prone mouse model and its blockade with IL21R.Fc reduces disease progression. J. Immunol. 178:3822–30 87. Young DA, Hegen M, Ma HL, Whitters MJ, Albert LM, et al. 2007. Blockade of the interleukin-21/interleukin-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis. Arthritis Rheum. 56:1152–63 88. Denny P, Lord CJ, Hill NJ, Goy JV, Levy ER, et al. 1997. Mapping of the IDDM locus Idd3 to a 0. 35-cM interval containing the interleukin-2 gene. Diabetes 46:695–700 89. King C, Ilic A, Koelsch K, Sarvetnick N. 2004. Homeostatic expansion of T cells during immune insufﬁciency generates autoimmunity. Cell 117:265–77 90. Yamanouchi J, Rainbow D, Serra P, Howlett S, Hunter K, et al. 2007. Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity. Nat. Genet. 39:329–37 91. Vollmer TL, Liu R, Price M, Rhodes S, La Cava A, Shi FD. 2005. Differential effects of IL-21 during initiation and progression of autoimmunity against neuroantigen. J. Immunol. 174:2696–701 92. Komiyama Y, Nakae S, Matsuki T, Nambu A, Ishigame H, et al. 2006. IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis. J. Immunol. 177:566–73

www.annualreviews.org • Interleukin-21

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:57-79. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

vi

Contents

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Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal,1,∗ D. Malo,2,∗ J.-F. Marquis,3 and P. Gros3,∗ 1

Department of Microbiology and Immunology, 2 Research Institute of the McGill University Health Center and Department of Human Genetics, and 3 Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6; email: [email protected]

Annu. Rev. Immunol. 2008. 26:81–132

Key Words

First published online as a Review in Advance on October 22, 2007

forward genetics, positional cloning, macrophage, NK cell, innate immunity

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090304 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0081$20.00 ∗

Authors contributed equally to this review.

Abstract Forward genetics is an experimental approach in which gene mapping and positional cloning are used to elucidate the molecular mechanisms underlying phenotypic differences between two individuals for a given trait. This strategy has been highly successful for the study of inbred mouse strains that show differences in innate susceptibility to bacterial, parasitic, fungal, and viral infections. Over the past 20 years, these studies have led to the identiﬁcation of a number of cell populations and critical biochemical pathways and proteins that are essential for the early detection of and response to invading pathogens. Strikingly, the macrophage is the point of convergence for many of these genetic studies. This has led to the identiﬁcation of diverse pathways involved in extracellular and intracellular pathogen recognition, modiﬁcation of the properties and content of phagosomes, transcriptional response, and signal transduction for activation of adaptive immune mechanisms. In models of viral infections, elegant genetic studies highlighted the pivotal role of natural killer cells in the detection and destruction of infected cells.

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INTRODUCTION

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Infectious diseases continue to be a major determinant of global health, exacerbated by recent factors, such as global population growth, an increase in the number and size of vulnerable populations, limited access to medical care in areas of endemic disease, and the human immunodeﬁciency virus (HIV) pandemic. Increased worldwide travel has further facilitated the export of several diseases to nonendemic areas. The impact of infectious diseases has been most severe in the developing world owing to the degradation of socioeconomic conditions, movement to new habitats, and, importantly, emergence of drug resistance and the lack of efﬁcacious vaccines leading to the remarkable resurgence of old foes, such as malaria and tuberculosis (TB) (1) (http://www.who.int). In addition to environmental factors, the health status of the host, and virulence determinants of the pathogen, clinical epidemiology and population studies as well as studies in twins have shown that host genetic factors play an important role, particularly in the onset but also in the progression of infection, the type of disease developed, and the ultimate outcome of infection with many pathogens (2, 3). In fact, infectious agents may have exerted the single most powerful evolutionary pressure on the human genome. Spectacular examples of the effect of infectious agents on the human genome include the protective effects of CCR5 mutations against HIV/AIDS (4) and the malaria-protective effect of heterozygosity at otherwise diseasecausing hemoglobinopathies, such as sickle cell anemia and thalassemias (5). Researchers have identiﬁed additional single-gene effects in humans that cause either selective or generalized immunodeﬁciencies, phenotypically expressed as an innate susceptibility to certain types of bacterial and viral infections (6). The study of these infrequent single-gene effects in humans is useful for better understanding the molecular pathogenesis of infection, but it also reveals normal host defense mechanisms that can potentially be used as novel targets for 82

Vidal et al.

drug discovery and therapeutic intervention in these diseases (7). However, linkage and association studies show that the genetic component of susceptibility to infection is usually complex and multigenic, which reﬂects the plurality of cell types and physiological and biochemical pathways involved in both the initial sensing of and the dynamic response to a pathogen (2). This complex host:microbe interaction interface gives rise to apparent genetic heterogeneity, incomplete penetrance, and variable expressivity that together reduce the power of standard genetic association or linkage studies. The laboratory mouse has proven to be extremely useful in the dissection of the genetic architecture of host defenses against many infectious diseases (8–11). Indeed, there exist excellent mouse models of experimental infection with many human pathogens, in which several aspects of the human disease are accurately reproduced: These include pathogenesis (tissues and cells involved, types and progression of lesions developed) and physiological responses (inﬂammation, immunity, etc.). These physiological responses can be studied in an environment in which pathogenassociated variables such as strain, virulence, dose, and route of infection can be carefully controlled. In addition, excellent immunological and biochemical reagents in the form of assays, markers, and antibodies are available to characterize innate and adaptive immune responses in these infection models. From a genetic standpoint, a number of inbred, recombinant, and naturally occurring or experimentally induced mutants [such as N-ethyl-N-nitrosourea (ENU)-induced mutants] exist that can be used to search for polymorphic alleles and major gene effects that affect onset, progression, host response, and ultimate outcome of infection. Additional mapping stocks such as recombinant inbred, recombinant congenic, and large multistrain intercross can sometimes be used to deconvolute multigenic effects into monogenic traits. With the sequencing and annotation of the mouse genome, good mapping tools are now

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available in the form of haplotype maps and informative markers, such as single nucleotide polymorphisms (SNPs) and simple sequence length polymorphisms (SSLPs). In addition, mRNA expression data are available for most genes, which facilitates the prioritization of candidate genes for positional cloning. Finally, germ line modiﬁcation in transgenic mice can be used to generate gain- or lossof-function alleles for validation of the role of individual genes. Generally, two major genetic approaches in the mouse have been used to identify host proteins and biochemical pathways that affect host response to infection with viral, parasitic, fungal, and bacterial pathogens. The ﬁrst is the reverse genetics approach in which the role of an individual gene is tested by directly infecting mice carrying a loss-of-function mutation (usually a deletion) at the corresponding locus. Usually, a possible role for a gene of interest is based on prior direct or indirect evidence that links the gene to pathogenesis or response to infection; researchers then examine the mutant for evidence supporting such a role. The second approach, which is the focus of this review, is the study of differential susceptibility of inbred mouse strains to infection with bacterial, parasitic, and viral pathogens. The major advantage of this socalled forward genetics approach is that the effect of the gene(s) on susceptibility is already validated in vivo. An obvious disadvantage of this approach is that the genetic effect may be complex, with individual monogenic contributions possibly difﬁcult to delineate. Nevertheless, this approach has been extremely successful in the study of infectious diseases and has revealed a treasure trove of genes, proteins, and signaling pathways that play critical roles in the immune response to a large number of infectious agents. We describe some of the most informative examples of such discoveries (summarized in Table 1 and Figure 1), with an emphasis on the implications of the discovered genes and proteins for our evolving understanding of innate or acquired immune defenses.

THE Ity-Lsh-Bcg LOCUS: REGULATION OF MACROPHAGE INTRACELLULAR IRON BY Nramp1/Slc11a1 Speciﬁc groups of pathogens penetrate host cells and proliferate, sheltered in the intracellular milieu. Certain microbes enter different cell types via an active microbe-driven invasion process, whereas others can survive and replicate after engulfment by professional phagocytes (macrophages, monocytes, and neutrophils). The intracellular environment provides a replicative niche rich in nutrients and sheltered from attacks by the host immune system. In the case of engulfment by professional phagocytes, intracellular pathogens have evolved mechanisms to escape or resist the microbicidal arsenal of these cells: These mechanisms include escape from the phagosome (Listeria), modulation of phagosome maturation (Mycobacterium, Legionella, Salmonella), and survival in fully mature phagolysosomes (Leishmania) (12–15). Understanding the genetic basis of differential susceptibility to infection with intracellular pathogens in inbred mouse strains has proven extremely informative for understanding the interface of host:pathogen interaction and identifying normal defense mechanisms of phagocytes that may fail in permissive hosts. The Ity-Lsh-Bcg locus constitutes one of the oldest and best studied examples of a single mutation in mouse with pleiotropic consequences on host defenses against unrelated intracellular pathogens (for historical reviews, please see References 16 and 17). Thirty years ago, several groups independently noted interstrain differences in susceptibility to infection with Salmonella enterica serovar Typhimurium (S. Typhimurium), Leishmania donovani, and Mycobacterium bovis (BCG). Segregation analyses showed that differences in susceptibility were controlled by a single gene, which was given the appellation Ity, Lsh, and Bcg. Concordance in differential www.annualreviews.org • Immunity to Infection in the Mouse

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Table 1

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Host resistance loci revealed by forward genetics approachesa

Pathogen(s)

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Locus (Gene)

Main cell type(s)

Protein function or biological process/mechanism of action

Reference(s)

Salmonella Typhimurium Leishmania donovani Mycobacterium bovis (BCG)

Ity-Lsh-Bcg (Nramp1)

M

Iron transporter/regulation of intraphagosomal iron

16, 17, 20

Salmonella Typhimurium

Lps (Tlr4)

M

Surface receptor for bacterial LPS/cellular recognition of LPS

63, 64, 70, 75

Salmonella Typhimurium

xid (btk)

B

Tyrosine kinase/tegulation of B cell development

60, 65, 66

Legionella pneumophila

Lgn1 (Birc1e)

M

NLR protein/intracellular antigen recognition

113, 114

Bacillus anthracis

Ltsx1 (Nalp1b)

M

NLR protein/sensing of anthrax toxin

125, 126, 128

Mycobacterium bovis (BCG) Salmonella Typhimurium Plasmodium chabaudi AS Mycobacterium tuberculosis

Myls (Icsbp/IRF8)

M

Transcriptional regulator/regulation of the IL-12 and IFN-γ pathway

130, 134, 135

Mycobacterium tuberculosis Listeria monocytogenes

sst1 (Ipr1)

M

Transcriptional regulator/transcriptional activation in response to intracellular pathogens

139, 144–146

Candida albicans Listeria monocytogenes

C5 (C5a)

unknown

Component of complement cascade/proinﬂammatory activity

156–158, 161, 162

Plasmodium chabaudi AS

Char4 (Pklr)

E

Pyruvate kinase/glycolysis in erythrocytes (role for ATP production)

167–170

Plasmodium chabaudi AS

Char9 (Vnn1/Vnn3)

E

Pantetheinases/production of the antioxidant cysteamine

167, 171

Toxoplasma gondii

Tyk2 (Tyk2)

M

Jak kinase/cellular signaling by cytokine receptors

180, 181

Orientia tsutsugamushi

Ric (Spp1)

M, T, NK

Phosphoprotein/recruitment of leukocytes and T cell polarization

187, 189, 190, 192, 193

Chlamydia trachomatis

Ctrq3 (Irgb10)

M and others

p47GTPase/mediator of the inhibitory effect of IFN-γ

197, 199

Orthomyxovirus (inﬂuenza)

Mx (Mx1)

M

GTPase/inhibition of viral genome transcription

206–209

Coronavirus (MHV)

Hv2 (Ceacam1)

EP

Transmembrane glycoprotein (with Ig domains)/adhesion molecule, signal regulatory protein

236–239

West Nile Virus (WNV)

Flv (Oas1b)

M

Oligoadenylate synthetase/part of the OAS/RNase L system of RNA decay pathway stimulated by type I IFN

257–259

Cytomegalovirus (MCMV)

Cmv1 (Ly49h)

NK

MHC class I receptor/recognition of infected cells by NK cell receptors

272, 274–276, 277–280

Staphylococcus aureus

Obl (Cd36 )

M

Scavenger receptor type B/regulation of the Tlr2/6-dependent signaling pathway

299

Vesicular stomatitis virus (VSV) Vaccinia virus

Lps2 (Trif )

M

Toll-receptor–associated activator of IFN/regulation of the Tlr3- and Tlr4-dependent signaling pathway

300, 305–308

Cytomegalovirus (MCMV)

Cpg1 (Tlr9)

M

Transmembrane receptor/recognition of pathogen-derived molecules

301, 309

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Table 1

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(Continued )

Pathogen(s)

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Locus (Gene)

Main cell type(s)

Protein function or biological process/mechanism of action

Reference(s)

Cytomegalovirus (MCMV) Staphylococcus aureus Listeria monocytogenes

3d (Unc93b)

M

Molecular mechanism unknown/regulation of the Tlr3, Tlr7 and Tlr9-dependent signaling pathway

295, 302, 310

Cytomegalovirus (MCMV) Listeria monocytogenes Vesicular stomatitis virus (VSV)

Domino (Stat1)

M

Transcription factor/activated by IFN

313, 315

Cytomegalovirus (MCMV)

Jinx (Unc13d)

NK, CTL

Membrane trafﬁcking/priming (fusion) of cytoplasmic vesicles

314, 316

a M, macrophages; E, erythrocytes; NK, natural killer cells; CTL, cytotoxic T lymphocytes; T, T cells; EP, epithelial cells; LPS, lipopolysaccharide; NLR, nucleotide-binding oligomerization domain (NOD)-like receptor; IL-12, interleukin-12; IFN-γ, interferon-γ; Jak, Janus kinase; p47GTPase, p47 family of IFN-γ-inducible GTPases; OAS, oligoadenylate synthetase; MHC, major histocompatibility complex; TLR, Toll-like receptor.

susceptibility to the three pathogens among inbred strains and in recombinant inbred strains (RIS) (bred from resistant and susceptible parents), together with cosegregation in progeny-testing experiments and colocalization of the three loci to the same proximal chromosome 1 domain, strongly suggested that Ity, Lsh, and Bcg were in fact the same locus (18, 19). In the three infection models, phenotypic expression of the locus is characterized by differential growth of the three pathogens in the spleen and liver, a phenomenon that can be reproduced in explanted macrophage populations ex vivo, which identiﬁed macrophages as responsible for the gene effect (17). The gene responsible for the ItyLsh-Bcg effect was one of the earliest genes to be isolated by positional cloning, which was in those days a laborious approach based on high-resolution linkage mapping, physical mapping by restriction enzyme analysis using pulsed-ﬁeld gel electrophoresis, and the creation of a transcript map of the minimal physical interval via delineation of CpG islands and isolation of splicing-competent exons (20). Based on its tissue speciﬁc expression in liver, spleen, and macrophages, a positional candidate, Nramp1 [natural resistance–associated macrophage protein 1, now annotated as so-

lute carrier family 11 member 1 (Slc11a1)] was selected from ﬁve candidates for further study. Nramp1 encodes a membrane phosphoglycoprotein with 12 putative transmembrane domains. Studies in inbred strains of mice showed that susceptibility to infections was associated with a single Gly169Asp (G169D) mutation in predicted TM4 of the protein (20). The G169D mutation impairs protein folding and processing, which results in the absence of mature Nramp1 polypeptide in the membrane compartment of susceptible cells (21). Researchers validated Nramp1 as the gene underlying Ity-Lsh-Bcg by creating a null mutation (knockout) and demonstrating that this otherwise resistant 129Sv mouse stock becomes susceptible to S. Typhimurium, L. donovani, and M. bovis (BCG) infections when Nramp1 is deleted (22). Also, transfer of the Nramp1G169 allele onto the C57BL/6J (Nramp1D169 ) background restores resistance to infection (23). Finally, the human gene, NRAMP1, was cloned and studied for a possible role in resistance and susceptibility to mycobacterial infections in humans (24). Brieﬂy, NRAMP1 maps on human chromosome 2q35, and researchers used numerous polymorphic variants within or outside the gene, including functional promotor www.annualreviews.org • Immunity to Infection in the Mouse

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during a TB outbreak in a Canadian Aboriginal family (26) and in pediatric TB cases from South Africa (27). Additional studies have shown that NRAMP1 is involved in susceptibility to two other common mycobacterial diseases, leprosy (28, 29) and Buruli ulcer (30).

polymorphisms, in case-control and familybased studies. NRAMP1 variants were consistently found to be associated with susceptibility to pulmonary TB in African and Asian populations but not in populations of European descent (25). In addition, direct genetic linkage data were obtained for NRAMP1

Chr. 1

A3

A5

B

C1

C2 C3

Stat1

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Chr. 2

A2 A3

A1

C5

E1 E2

D

Nramp1 B

C1

C3

E2

F

H1 H2 H3 H4

H6

H1 H2

H4

Ipr1 E1

D

E2 E3

E5 F2 F3

H3

C5a Chr. 3

A1 A2

A3

B

C

E1

D

E3

F1

F2

G1 G2 G3 H1

F3

H2

H3

H4

Pklr Chr. 4

A1 A2

A3

B2 B3

A5

C1 C2

C3

C5 C6

C7

D1

E1 E2

D3

Tlr4 Chr. 5

A1 A2

B1 B2 B3 C1

A3

D

Chr. 6

A2

A1

A3

B1

E2 E3 E4 E5

E1

Cd36

F

B3

B2

C1

Oas1b

D1 D2 D3 E1 E2 E3 F1 F2 F3

C3

Ly49p Chr. 7

A1

A2

C

A3 B1 B2 B3 B4 B5

D2

G2 G3

G1

Spp1

E1

E3 F1

F2

G3

G1

Ly49h F3

F4

F5

Ceacam1 Chr. 8

A2

A3

Chr. 9

A1 A2 A3

A4

B2 B3.1 B3.2 B3.3 C1 C2

C3 C4 C5

D1

D3

E1

E2

Icsbp/IRF8 B

C

D

E4

E1

F2 F3 F4

F1

Tyk2 Chr. 10

A1

A2

A3 A4

Tlr9

B1

B3

B2

B4

C1

C2

C3

D1

D2

D3

Vnn1/Vnn3 Chr. 11

A1 A2

A4

A5

B1.1 B1.2 B1.3 B2

Irgb10 Chr. 13

A2 A3.1

A1

A5

A3.3 A4

B1

B3

B4

C

B5

Nalp1b B2

B3

D

E1

E2

Unc13d

C1

C3

D1

Birc1e Chr. 16

A2 A3

B1

B2

B3

B4

B5

C1.1 C1.2 C1.3 C2 C3.1

C3.2

C3.3

C4

Mx1 Chr. 17

A2

A1

B1

B2

B3

C

D

E1.1 E1.2 E1.3 E2

A

B

E4

E5

Trif

H2-D Chr. 19

E3

C1

C2

C3

D1

D2

D3

Unc93b Chr. X

A2

A4

A5

A6

B

C1 C2 C3

D

E1 E2 E3 F1

F2 F3 F4 F5

Btk 86

Vidal et al.

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The Slc11a1 protein is present in the membrane of (a) Lamp-1+ lysosomes of macrophages and monocytes and (b) gelatinase+ tertiary granules of neutrophils (31, 32). Upon phagocytosis of inert particles or live microbes, Slc11a1 is rapidly recruited to the membrane of the maturing phagosome (31, 32). Clues about the function of Slc11a1 at the phagosomal membrane came from the discovery that the close mammalian relative Slc11a2 (Nramp2, DCT1, DMT1; ∼75% similarity to Slc11a1) functions as a pH-dependent iron (Fe2+ ) transport system (33, 34). Slc11a2 is required for the acquisition of nutritional iron at the duodenum brush border and acts as a transporter of transferrin-associated iron across the membrane of recycling endosomes in many cell types. Mutations in mouse and human (35) Slc11a2/Nramp2 cause a severe form of microcytic anemia (35, 36). Subsequently, microﬂuorescence imaging studies in live macrophages using inert particles labeled with metal-sensitive ﬂuorophores, together with studies in transfected CHO cells, showed that Slc11a1 (Nramp1) similarly functions as a metal efﬂux pump for Mn2+ and Fe2+ ions at the phagosomal membrane (37, 38). Transport occurs down a pro-

ton gradient generated by the concanamycinsensitive vacuolar H+ /ATPase. Therefore, Nramp1 and Nramp2 transport metal ions by the same mechanism but individually perform this activity in a cell type– and subcellular compartment–speciﬁc fashion. Consequently, the antimicrobial effect of Nramp1/Slc11a1 against different infectious agents can be explained by its capacity to restrict metal ions from the phagosomal space. The mechanism by which Nramp1mediated iron restriction negatively affects intracellular microbial survival/growth was investigated using phagosomes containing Mycobacterium, Salmonella, and Leishmania. Using M. bovis (BCG) and Mycobacterium avium as test pathogens, investigators showed that Nramp1 recruitment to the phagosomal membrane abrogates the ability of mycobacteria to block phagosome maturation, which causes increased acidiﬁcation, enhanced fusion to lysosomes, augmented mycobacterial cell damage, and reduced intracellular replication (39, 40). In the case of Salmonellacontaining vacuoles (SCV), recruitment of Nramp1 similarly antagonizes the ability of Salmonella to modulate phagosome maturation, which causes enhanced association

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1 Chromosomal location of mouse genes affecting susceptibility to infections and identiﬁed by forward genetics. The chromosomal position of host resistance-associated genes affecting susceptibility to infection with bacterial, viral, and parasitic pathogens is identiﬁed with respect to relevant mouse chromosomes. Map positions have been determined using the Mouse Ensembl annotation (www.ensembl.org/Mus musculus/index.html). The position of cytogenetically identiﬁable chromosomal bands is indicated. The chromosomes are not drawn to scale, and the exact position for each gene may differ slightly depending on the source of information. See text for details. [Abbreviations: Stat1, signal transducer and activator of transcription 1; Nramp1, natural-resistance-associated macrophage protein 1; Ipr1, intracellular pathogen resistance 1; C5a, complement component 5a; Pklr, pyruvate kinase liver and red blood cell; TLR, Toll-like receptor; Cd36, Cd36 antigen; Spp1, secreted phosphoprotein 1; Oas1b, 2 -5 oligoadenylate synthetase 1B; Ly49p, killer cell lectin-like receptor, subfamily A, member 16; Ly49h, killer cell lectin-like receptor, subfamily A, member 8; Ceacam1, CEA-related cell adhesion molecule 1; Icsbp, interferon (IFN) consensus sequence-binding protein; IRF8, IFN regulatory factor 8; Tyk2, tyrosine kinase 2; Vnn1/Vnn3, vanin 1 and 3; Irgb10, iron-regulated virulence protein 10; Nalp1b, NACHT, LRR, and Pyrin domain–containing 1b; Unc13d, unc-13 homolog D; Birc1e, baculoviral IAP repeat-containing 1e; Mx1, myxovirus resistance 1; H2-D, histocompatibility 2, D region; Trif, Toll receptor–associated activator of IFN; Unc93b, unc-93 homolog B; Btk, Bruton agammaglobulinemia tyrosine kinase.] www.annualreviews.org • Immunity to Infection in the Mouse

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of early endosomal proteins (e.g., EEA1, a ﬂuid phase marker) and the late endosomal protein, the mannose-6-phosphate receptor (M6PR) (41). Furthermore, the addition of membrane-permeant iron chelators to Nramp1-negative macrophages can recapitulate the Nramp1 effect and stimulate recruitment of M6PR and EEA1 to SCVs (42). Salmonella responds to Nramp1-mediated iron depletion by activating transcription of several virulence genes that map within the Salmonella pathogenicity island 2 (SPI2) region, including ssrA and sseJ (43). An adequate supply of iron is essential for intracellular survival of Salmonella, and iron stimulates intracellular growth of this bacterium (44). The Salmonella genome encodes several high- and low-afﬁnity transporters for ferric and ferrous iron ( fepBCDG, sitA-D, FeoABC, CorAD, and the Nramp homolog MntH ), and several of these transporters are essential for Salmonella virulence in vivo and for replication in Nramp1-negative macrophages (45, 46). Conversely, the presence of intracellular Salmonella is sensed by macrophages, which respond by activating an iron-restriction/extrusion pathway with enhanced expression of ferroportin (iron exporter), lipocalin 2, and heme oxygenase mRNAs (44). On a related note, Huynh and coworkers (47) recently identiﬁed the LIT1 protein as the major Fe2+ import system of Leishmania; LIT1 is uniquely expressed at the plasma membrane of the intracellular form of the parasite (amastigote), and it is essential for virulence in vivo and in macrophages ex vivo. LIT1 expression is induced in response to low iron levels, and LIT1 is differentially expressed in Nramp1positive (higher expression) and Nramp1negative (lower expression) macrophages (47). These studies highlight the critical role that iron plays at the interface of host:pathogen interaction, where metal transporters such as Nramp1 and ferroportin act as major defenses to restrict intracellular access to this essential nutrient.

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THE Lps LOCUS: CELLULAR RECOGNITION OF BACTERIAL LIPOPOLYSACCHARIDE BY TOLL-LIKE RECEPTOR 4 AND INNATE IMMUNITY TO SALMONELLA INFECTION Salmonella is a ubiquitous, facultative intracellular, Gram-negative bacterium that causes two major diseases in developed and developing countries: typhoid fever and salmonellosis. Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever and affects 17 million people annually, with 200,000 associated deaths. Salmonellosis is one of the most common and widely distributed food-borne diseases (http://www.cdc.gov). The frequency of multidrug-resistant nontyphoidal Salmonella (NTS) is increasing in developing countries; the disease presents as bacteremia (invasive NTS) often without diarrhea (48). Several speciﬁc human populations are at increased risk of Salmonella infection, including patients with sickle cell anemia (49), chronic granulomatous disease (50), and Plasmodium falciparum infection (51). In addition, humans who harbor mutations in genes that regulate the activation of phagocytes (IFNGR1, IFNGR2, IL12B, IL12RB1, and STAT1) are highly susceptible to recurrent Salmonella infections (reviewed in 52). Increased incidence of Salmonella infections also occurs in individuals with immunodeﬁciencies such as common variable immunodeﬁcieny (CVID), X-linked agammaglobulinemia (XLA), major histocompatibility complex (MHC) class II deﬁciency, HIV infection, and ectodermal dysplasia with immunodeﬁciency (reviewed in 53). Several mouse models of Salmonella infection have been used for the study of host:pathogen interactions (54–57). The most widely used model is intravenous infection with S. Typhimurium, which causes systemic disease resembling typhoid fever. Response to this infection involves the activation of both the innate and adaptive immune responses of the host; genetic analysis in the

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Salmonella model has successfully identiﬁed key host genes and proteins participating in these two pathways. Sublethal systemic infection with S. Typhimurium is characterized by four distinct phases of infection: rapid clearance of the bacteria from the bloodstream (phase 1), which requires complement and the production of reactive oxygen species (ROS); sheltering of some bacteria in macrophages and polymorphonuclear leukocytes from the spleen and the liver where the bacteria initiate a replicative phase (phase 2); stimulation of the innate immune response pathways by Salmonella products, such as lipopolysaccharide (LPS), and priming of the adaptive innate immune response [by tumor necrosis factor α (TNF-α), IFN-γ, interleukin (IL)-12, IL-18, and Toll-like receptor 4 (Tlr4)], resulting in dampening of the infection (phase 3); and activation of antigen-speciﬁc immunity and clearance of the infection (phase 4) (53). Inbred strains of mice show differential susceptibility to infection with Salmonella (reviewed in 58). Strains such as C57BL/6J, C57BL/10J, BALB/cJ, FVB, and DBA/1J are extremely susceptible and succumb within 5– 6 days post infection owing to their inability to suppress bacterial growth during the early innate immune response phase (phase 2) (22; D. Malo, unpublished data). C3H/HeJ mice show a similar degree of susceptibility, although they are able to survive the second phase of infection. However, C3H/HeJ mice present with high bacterial load in spleen and liver later during infection because of an inadequate adaptive innate immune response. Strains A/J, DBA/2J, and C3H/HeN present an intermediate susceptibility phenotype and show increased survival time (most noticeable with lower infectious inoculum) but cannot survive beyond the transition of innate to acquired immune defenses (phase 3). CBA/J mice show increased survival times and are able to survive the ﬁrst three phases of infection but ultimately succumb to infection because of their inability to mount an appropriate antigen-speciﬁc immunity. Finally, substrains of 129 inbred mice are ex-

tremely resistant, although they are unable to clear the infection, and they develop a chronic carrier state (56, 58). Investigators studying the natural variation of the host response to Salmonella infection in spontaneous mouse mutants (Ity, xid, Lps, and Ity4) (59–62) identiﬁed major novel host response pathways (Nramp1Ity , btkxid , Tlr4Lps , and PklrIty4 ) (20, 62– 66). The classical inbred strains, C57BL/6J, C57BL/10J, BALB/cJ, FVB, and DBA/1J, are all extremely susceptible to infection because of a loss-of-function mutation in the Nramp1/Slc11a1 gene (see the section on ItyLsh-Bcg), a major regulator of Salmonella susceptibility in mice. Mice carrying a mutant allele at Nramp1 all die early independently of their genetic background; the Nramp1 effect can be worsened in some cases by the presence of other Salmonella susceptibility alleles such as Tlr4Lps or PklrIty4 (see below) (62, 67, 68). The C3H/HeJ strain is exquisitely susceptible to infection with S. Typhimurium and other Gram-negative bacteria (59, 69, 70). This susceptibility is independent of Nramp1 alleles (C3H/HeJ has a resistance Nramp1G169 allele) but is linked to an aberrant response to the immunostimulatory properties of LPS. Sultzer (71) ﬁrst described the Lps (lipopolysaccharide response) locus in C3H/HeJ mice in 1968, and observed that C3H/HeJ mice are 20 to 38 times more resistant than A/HeJ mice to the toxic effects of systemic administration of LPS. A defective LPS response in C3H/HeJ mice affects several cell types, including B cells, T cells, macrophages, and ﬁbroblasts. Two allelic forms were recognized for Lps: a normal allele, Lpsn , and a defective allele, Lpsd (72). Other strains of mice (C57BL/10ScNCr and C57BL/6J.KB2mnd ) present LPS hyporesponsiveness as well (73, 74). F1 progeny from crosses between C3H/HeJ and C57BL/10ScNCr or between C3H/HeJ and C57BL/6J.KB2-mnd were also LPS hyporesponsive, which indicates that the three parental strains harbor a genetic defect in the same gene or in the same pathway. www.annualreviews.org • Immunity to Infection in the Mouse

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LPS hyporesponsiveness of C3H/HeJ mice is inherited as an autosomal codominant trait, whereas the LPS-hyporesponsive phenotype of C57BL/10ScCr mice is fully recessive (reviewed in 75). The response to Salmonella infection was inherited as a single dominant trait in all crosses performed between Lpsd and Lpsn mice. Using a variety of RIS, segregating backcrosses and congenic mice, the Lps locus was mapped to mouse chromosome 4 (reviewed in 75). A positional cloning approach was used by two research groups to identify the gene underlying the Lps phenotype (63, 64). Different subphenotypes were used to clone the Lps locus: the LPS-mitogenic response of splenocytes (63, 64), the response to Salmonella infection (64), and TNF production by macrophages (63). High-resolution linkage and physical mapping narrowed the Lps interval to 1.8 Mb (70; Ensembl mouse m36) that harbored an excellent positional candidate, Tlr4, an important component of the signal transduction initiated by LPS in humans in vitro (76). Support for Tlr4 underlying Lps came from the identiﬁcation of independent mutant alleles at Tlr4 in mouse strains defective in LPS response: C3H/HeJ mice present a single missense mutation resulting in a proline to histidine substitution at codon 712 within the signaling domain of Tlr4 (63, 64); C57BL/10ScCr mice have no Tlr4 transcripts (63, 64), as a consequence of a 75-kb chromosomal deletion encompassing the whole Tlr4 gene (77); and the mutation identiﬁed in C57BL/6.KB2-mnd Tlr4 mice consists of a complete deletion of exon II owing to a splicing defect, which results in a mutant variant consisting of only the ﬁrst 31 N-terminal residues of the protein (wildtype Tlr4 contains 835 residues) (74). Conﬁrmation of the role of Tlr4 in LPS hyporesponsiveness and Salmonella susceptibility was obtained through the creation and study of Tlr4-deﬁcient mice and Tlr4 transgenic mice (67, 68, 78, 79). TLR family members are evolutionarily conserved, type 1 transmembrane receptors

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characterized by an extracellular leucine-rich repeat (LRR) domain and an intracellular domain similar to the intracellular domain of the IL-1 receptor, the TIR (Toll/IL-1 receptor) domain. The ﬁrst identiﬁed member of this family, Drosophila Toll, is involved with innate immunity to fungal infection through the induction of an antifungal peptide. In mammals, 12 TLRs have been identiﬁed and classiﬁed by their ability to sense speciﬁc pathogen-associated molecular patterns (PAMPs) in vitro (reviewed in 80). Several TLRs are essential for defense against different pathogens, as shown by studies in TLR-deﬁcient mice. Tlr4 recognizes LPS with the cooperation of LPS-binding protein and coreceptors CD14 and MD2 (now annotated Ly96, lymphocyte antigen 96). Tlr4 triggers innate immunity through the activation of two signaling pathways: (a) nuclear factor-κB (NF-κB) signaling via the TIR domain–containing adapter protein (TIRAP) and myeloid differentiation primary response gene 88 (MyD88), which results in the activation of IL-1 receptor–associated kinases (IRAKs) and further recruitment of TNF receptor–associated factor 6 (TRAF6), and (b) a MyD88-independent pathway involving adapter proteins TRIF (Toll receptor– associated activator of IFN) and TRAM (TRIF-related adapter molecule), which results in the activation of IFN regulatory factor 3 (IRF3), the induction of type I IFNs, and a delayed NF-κB response (80). Recognition of LPS by TLR4 leads to the induction by macrophages of several proinﬂammatory cytokines, including IL-1, IL-6, IL-8, IL-12, chemokines, costimulatory molecules (CD80 and CD86), MHC class II, and nitric oxide synthase 2 (NOS2). Induction of CD80/CD86 and IL-12 by TLRs contributes to the initiation of adaptive immunity and the induction of T helper 1 (Th1) effector responses (81). TLR4 gene polymorphisms are associated with susceptibility to various infectious and noninfectious diseases (reviewed in 82, 83). Two polymorphisms, Asp299Gly and Thr399Ile, were originally associated with

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the lung response to inhaled LPS in healthy human volunteers (84). The Asp299Gly and Thr399Ile TLR4 alleles are associated with an increased risk of Gram-negative infection (85), sepsis (86, 87), severe inﬂammatory response syndrome (88), severe malaria (89), brucellosis (90), and respiratory syncytial virus disease (91). The same TLR4 alleles are protective against Legionnaire’s disease (92). Studies in mice bearing inactivating mutations for individual members of the TLR family have shown that other members of the TLR family likewise play a critical role in the host response to a broad range of bacterial, viral, fungal, and parasitic infections in TLRdeﬁcient mice (reviewed in 80, 93) and in speciﬁc human populations (reviewed in 82, 83). In humans, genetic variants in TLR2 are associated with susceptibility to TB (94) and the clinical manifestations (paucibacillary versus multibacillary) of leprosy (95, 96). A common mutation within TLR5 is associated with an increased susceptibility to Legionella pneumophila, the causative agent of Legionnaire’s disease (97). Heritable defects of the human TLR signaling pathway have been identiﬁed in patients suffering from recurrent infection and carrying mutations within the genes NEMO (now annotated IκBkγ, inhibitor of kappaB kinase gamma), IKBA (now annotated NFκBiα, nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha), and IRAK4 (6).

THE X-LINKED IMMUNODEFICIENCY LOCUS: B CELL DEVELOPMENT AND HOST RESPONSE TO SALMONELLA INFECTION CBA/N mice do not survive the late phase of Salmonella infection because they are defective in the humoral immune response against bacterial antigens (60). CBA/N mice carry the xid mutation (x-linked immunodeﬁciency), which is phenotypically expressed as a reduced number of B cells (50% of the normal B cell num-

ber), decreased serum IgM and IgG3 levels, normal levels of IgG1, IgG2a, and IgG2b, and a compromised T cell–independent immune response (98). The susceptibility of xid mice is recessive and maps to the X-chromosome; hemizygous males and homozygous females (for the xid locus) are susceptible to Salmonella infection whereas heterozygous females are fully resistant (60). The transfer of speciﬁc anti-Salmonella antibodies restores resistance to infection in affected xid males, which shows the importance of circulating antibodies in resistance to S. Typhimurium during the late phase of infection (99). The xid mutation of CBA/N mice was cloned via a positional candidate approach several years after it was ﬁrst described (65, 66). The xid mutation maps to a region of mouse chromosome X that is homologous to the human chromosomal region carrying the gene involved in XLA or Bruton’s agammaglobulinemia. Patients with XLA suffer from recurrent and persistent bacterial infections caused by Pseudomonas spp., Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus inﬂuenzae early in life. Lessfrequent intestinal infections with Salmonella spp. also occur in XLA patients. Male patients with XLA present a more severe immunodeﬁciency compared with the xid mouse, have less than 1% of the normal number of B cells, present panhypogammaglobulinemia [absence of immunoglobulins (Igs) of all classes], and fail to make antibody to all antigens (reviewed in 100). The gene responsible for the human disease, Bruton’s tyrosine kinase (BTK), was identiﬁed by positional cloning as a tyrosine kinase expressed during B cell development (101, 102). In humans, mutations within BTK occur in XLA families (101, 102). Soon after the human discovery, a mutation within the gene encoding btk was shown to be responsible for the xid phenotype in CBA/N mice (65, 66). The role of btk in xid was validated in vivo through the creation of a deletion allele; this allele causes a phenotype similar to xid and less severe than XLA in humans, possibly owing to www.annualreviews.org • Immunity to Infection in the Mouse

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the presence in the mouse of a btk-related kinase, Tec, that may compensate for the loss of btk function (103). Btk is a member of the Tec family of nonreceptor cytoplasmic tyrosine kinases that are highly expressed in most cells of the hematopoietic lineage (104). btk is expressed at all stages of B cell differentiation from pro to mature B cells, with the exception of plasma cells. Btk is also expressed in erythroid precursors, myeloid cells, mast cells, and megakaryocytes, but not in T or natural killer (NK) cells (101, 102). btk encodes a tyrosine kinase that possesses ﬁve different domains; these include the pleckstrinhomology (PH), the Tec-homology (TH), the Src homology 1 (SH1, known as the kinase domain), SH2, and SH3 domains (105). The expression of btk is critical for the proliferation, differentiation, survival, and apoptosis of B-lineage cells and is a major component of the B cell receptor (BCR) signalosome (105, 106). All domains of the btk protein bind a variety of interacting partners, which suggests its involvement in multiple signaling pathways (reviewed in 105). Notably, btk is involved in the activation of the transcription factor NFκB through the phosphorylation of the p65 subunit of the NF-κB complex in response to BCR engagement and to LPS (107, 108). Btk is also involved in the negative regulation of TLR signaling and, more speciﬁcally, in the phosphorylation of the adapter protein TIRAP after TLR2 and TLR4 stimulation (109, 110), which allows the interaction of TIRAP with SOCS-1 (suppressor of cytokine signaling 1). The interaction of TIRAP with SOCS-1 results in TIRAP polyubiquitination and subsequent degradation (110). Lindvall and colleagues (105) reported 554 different mutations in XLA patients that involve all domains of the BTK gene as well as noncoding sequences (http:// bioinf.uta.fi/BTKbase). In xid mice, a missense mutation at a conserved arginine residue (R28C) within the PH domain of btk prevents its ability to translocate to the plasma membrane and trigger signaling that regulates B cell survival and growth (111).

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THE LGN1 LOCUS: INTRACELLULAR RECOGNITION OF LEGIONELLA BY BIRC1E, A MEMBER OF THE NOD-LIKE RECEPTOR FAMILY Legionella pneumophila is a Gram-negative bacterium that causes a severe form of pneumonia called Legionnaire’s disease. L. pneumophila is a strict intracellular pathogen that survives and replicates in human macrophages, residing in a replicative phagosome that does not mature into a phagolysosome but rather acquires functional and biochemical characteristics of the endoplasmic reticulum (ER). These characteristics include the presence of ribosomes at the phagosomal membrane, retention of the ER markers calnexin and glucose-6-phosphatase, and the absence of lysosomal markers (e.g., Lamp1) (112). Macrophages from most inbred strains of mouse are resistant to infection with L. pneumophila (Philadelphia 1), with the exception of thioglycolate-elicited A/J macrophages that are permissive to the replication of L. pneumophila ex vivo (reviewed in 113). Studies in informative crosses between A/J and other inbred strains showed that susceptibility in A/J is controlled by a single locus on mouse chromosome 13, designated Lgn1. The minimal genetic interval (0.32 cM) is a highly complex and duplicated region containing several intact and rearranged copies of the Birc1 gene (113). Birc1 proteins are expressed in macrophages and are upregulated following macrophage phagocytosis of inert particles or live Legionella or Salmonella. Two intact gene copies, Birc1b and Birc1e, are contained within the minimal physical interval of Lgn1 (113). Functional complementation studies in transgenic mice, using large overlapping cloned segments from the region [bacterial artiﬁcial chromosome (BAC) clones], showed that Birc1e gene transfer to otherwise permissive A/J mice and overexpression of the Birc1e gene was sufﬁcient to restore resistance and restrict L. pneumophila replication in macrophages derived from the transgenic mice. Parallel

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studies showed that morpholino-based antisense inhibition of Birc1e can partially reverse the nonpermissiveness of macrophages from mouse strains containing a transgenic copy of Birc1e (114, 115). Together, these results strongly suggested that Birc1e is the gene underlying the Lgn1 defect. The complexity of the genomic region overlapping Lgn1 has so far precluded the creation and testing of a lossof-function mutant at Birc1e or at other Birc1 family members. The Birc1 gene family was initially annotated as neuronal apoptosis inhibitory protein (Naip) on the basis of (a) a possible implication of Naip in spinal muscular atrophy as a regulator of neuronal apoptosis and (b) the presence of baculovirus inhibitor of apoptosis protein (IAP) repeat (BIR) domains in the predicted Naip polypeptide, which interact with cellular caspases (116). The role of Naip5 in macrophage function remained unknown for many years. The recent reclassiﬁcation of Naip/Birc proteins as part of the nucleotidebinding oligomerization domain (NOD)-like receptor (NLR) family provided important clues to the possible function of Birc1e in macrophage defenses against infections. NLRs are a group of 23 structurally related cytosolic proteins (117, 118) that have several features in common: (a) a nucleotide binding domain (for oligomerization), (b) LRRs that act as recognition motifs for PAMPs in other proteins such as TLRs, and (c) a protein:protein interaction module that is speciﬁc for each member or subgroup of this family (e.g., BIR domains in Birc1). The NLR family [also known as the nucleotide binding site (NBS)-LRR or CATERPILLAR family] includes NOD1/NOD2 [also known as CARD (caspase recruitment domain)4/CARD15], which sense bacterial peptidoglycan; IL-1βconverting enzyme (ICE)-protease-activating factor (Ipaf ) (also known as CARD12), which interacts with intracellular ﬂagellin from certain Gram-negative bacteria; cryopyrin [also known as NALP3 (nacht, LRR, and Pyrin domain–containing 3), which binds bacterial RNA; and others that function as intracellu-

lar sensors of bacterial products (117, 118). Mutations in NLR family members are associated with dysregulated inﬂammation in certain chronic conditions such as Crohn’s disease (NOD2), Blau syndrome (NOD2), atopic eczema and asthma (NOD1), familial cold inﬂammatory syndrome [cold autoinﬂammatory syndrome 1 (CIAS1)], and bare lymphocyte syndrome [MHC class II transactivator (CIITA)] (117, 118). Recent studies have shed considerable light on the mechanism of action (the ligand and cellular signaling pathways) of Birc1e in macrophage defenses against Legionella. Infection of macrophages with Legionella pneumophila induces caspase-1-dependent cell death, a protective mechanism that restricts intracellular replication and requires a functional copy of Birc1e (Birc1e is seemingly impaired in A/J cells) (119). Additional studies by Nunez and colleagues (120) pointed to further complexity in the activation of caspase-1 in macrophages following infection with L. pneumophila (in addition to Birc1e). Activation of caspase-1 is dependent on the transfer of Legionella products to the cytosol via a functional type IV secretion system, and macrophages deﬁcient in caspase-1 (Casp1−/− ) or deﬁcient in Ipaf (Card12−/− ) are more permissive to L. pneumophila infection than control C57BL/6J (B6) macrophages (119). These observations support a model in which recognition of Legionella products by the LRR domain of Birc1e causes activation of the Ipaf-containing inﬂammasome, which results in bacteriostatic activity and ultimately cell death. Recent genetic analyses of L. pneumophila mutants strongly suggest that ﬂagellin may be the bacterial protein recognized by Birc1e: Bacterial ﬂagellin mutants grow in otherwise nonpermissive B6 macrophages and do not induce cell death (121, 122). Finally, results from studies of L. pneumophila– containing phagosomes formed in wild-type and Birc1e-deﬁcient macrophages suggest even more complexity in the mechanism of action of Birc1e. Indeed, the presence of Birc1e is associated with a reduced acquisition of ER www.annualreviews.org • Immunity to Infection in the Mouse

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markers (calnexin) and an enhanced acquisition of lysosomal markers (Lamp1, Cathepsin D) in primary macrophages (123). The Birc1e effect on phagosome maturation is very rapid, occurring within the ﬁrst hour of infection, which raises the possibility that, in addition to caspase-1, the sensing of bacterial products by Birc1e may affect other early protein targets important in antagonizing the intracellular survival strategy of L. pneumophila. The identity of such targets is unknown but of great interest.

LTXS1: SENSING OF ANTHRAX TOXIN BY THE NOD-LIKE RECEPTOR PROTEIN NALP1B Bacillus anthracis is a Gram-positive bacterium that causes anthrax. Pathogenesis of B. anthracis infection involves the production of several soluble factors by the bacterium; these include the protective antigen (PA), the edema factor (EF), and the lethal factor (LF), which assemble to form the edema toxin (ET) (PA + EF) and the lethal toxin (LeTx) (PA + LF). LeTx causes necrosis, is highly toxic for macrophages, and induces rapid death in systemic anthrax infection (124). Inbred mouse strains differ in their degree of susceptibility to LeTx-induced macrophage necrosis: Strains such as AKR/J, C57BL/6J, A/J, P/J, NOD/LtJ, and SJL/J are resistant, and strains such as BALB/cJ, C3H/HeJ, CBA/J, FVB/NJ, SWR/J, and NON/LtJ are susceptible (125). Typically, C3H macrophages are lysed by LeTx concentrations 100,000 times lower than those required to lyse A/J macrophages, and it was therefore suggested that A/J cells lack the LeTx target (124). Initial mapping studies in informative BXH and AKXL RIS and in [B6 × C3H] F1 × B6 backcross mice showed that the differential response to LeTx is controlled by a single gene, designated Ltxs1, that was regionally assigned to the central portion of mouse chromosome 11 (HSA17) (126). Additional high-resolution linkage and physical mapping 94

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studies delineated the Ltxs1 interval to a 0.51 cM segment that contained 14 known genes and 5 unannotated sequences (125). Dietrich and coworkers (127) initially identiﬁed the kinesin-like motor protein Kif1C as responsible for the Ltxs1 gene effect on the basis that (a) Kif1C was the only gene in the minimal interval that showed polymorphic differences between responsive and unresponsive mouse strains, (b) alteration of the subcellular distribution of the Kif1C protein by brefeldin A increased susceptibility to LeTx, and (c) ectopic expression of the resistance allele of Kif1C in susceptible macrophages increased survival following LeTx exposure. Additional recent studies by Dietrich and coworkers (128) excluded Kif1C as a candidate and provided convincing evidence that an adjacent positional candidate, the NLR protein–encoding gene Nalp1b, is in fact responsible for the Ltxs1 effect. Nalp1b is part of a cluster of three adjacent paralogs (with Nalp1a and Nalp1c), and studies in macrophages from the susceptible strain 129S1 showed that of the three only Nalp1b was transcribed in that strain. Sequencing the Nalp1b gene in 18 mouse strains revealed a complex pattern of coding polymorphisms in the Nalp1b protein that can be grouped into four major alleles (128). LeTx-susceptible strains uniformly harbored the same allele (allele 1), whereas resistant strains carried either alleles 2, 3, or 4. Validation of the Nalp1b gene as responsible for Ltxs1 came from (a) the demonstration that transgenic mice from a B6 background (resistant) carrying a BAC clone containing the susceptible allele from 129S1 became susceptible to LeTx, whereas (b) treatment of 129S1 macrophages with antisense morpholino oligonucleotides against Nalp1b increased resistance to LeTx (128). Generating a death signal through Nalp1b in response to LeTx appears to involve caspase-1. Indeed, treatment of macrophages from susceptible strains but not resistant strains activates caspase-1, and macrophages from 129S1 (susceptible) mice carrying a null mutation at caspase-1 (Casp1−/− ) become resistant to

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cell lysis by LeTx. These results suggest a model for the cytoxicity of LeTx in susceptible strains in which the intracellular toxin interacts with Nalp1b, which results in the activation of caspase-1 and the induction of cell death. Although the mechanism by which LeTx activates Nalp1b remains unknown (PAMP emulation, direct proteolytic processing by the toxin, etc.), these studies provide another clear illustration of the role of NLR proteins as cytoplasmic sensors of bacterial products.

MYLS: PLEIOTROPIC EFFECTS OF ICSBP/IRF8 MUTATION ON MYELOPROLIFERATION AND SUSCEPTIBILITY TO INTRACELLULAR INFECTIONS As described above, susceptibility to infection with low doses of M. bovis (BCG) is determined in inbred mouse strains by alleles at the Nramp1 (Slc11a1) locus; Nramp1G169 is associated with resistance and Nramp1D169 with susceptibility (16). Notable exceptions include (a) the wild-derived strain Mus spretus, whose intermediate level of resistance (despite a Nramp1G169 haplotype) is caused by a complex set of modiﬁers (129), and (b) the BXH2 strain (130). BXH-2 is a RIS derived from C3H/HeJ and C57BL/6J parents, known to develop a chronic myelogenous leukemia by a two-step mutagenesis process (131). The ﬁrst hit is an inherited, predisposing mutation speciﬁc to BXH-2 that causes myeloproliferation, whereas the second hit results from retroviral-mediated insertional mutagenesis that causes the expansion of clonal tumors (132). The second hit has been well characterized, including the nature of the replicationcompetent B-ecotropic virus, the multiple sites of integration, the study of which has unveiled a number of novel tumor suppressors and oncogenes altered by the virus (133). By monitoring splenomegaly and the inﬁltration of GR1+ /Mac1+ neutrophil precursors in spleen, liver, and lymph nodes as phenotypic markers, investigators showed that the BXH-

2-speciﬁc myeloproliferation trait is inherited as a single recessive trait (designated Myls) that maps to the distal portion of chromosome 8 (130). Additional genetic and physical mapping experiments identiﬁed IFN consensus sequence-binding protein 1 (Icsbp1), also known as IRF8, as a strong positional candidate. Icsbp1 is a transcriptional regulator that heterodimerizes with other members of the IRF family and plays an important role in activation of IFN-γ-responsive genes that bear internal ribosome entry site (IRES) regulatory sequence elements. BXH-2 mice carry a R294C mutation within the predicted IRFassociation domain of the protein. On the basis of previous reverse genetics studies showing that Icsbp1 acts as a negative regulator of the granulocyte lineage, investigators proposed that the R294C mutation is pathogenic. Subsequently, Turcotte and coworkers found the R294C allele to be associated with a failure of BXH-2 splenocytes to produce IL-12 and IFN-γ in response to activating stimuli (134). Despite a C3H/HeJ-derived Nramp1G169 allele, BXH-2 mice are susceptible to M. bovis (BCG) infection, and experiments in informative F2 crosses derived from BXH-2 showed that homozygosity for the mutant Irf8C294 allele is associated with increased BCG replication early in infection (130, 134). Strikingly, BXH-2 cannot resolve M. bovis (BCG) infection in spleen and liver late in infection. The associated uncontrolled replication of the otherwise avirulent M. bovis (BCG) is associated with the complete absence of granuloma formation in infected BXH-2 tissues (135). Additional preliminary data also indicate that BXH-2 mice are extremely susceptible to pulmonary TB following an intravenous challenge with highly virulent Mycobacterium tuberculosis H37Rv (J.F. Marquis and P. Gros, unpublished data). The effect of the Irf8C294 mutation is pleiotropic because BXH-2 mice show collateral susceptibility to infection with the unrelated pathogens S. Typhimurium and Plasmodium chabaudi (135). In the case of P. chabaudi, although BXH-2 mice can clear www.annualreviews.org • Immunity to Infection in the Mouse

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the initial burst of parasitemia, they fail to mount a long-term protective immune response as the animals develop multiple waves of recurring parasitemia late in the infection. These ﬁndings together suggest that Icsbp1 plays a critical role in both innate and acquired immune responses to intracellular pathogens.

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SUSCEPTIBILITY TO PULMONARY TUBERCULOSIS (SST1): ROLE OF THE IPR1 GENE IN MACROPHAGE RESPONSE TO MYCOBACTERIUM TUBERCULOSIS TB is caused by pulmonary infection with the bacterial pathogen Mycobacterium tuberculosis. TB remains a major global health issue, with an estimated 32% of the world’s population currently or previously infected (136), 8 million new cases per year (137), and 1–1.5 million deaths annually. Despite high infection rates, only 5%–10% of infected individuals have a lifetime risk of developing active disease, which suggests a possible role for genetic factors in human susceptibility to TB. Direct evidence for a genetic component includes the following: (a) epidemiological data indicating sex and racial differences in susceptibility, (b) geographical distribution and familial aggregation of disease, (c) population studies in endemic areas of disease and during ﬁrst contact epidemics, and (d ) concordance rates in mono- and dizygotic twins (for a comprehensive review, see 138). Numerous case control studies pointed to several gene variants that contribute to the risk of TB, and whole-genome scans identiﬁed suggestive linkages on several chromosomes, including 8, 11, 15, 20, and X (138). The complex genetic component of susceptibility to TB has been extensively studied in mouse models of infection for the past 60 years. Brieﬂy, the efﬁcacy of the host response to pulmonary TB is under complex genetic control in the mouse, with a broad spectrum of disease severity observed among different strains (138). Genetic 96

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studies by whole-genome scanning have located a number of TB susceptibility loci, but so far only a single locus (Ipr1) has been identiﬁed (139) and is reviewed herein. There is an additional enormous body of published work on the effect of individual gene knockouts on the susceptibility to pulmonary infection with M. tuberculosis, using a reverse genetics approach (140–142); this work is not discussed here. Surveys of inbred strains for susceptibility to M. tuberculosis (H37Rv), as measured by the extent of pulmonary replication and overall survival following intravenous or aerosol infections, classiﬁed the CBA, DBA/2, C3H, and 129/Sv strains as susceptible and the C57BL/10, C57BL/6, and BALB/c strains as more resistant (143). In a number of F1 hybrids tested (B6D2, CD2, CB6, and B6129), resistance is inherited in a dominant fashion. Susceptibility is generally associated with a failure to contain bacterial growth in the lungs, an inﬂammatory reaction causing consolidation of the lungs, and ultimately death. Resistant strains limit bacterial growth and can prevent massive tissue injury (142). Kramnik and colleagues (144) recently characterized the unique supersusceptibility of the C3HeB/FeJ strains to infection with M. tuberculosis Erdman (106 bacilli, i.v.). Studies in informative C3HeB/FeJ × C57BL/6J F1 and F2 mice led to the localization of a major gene effect on central chromosome 1 [(LOD) = 10.4], designated sst1. Studies in congenic C3HeB/FeJ mice with B6 sst1r alleles (C3H.B6-sst1) showed that sst1 exerts pleiotropic effects on other intracellular pathogens and also confers increased protection in vivo against Listeria monocytogenes, and explanted sst1 congenic macrophages show increased IFN-γ-dependent listericidal activity in vitro (145). Resistance is associated with an increased capacity of macrophages to restrict replication of M. tuberculosis in vitro, linked to an induction of apoptosis in M. tuberculosis– infected resistant macrophages. Scrutiny of the sst1 genetic interval for positional candidates expressed in macrophages identiﬁed a

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transcript, designated intracellular pathogen resistance 1 (Ipr1), which is present in resistant macrophages and whose expression is induced upon M. tuberculosis infection but is absent from susceptible cells (139). The sst1 genetic interval includes part of a large unstable repeat of ampliﬁed DNA that contains several rearranged copies of Ipr1-related sequences. In C3HeB/FeJ, this ampliﬁcation may have caused rearrangement(s) of the Ipr1 gene, leading to the absence of mRNA expression. The Ipr1 candidacy for sst1 was validated by showing that expression of the full-length Ipr1 transcript in transgenic C3HeB/FeJ mice can partially suppress M. tuberculosis replication in the lungs in vivo, and in macrophages in vitro (139). Ipr1 codes for IFN-induced protein 75 (Iﬁ75), a protein with several sequence motifs that indicate a nuclear localization and transcriptional regulatory activity. Iﬁ75 is a relative of the human protein SP110, a transcription factor regulated by IFN that interacts with certain viral proteins, including proteins from hepatitis C virus (HCV) and Epstein-Barr virus (146). These ﬁndings suggest that Iﬁ75 participates in transcriptional activation in macrophages in response to intracellular pathogens. In humans, loss-of-function mutations in SP110 cause veno-occlusive disease with immunodeﬁciency (VODI), an autosomal recessive disorder characterized by severe hypogammaglobulinemia, combined B cell and T cell immunodeﬁciency, absent lymph node germinal centers, absent tissue plasma cells, and hepatic vascular occlusion and ﬁbrosis (147). Evidence for an association of SP110 alleles with susceptibility to TB has been obtained from studies of families from different areas of Western Africa, including Gambia, Guinea-Bissau, and the Republic of Guinea (148). Additional case-control studies with populations from West Africa (149), Russia (150), and South Africa (151) have failed to provide evidence for an association of Sp110 variants with susceptibility to TB.

DEFICIENCY IN THE C5 COMPONENT OF COMPLEMENT AND SUSCEPTIBILITY TO SYSTEMIC INFECTION WITH CANDIDA ALBICANS In humans, Candida albicans exists as a harmless commensal organism in the gastrointestinal and genitourinary tracts. However, C. albicans can also cause infections in immunocompromised individuals. Such infections can be superﬁcial and limited to mucocutaneous candidiasis or can take the form of severe acute invasive candidiasis after bloodstream dissemination of the organism (152, 153). Researchers have described mouse models of mucocutaneous and acute infections and conducted genetic studies in inbred mouse strains in each model. The LD50 (lethal dose, 50%) for most C. albicans isolates introduced intravenously in immunocompetent mice is between 104 and 106 blastospores, depending on the strain of C. albicans, the growth conditions used to prepare the inoculum, and the genetic makeup of the murine host (154, 155). Candida albicans introduced by the intravenous route replicates in the spleen, liver, kidneys, and brain; inbred mouse strains are either resistant (C57BL/6J, BALB/cJ, CBA/J, and DBA/1) or susceptible (A/J, DBA/2J, NZB/J, and AKR/J) to infection, as measured by the extent of fungal replication, type and extent of tissue damage, and overall survival time (156). These studies further identiﬁed a partial correlation between the complement competence status (C5a) and susceptibility to infection (156). Recent genetic studies in A/J and C57BL/6J mice showed that extreme susceptibility in A/J mice is associated with the absence of an inﬂammatory response in infected tissues and very early death. Interestingly, A/J mice succumb to the acute infection within 48 h but display fungal loads in infected tissues that are considerably lower than those detected in moribund, resistant B6 mice three weeks after infection. In informative (A/J × B6) F2 mice, susceptibility is www.annualreviews.org • Immunity to Infection in the Mouse

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recessive and inherited as a monogenic trait that was mapped by whole-genome scan to the proximal part of chromosome 2 (157) in the vicinity of the structural gene for the C5 component of complement. Up to 40% of inbred strains carry an ancestral mutation consisting of a 2-bp deletion near the 5 end of the mRNA, which introduces a premature stop codon 4 bp downstream of the deletion (158). This mutation leads to the production of a truncated and nonfunctional 216 amino acid translation product. This truncated protein is not secreted. Thus, C5 deﬁciency in mice is associated with severe susceptibility to acute C. albicans infection. C5 is proteolytically processed to C5a, b, and c, which react with opsonized microbes to form a membrane attack complex that creates pores in the membrane of invading microbes following the binding of antibodies. In addition, C5a acts as a major chemoattractant to recruit neutrophils and macrophages to the site of infection, a response that is impaired in C5-deﬁcient mice (157, 159). Analysis of the proﬁle of cytokines during infection of A/J and B6 mice shows a pattern of extreme inﬂammatory and allergic response, which suggests unregulated production of proinﬂammatory molecules including TNF-α, IL-6, monocyte chemotactic protein 1 (MCP1), macrophage inﬂammatory protein 2 (MIP2), tissue inhibitor of metalloproteinase 1 (TIMP1), and KC. Transcript-proﬁling studies showed that this dysregulated inﬂammatory response is associated with severe cardiomyopathy (elevated creatine kinase and cardiac troponin I), hypoglycemia, and rapid death (160). In addition, C5 deﬁciency is associated with susceptibility to other types of infections, including infection by the intracellular pathogen L. monocytogenes. Indeed, the Lr1 locus previously shown to control interstrain difference in susceptibility to L. monocytogenes in A/J, C57BL/6J, and AXB/BXA recombinant inbred mice (161) was attributed to C5 deﬁciency (162). Finally, we observed that C5 deﬁciency in A/J mice and related strains has a positive effect on susceptibility to cerebral

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malaria caused by Plasmodium berghei (K. Kain and P. Gros, unpublished data), highlighting the detrimental impact of the host inﬂammatory response on the pathogenesis of cerebral malaria.

BLOOD-STAGE REPLICATION OF THE MALARIA PARASITE PLASMODIUM CHABAUDI: POSITIONAL CLONING OF THE CHAR4 AND CHAR9 RESISTANCE LOCI Malaria is caused by infection with members of the protozoan parasite family Plasmodium. Close to half a billion cases of malaria are believed to occur each year, with one million reported fatalities, mostly in young children from countries in sub-Saharan Africa. Severe anemia and cerebral malaria are major disease manifestations of blood-stage malaria, especially in Africa, where transmission rates are high. There is no effective vaccine against malaria, a problem exacerbated by the appearance of drug resistance in the Plasmodium parasite and insecticide resistance in the insect vector (163). Malaria is one of the clearest manifestations of host genetic factors inﬂuencing disease pathogenesis, with important threeway interactions between host genes, the environment, and the malaria parasite. Wellknown normal or disease-associated alterations in certain erythrocyte proteins can protect against malaria in humans, and positive selection by the parasite for heterozygosity at these variants occurs in endemic areas (coevolution). Celebrated examples include disease-causing alleles of thalassemias, sickle cell anemia, glucose-6-phosphate dehydrogenase (G6PD) deﬁciency, and a few others. For a more comprehensive description of the genetic component of susceptibility to malaria in humans, the reader is referred to recent comprehensive reviews on this subject (5, 164). The genetic control of susceptibility to malaria has been studied in mouse via the use of two infection models: P. chabaudi

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AS and P. berghei, which respectively mimic the blood-stage and cerebral phases of the disease. Although linkage mapping studies have detected multiple loci that affect susceptibility to either type of infection (165), the genes underlying these effects have been identiﬁed in only two cases, which are reviewed below. Infection of mice with P. chabaudi– parasitized erythrocytes mimics several pathophysiological aspects of the blood-stage infection in humans, including host response, genetic control of parasitemia, and ultimate outcome of infection. Using levels of parasitemia at the peak of infection, investigators have classiﬁed inbred strains of mice as either resistant (C57BL/6J) or susceptible (A/J, C3H/HeJ, SJL) to blood-stage replication of P. chabaudi (165). Susceptibility is associated with muted inﬂammatory and erythropoietic responses and a decreased survival time. Whole-genome scans conducted in backcross and F2 mice bred from resistant and susceptible parents, using peak parasitemia and mortality as quantitative phenotypes, showed that the genetic control of interstrain differences is extremely complex, with as many as nine detected Chabaudi resistance loci (Char), including major contributions from distal chromosome 9 (Char1), central chromosome 8 (Char2), and chromosome 17 (Char3, H-2 locus) (165). This complex genetic trait has been studied in a set of AcB/BcA reciprocal recombinant congenic strains (derived from A/J and B6 mice by systematic inbreeding of a second backcross) (166). By virtue of the breeding scheme used in their derivation, individual AcB/BcA strains carry a small portion (12.5%) of one parental genome ﬁxed as a set of congenic segments on the genetic background (87.5%) of the other parental strain. Of a subset of 18 AcB/BcA strains tested for susceptibility to infection with P. chabaudi AS, AcB55 and AcB61 mice showed a discordant phenotype; these mice were very resistant to P. chabaudi infection despite carrying A/J-derived susceptibility alleles at Char1 and Char2 (165). Linkage studies in informative

[AcB55 X A/J] F2 mice localized a strong gene effect (Char4) on chromosome 3 (LOD = 6.57) that regulates peak parasitemia following infection (167). Phenotypic characterization of AcB55 and AcB61 strains showed that resistance to malaria in these strains is associated with splenomegaly, elevated reticulocyte numbers in peripheral blood, and an elevated number of Ter119+ cells in the bone marrow, which suggests enhanced constitutive erythropoietic activity in both strains (168). Combined analysis of the level of blood stage parasitemia at the peak of infection and reticulocyte numbers showed that both traits are regulated by the same locus (Char4), which suggests that the two traits are physiologically related. The transcript map on the chromosomal region contained a strong positional candidate, liver- and red cell–speciﬁc pyruvate kinase (Pklr), on the basis of both its essential role for ATP production in erythrocytes and the fact that mutations in PKLR cause hemolytic anemia in humans. Sequencing revealed the presence of an isoleucine to asparagine substitution at residue 90 (I90N) of the Pklr protein in malaria-resistant AcB55 and AcB61 strains; this is a mutation that was previously described in a human case of pyruvate kinase (PK) deﬁciency (169). Recently, a second mutant allele was identiﬁed at the pklr locus in a CBA/N mouse genetic background (CBA/N-Pkslc ), G338D. Similar to the I90N allele, the G338D allele not only abrogates PK enzymatic activity and causes severe hemolytic anemia, but also confers dramatic protection against P. chabaudi infection (170). These ﬁndings show that deﬁciency in the erythrocyte PK protects against malaria. The mechanistic basis of the protective effect is related to (a) a reduced half-life of PK-deﬁcient erythrocytes in vivo and (b) increased phagocytosis of uninfected and Plasmodium-infected erythrocytes in an Fc receptor–independent fashion (170). Therefore, the protective effect of PK deﬁciency is mechanistically related to the effect previously proposed for G6PD deﬁciency and β-thalassemias. Interestingly, the malaria-protective effect of the www.annualreviews.org • Immunity to Infection in the Mouse

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PK deﬁciency–induced hemolytic anemia has the opposite effect on susceptibility to acute infection with S. Typhimurium (170a). In this case, perturbations in either homeostasis of iron stores or hemopoietic cell composition (constitutive erythropoiesis) or both contribute to reduce host defenses or enhance replication of this unrelated pathogen. During the positional cloning of the Char4 locus, linkage analysis in [AcB55 × A/J] F2 mice revealed a second, albeit more modest, genetic contribution of chromosome 10 (D10Mit189) that is localized to a 14-Mb C57BL/6J-derived congenic segment ﬁxed in the AcB55 strain (167). C57BL/6J alleles at this locus are protective (reduced peak parasitemia), are inherited in a codominant fashion, and show an additive effect with Char4. This locus was given the designation Char9 and was recently identiﬁed by positional cloning (171). The B6-derived 14-Mb Char9 congenic segment contains 77 predicted genes that were characterized with respect to (a) tissue-speciﬁc expression, (b) the presence of strain-speciﬁc alterations in the level of gene expression, and (c) strain-speciﬁc polymorphic variants in coding and regulatory regions of positional candidates. Vanin 1 (Vnn1) and Vnn3 were identiﬁed as the likely candidates responsible for Char9. Vnn1/Vnn3 map within a conserved haplotype block, and their expression is strictly cis-regulated by this haplotype. The absence of Vnn messenger RNA expression and the lack of pantetheinase protein activity in tissues are associated with susceptibility to malaria and are linked to a complex rearrangement in the Vnn3 promoter region (171). Vanin genes code for pantetheinases, a group of enzymes involved in the production of cysteamine, a key regulator of host responses to inﬂammatory stimuli. Passive administration of cysteamine in vivo partially corrects susceptibility to malaria in A/J mice, as measured by reduced blood parasitemia and decreased mortality (171). These studies suggest that pantetheinase is critical for the host response to malaria. Indeed, pantetheinase activity and cystamine production

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may be required to mount an early and beneﬁcial inﬂammatory response against the infectious agent, which includes the production of protective type I cytokines such as IL-12. Conversely, elimination of pantetheinase activity in Vnn1−/− mice protects against the pathogenic inﬂammatory response in the gut caused by either chronic exposure to nonsteroid anti-inﬂammatory drugs or infection with Schistosoma mansoni (172). Finally, these results raise the possibility that cysteamine may be a valid, hostbased molecule for therapeutic intervention in malaria, either alone or in combination with current parasite-based antimalarial drugs such as meﬂoquine.

THE JANUS KINASE TYROSINE KINASE 2 AND ITS ROLE IN RESISTANCE TO TOXOPLASMA GONDII INFECTION Toxoplasma gondii is an obligate intracellular protozoan. Toxoplasmosis is an important opportunistic infection in pregnant women (causing abortion and fetal abnormalities) and immunocompromised individuals (causing ocular and brain necrosis during reactivation of toxoplasmosis). In immunocompetent hosts, toxoplasmosis is the most common cause of infection-associated eye disease worldwide (173). Domestic cats are the host and the main reservoir of the parasite, although most warm-blooded animals as well as ﬁsh and reptiles can be carriers of Toxoplasma. Human contaminations occur principally through ingestion of contaminated food and fecal contamination of hands or congenitally by transplacental transmission (reviewed in 174). During the acute phase of infection, Toxoplasma tachyzoites replicate in macrophages and dendritic cells (DCs). During this early phase of infection, the cytokine IL-12 is essential to trigger IFN-γ-dependent immune responses and for the development of a parasite-speciﬁc Th1 response (175–177). The mouse strain B10.Q-H2q/SgJ is extremely susceptible to infection with T. gondii,

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and these mice succumb within two weeks after systemic infection owing to an inability to control parasite replication in macrophages (178, 179). In B10.Q-H2q/SgJ, susceptibility is associated with a general defect in the production of IFN-γ early during infection in vivo and with the hyporesponsiveness of splenocytes to IL-12 in vitro with respect to IFN-γ production (178). An F1 cross between susceptible B10.Q-H2q/SgJ and the related but resistant substrain B10.QH2q/Ai demonstrated that the susceptible phenotype is recessive (179). Linkage mapping in (B10.Q-H2q/SgJ × C57BL/6J)F1 × B10.Q-H2q/SgJ backcrossed mice conﬁrmed that susceptibility to infection with Toxoplasma is recessive and controlled by a single gene located on the centromeric part of chromosome 9 (HSA19) (180). Yap and colleagues (180) used a positional-candidate approach to identify tyrosine kinase 2 (Tyk2), a Janus kinase involved in cellular signaling by cytokine receptors as a strong candidate for the chromosome 9 locus (181). Sequencing of the Tyk2 gene led to the identiﬁcation of a strain-speciﬁc missense mutation (E775K) in B10.Q-H2q/SgJ mice. The E775K mutation results in the complete loss of Tyk2 function, most likely owing to instability of the E775K Tyk2 protein variant. The mutation in Tyk2 was used for mapping in the segregating backcross panel with the mutation in Tyk2 and showed tight linkage to the disease phenotype (LOD score of 53.3). Tyk2-deﬁcient mice were subsequently created and shown to be susceptible to infection with Toxoplasma; their response is characterized by impaired IFN-γ production and high parasite replication. More importantly, mice issued from a cross between B10.Q-H2q/SgJ and Tyk2-deﬁcient mice remained susceptible to infection, which conﬁrms that Tyk2 is the gene underlying the immune defect of B10.Q-H2q/SgJ mice (180). The family of Janus kinases consists of four members: Jak1, Jak2, Jak3, and Tyk2. Mutations in Jak3 cause severe combined immunodeﬁciency (SCID) in humans (182). Different Jak kinases associate constitutively with

the cytoplasmic domain of speciﬁc cytokine receptors (reviewed in 183). After activation of the receptor by ligand binding, Jak kinases phosphorylate the receptor and trigger the recruitment of signal transducer and activator of transcription proteins (STATs) and other adapter molecules. Activation of Tyk2 is involved in the signaling of several cytokines, including IL-12, IL-23, and type 1 IFNs (184). The critical role of TYK2 in humans was established by studies of a rare case of TYK2 deﬁciency in a patient presenting unusual susceptibility to various bacterial (S. aureus, atypic mycobacteria, nontyphi Salmonella), viral (herpes simplex infection), and fungal (oral candidasis) infections (185). The patient also suffered from atopic dermatitis with elevated levels of IgE. This patient displayed several cellular defects in cytokine signaling pathways, which explains his susceptibility to different classes of pathogens; the defects included alterations in signaling through type 1 IFN, IL-6, IL-10, IL-12, and IL-23. These studies in mouse and human demonstrated the pleiotropic role of Tyk2 in the innate and acquired immune responses to a variety of pathogens.

THE RIC LOCUS: RESISTANCE TO ORIENTIA TSUTSUGAMUSHI Orientia (formerly Rickettsia) tsutsugamushi is an obligate intracellular Gram-negative bacterium causing Scrub typhus, an acute, febrile, infectious disease zoonotic across southeast Asia and the western Paciﬁc region (186). Approximately one million cases of Scrub typhus occur annually (http://www.cdc.gov). Humans acquire the disease when O. tsutsugamushi is transmitted by feeding trombiculid mites. The bacteria multiply at the inoculation site, causing local inﬂammation and eschars with regional lymphadenopathy, before progressing within a few days to cause a systemic disease involving the lungs, heart, liver, spleen, and CNS. Resistance to the lethal effects of acute systemic infection with O. tsutsugamushi was www.annualreviews.org • Immunity to Infection in the Mouse

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studied in inbred mouse strains. Groves & Osterman (187) classiﬁed mice as resistant (AKR/J, BALB/c, C57BL/6J, C57L/J, and SWR/J) or susceptible (A/HeJ, CBA/J, DBA/1J, DBA/2J, SJL/J, and several C3H substrains) to infection on the basis of LD50 after intraperitoneal infection. Susceptible mice develop local and systemic bacterial growth during the ﬁrst week of O. tsutsugamushi infection and die within 10–12 days, whereas resistant mice show low microbial replication and complete survival after infection. Macrophage-mediated cellular immunity is essential for the resolution of infection in resistant strains (188). Genetic analyses in F1 and F2 crosses between resistant BALB/c and susceptible C3H/He progenitors showed that resistance to infection is determined by a single dominant gene, designated Ric (187, 189). Groves and colleagues mapped the Ric locus to a 45-Mb region of chromosome 5 using RIS of the BXH and CXS series (189). During the study of the molecular defect underlying Ric, a positional candidate, Eta1 (early T lymphocyte activation 1; also known as osteopontin and renamed Spp1 for secreted phosphoprotein 1), was identiﬁed. Eta1 expression is strongly induced during O. tsutsugamushi infection in vivo and is associated with inhibition of bacterial replication (190). Eta1 is a secreted, integrin-binding glycophosphoprotein with multiple functions that is expressed in several tissues during inﬂammation. Eta1 is produced by activated macrophages, T cells, and NK cells (191). More recent work using mice deﬁcient in Eta1 indicated that Eta1 contributes to host resistance to virus (rotavirus and herpes simplex virus 1) and bacteria (L. monocytogenes) (192, 193) and correlates with the clinical outcome of mycobacterial infection in humans (194). The proinﬂammatory functions of Eta1 include recruitment of leukocytes to sites of inﬂammation and polarization of T cells (195). Although these studies clearly identiﬁed a critical role for Eta1 in the host response to certain intracellular pathogens, whether it is the primary molecular defect underlying the

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Ric mutation remains to be established. Additional high-resolution linkage studies, mutational analysis in O. tsutsugamushi permissive strains, and complementation testing with Eta1-deﬁcient mice are required to clarify this point.

THE CTRQ3 LOCUS: ROLE OF THE P47GTPases IN SUSCEPTIBILITY TO CHLAMYDIA TRACHOMATIS Chlamydia trachomatis is an obligate intracellular pathogen that causes a variety of diseases in humans; different serotypes infect the ocular or genital mucosa. Chlamydia has a dual life cycle that involves an extracellular elementary body, a metabolically quiescent but infectious form, and a reticular body that replicates inside host cells. In host cells, Chlamydia replicates inside a specialized vacuole known as the inclusion (196). The variability in disease frequency and disease severity in people infected with Chlamydia suggested a possible contribution of genetic factors. Several infection models were developed in mice using either C. trachomatis (muco/cutaneous or systemic model) or C. pneumoniae (pulmonary model) (197, 198). In an intravenous infection with high doses of C. trachomatis, inbred mouse strains show different levels of susceptibility as determined by the extent of transient replication in the spleen (197). Typically, C57Bl/6J mice support 10-fold less replication than C3H mice, as measured by the amount of Chlamydia DNA (determined by PCR-based methods). The different degree of susceptibility to C. trachomatis infection in vivo can be partially reproduced ex vivo using mouse primary ﬁbroblasts (MEFs), which suggests the involvement of a cell-autonomous mechanism of defense. Whole-genome scan experiments in F2 mice derived from C57BL/6J and C3H/HeJ, using spleen chlamydial load 29 h postinfection, suggested a multigenic control with three quantitative trait loci (QTLs). These QTL reach signiﬁcance on chromosomes 2 (Ctrq1), 3 (Ctrq2), and 11 (Ctrq3) (197). A

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congenic mouse line carrying a 30 Mb chromosome 11 segment harboring Ctrq3 from C3H/HeJ (susceptible), transferred onto the resistant genetic background of C57BL/6J, was more permissive to C. trachomatis replication than were B6 controls. Therefore, the Ctrq3 locus can contribute to susceptibility independently of the Ctrq1 and Ctrq2 QTLs (197). Subsequent studies in subcongenic lines narrowed the Ctrq3 interval down to a 1.2 Mb segment (199). This segment contains 18 annotated genes, including two members of the p47 family of IFN-γinducible GTPases (p47GTPases), IFN-γinduced GTPase (Igtp), and iron-regulated virulence protein 10 (Irgb10). p47GTPases comprise a group of 23 related proteins that play an important role in the innate defense repertoire of macrophages and other cell types. p47GTPases are structurally deﬁned by the presence of a nucleotidebinding domain (G domain) that confers GTP binding and hydrolysis properties on these proteins. The expression of p47GTPases is inducible (50- to 100-fold) by IFN-γ through STAT1-mediated transcriptional activation. P47GTPases bind to different endomembrane compartments and quickly relocalize to pathogen-containing vacuoles in macrophages and other cells upon infection (200). Recent reverse genetics studies showed that mice lacking members of this family become dramatically susceptible to infection with certain intracellular bacteria, including Mycobacterium, Salmonella, and Listeria, as well as protozoans such as T. gondii and Leishmania major, and viruses such as Coxsackie B3 and vesicular stomatostatis virus (VSV) (200). Expression studies showed that although constitutive and IFN-γ-inducible mRNA expression of Igtp is similar in both B6 and C3H mice, Igrb10 expression in response to IFN-γ was 20-fold higher in B6 than in C3H mice, which suggests that diminished expression of Igrb10 may be responsible for C. trachomatis susceptibility in C3H/HeJ mice (199). Addi-

tional experiments showed that overexpression of Irgb10 in MEFs from C3H/HeJ mice in the presence of IFN-γ caused increased resistance to C. trachomatis, whereas RNAimediated inhibition of Irgb10 in C57BL/6J cells rendered them more permissive to C. trachomatis replication (199). These results are supported by recent RNAi studies showing that another p47GTPase, IFN-inducible GTPase 1 (Iigp1), is a critical component of the inhibitory effect of IFN-γ on the growth of human C. trachomatis L2 in murine epithelial cells in vitro (201). Together, these results highlight the role of IFN-γ and p47GTPase signaling in effective host defenses against Chlamydia (see Figure 2).

THE MYXOVIRUS RESISTANCE (MX) LOCUS: IFN-INDUCIBLE GTPases OF THE DYNAMIN SUPERFAMILY AND RESISTANCE TO ORTHOMIXOVIRUS (INFLUENZA) INFECTION Inﬂuenza viruses (members of the orthomyxovirus family) cause upper respiratory tract infections in humans and various domestic animals; these include chickens (fowl plague), horses (equine inﬂuenza), and pigs (swine inﬂuenza). In nature, wildfowl and shorebirds may form the virus reservoir. According to the World Health Organization (WHO) (http://www.who.int/en/), in an inﬂuenza epidemic 5% to 15% of the population is affected, including 3–5 million cases of severe illness and between 250,000 to 500,000 deaths every year. The elderly, young children, and people with compromised health status are at high risk for serious complications. Highly pathogenic inﬂuenza of certain subtypes can cause global outbreaks. Three inﬂuenza pandemics have occurred in the twentieth century: the Spanish ﬂu in 1918 resulted in approximately 50 million deaths worldwide, the Asian inﬂuenza in 1957 resulted in more than 1 million deaths, and the Hong Kong inﬂuenza in 1968 www.annualreviews.org • Immunity to Infection in the Mouse

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S. aureus?

LRR TLR4

MyD88 Tirap TIR Tram Trif

LRR TLR6 TIR

1 Early response (inflammatory cytokines) 2 Late response (lFN-β)

2

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Pathogen

NK cells T cells

Irgb10 NF-κB

Igtp

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Cytokines

Tyk2

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pro-IL-1β

?

IL-1β

STAT1 STAT2

Legionellacontaining vacuole

Nalp1b Bacterial growth restriction Caspase 1

IFN-β STAT1

Flagellin

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STAT1 STAT1

-β IFN

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Nr

am

p1

Fe2+

CD36 TLR2 LRR Phagosome

MyD88 Tirap TIR

Lysosome

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IFN-β ?

NUCLEUS

Mtb MACROPHAGE

Figure 2 Central role of the macrophage in genetic studies of infection susceptibility loci. Schematic representation of several major biochemical, physiological, and signaling pathways relevant to macrophage defenses against infectious agents. Genes and proteins involved play a key role in these pathways; mutations identiﬁed by forward genetics as causing alterations in resistance to infections are shown in red. See text for details. [Abbreviations: Nramp1, natural-resistance-associated macrophage protein 1; LRR, leucine-rich repeat; MyD88, myeloid differentiation primary response gene 88; Tirap, Toll receptor-associated activator of interferon; Trif, Toll/IL-1 receptor (TIR) domain–containing adapter-inducing interferon-β; Tram, TRIF-related adapter molecule; Icsbp, IFN consensus sequence-binding protein; IRF, IFN regulatory factor; Mtb, Mycobacterium tuberculosis; Nalp1b, nacht, LRR, and Pyrin domain–containing 1b; Birc1e, baculoviral IAP repeat-containing 1e; Ipaf, IL-1 β-converting enzyme–protease-activating factor; JAK, Janus kinase; STAT, signal transducer and activator of transcription; Tyk, tyrosine kinase; CD36, CD36 antigen; Irgb10, iron-regulated virulence protein 10; Igtp, IFN-γ-induced GTPase; Ipr1, intracellular pathogen resistance 1; Iﬁ75, IFN-induced protein 75.]

was responsible for approximately 700,000 deaths (202). More recently, the emergence of the highly pathogenic avian H5N1 inﬂuenza virus, which is transmitted directly from birds 104

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to humans, highlights the threat of future inﬂuenza outbreaks and pandemics. Genetic studies in mouse models of inﬂuenza were used successfully to identify

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major host determinants of susceptibility and resistance to infection. Most laboratory mouse strains (C57BL/6J, C3H/HeJ, BALB/cJ CBA/J, CE/J, I/LnJ, and PERA/Ei) are extremely susceptible to intranasal infection with mouse-adapted strains of inﬂuenza virus, with the exception of the A2G strain and several wild-derived inbred strains (SL/NiA, T9, and CAST/Ei) (203–205). Resistance of A2G mice is speciﬁc for inﬂuenza and is controlled by a single dominant gene named Mx (myxovirus resistance locus); the protective effect of Mx is mediated by type 1 IFNs (206). The Mx locus has two phenotypic alleles: Mx+ (resistant, dominant) and Mx− (susceptible, recessive) (207). A candidate gene for Mx was identiﬁed as a protein that was differentially expressed (determined by 2D-gel electrophoresis) in Mx+ and Mx− cells in response to stimulation with type 1 IFN (208). A nuclear protein of 72 kDa, named Mx1, was inducible by type 1 IFN and present only in cells carrying the wild-type allele at Mx but not in cells of the Mx− phenotype. Staeheli and colleagues (207) further cloned the Mx1 gene, mapped it to mouse chromosome 16, and showed that it belongs to the superfamily of dynamin-like large GTPases. Linkage studies in informative backcross progeny issued from crosses between different laboratory strains carrying the Mx− allele established that susceptibility to inﬂuenza maps to the 8 Mb at the distal end of chromosome 16 where the Mx1 gene was previously localized (209). Mutational analyses revealed that the Mx− defect in BALB/cJ, C57BL/6J, and C3H/HeJ mice is caused by deletion of exons 9–11 and some ﬂanking sequences, which results in a truncated, nonfunctional Mx1 protein. The Mx− phenotype of CBA/J, CE/J, I/LnJ, and PERA/Ei mice is caused by a nonsense mutation in exon 10 that converts codon 389 (Lys) to a termination codon, which prevents synthesis of functional Mxl (210). Mx1 is a nuclear protein whose expression is stimulated by IFN-α/β or by viral infection. Mx1 contributes to speciﬁc resistance against the inﬂuenza virus by acting at an early stage of the

viral infection cycle to inhibit primary transcription of the virus, probably through inhibition of the viral PB2 polymerase subunit (211, 212). Mx homologs are present in all eukaryotes examined, including humans, cattle, pigs, rats, horses, hamsters, chicken, ﬁsh, and yeast. In mice, a second Mx gene (Mx2), closely linked to Mx1 on chromosome 16, was identiﬁed and shown to be nonfunctional in classical inbred mouse strains (the Mx2 mRNA carries an insertion at position 1366, which causes a translational frameshift) (213). However, in wild-derived mice, Mx2 is expressed following exposure to type 1 IFN or viral infection and localizes to the cytoplasm, where it can inhibit VSV replication (214). In humans, two Mx proteins (MxA and MxB) were identiﬁed as GTPases and map to the region of chromosome 21 homologous to mouse chromosome 16. Only MxA has detectable antiviral activity and, in contrast to Mx1, inhibits replication of a spectrum of viruses, including members of the orthomyxoviruses (inﬂuenza and Thogoto virus), rhabdoviruses (VSV), paramyxoviruses (Measles virus), and picornaviruses (coxsackie virus), among others (reviewed in 215). The essential role of Mxl in inﬂuenza resistance has been demonstrated in vitro and in vivo. Transfection of the wild-type Mx1 allele into Mx− cells transforms them from a sensitive to a resistant phenotype (207). In addition, the resistant phenotype of Mx+ cells can be abolished by the use of an anti-Mxl antibody (216). In vivo, transgenic mice that express mouse Mx1 or human MxA are fully protected against inﬂuenza infection (217, 218).

THE HV2 LOCUS: CEACAM1B AND THE CONTROL OF CORONAVIRUS ENTRY INTO THE HOST CELL With a single-stranded positive-sense RNA genome of 30–32 kb (219), coronaviruses are the largest of all the RNA viruses. They have a broad host range and cause a variety of diseases. In humans, coronaviruses www.annualreviews.org • Immunity to Infection in the Mouse

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cause respiratory disease and, to a lesser extent, infections of the digestive tract and neurological syndromes. Human coronaviruses OC43 (HCoV-OC43) and HCoV-229E are the causative agents of the common cold (220); the more recently identiﬁed agents HCoV-HKU1 and HCoV-NL63 cause more severe, although rarely fatal, infections of the upper and lower respiratory tract (221–223). Severe acute respiratory syndrome (SARS)CoV causes a life-threatening pneumonia (224) and is the most pathogenic human coronavirus identiﬁed to date (225, 226). SARSCoV may reside in an animal reservoir (227, 228) and recently initiated an epidemic in humans through zoonotic transmission (228). Coronaviruses also cause economically important diseases of livestock, poultry, and laboratory animals (229). In mice, several strains of the prototype coronavirus, mouse hepatitis virus (MHV), with different degrees of pathogenicity and tissue tropism, have been used to study infection in vivo. For example, the A59 strain of MHV is hepatotropic (230), and the JMH strain induces encephalitis and sometimes demyelination (231). The MHV3 strain induces fatal hepatitis (232). More recently, researchers found that respiratory infection with the MHV1 strain produces severe pneumonia accompanied by tissue destruction, mainly in the lung (233). Several pathophysiological features are common between mice infected with MHV and patients infected with SARS-CoV and are consistent with immunopathological disease: These include the propensity of viruses to infect macrophages and DCs and an increased systemic concentration of chemokines and cytokines (234). In mice, activated macrophages are present at sites of inﬂammation and participate in tissue destruction (234). Inbred mouse strains are susceptible to experimental infection with MHV, although the SJ/L strain can sustain a 10,000-fold higher infectious dose (235). Examination and segregation analysis of several inbred strains showed that resistance is recessive and that expression of disease resistance is controlled by

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a single autosomal gene acting at the level of the infected cell (236). Following virus replication in macrophages, this resistance trait was mapped to mouse chromosome 7 at the Hv2 locus (237, 238), now designated carcinoembryonic antigen–related cell adhesion molecule 1 (Ceacam1). More recently, investigators showed that Ceacam1 controls survival differences between SJ/L and BALB/c mice (239). In the SJ/L strain, the inability of MHV to bind the host cell membrane suggested that resistance to infection is due to the absence of a functional receptor for the virus (240); the receptor was ﬁnally identiﬁed biochemically (241). CEACAM1 is a member of the carcinoembryonic antigen (CEA) family in the Ig superfamily (242). Mouse CEACAM1 isoforms are transmembrane glycoproteins with either two or four Ig domains that are produced by alternative splicing of the primary transcript (243). An immunoreceptor tyrosine-based inhibitory motif (ITIM) is present in the cytoplasmic domain of CEACAM1 and confers immunomodulatory function to this molecule (244). The expression of CEACAM1 glycoproteins is widespread not only on the apical membranes of epithelial cells in the gastrointestinal and respiratory tracts (245), which are the main targets of MHV replication, but also in small vascular endothelial cells, hematopoietic cells, and glial cells of the nervous system. In these tissues, CEACAM1 performs many important cellular functions. It may act as a cell adhesion molecule, an angiogenic factor, a tumor suppressor, and/or a signal regulatory protein (246). In inbred mice, there are two Ceacam1 alleles; susceptible strains carry the Ceacam1a allele. Expression of CEACAM1a in human and hamster cells is sufﬁcient to overcome the species barrier to MHV infection, which supports the role of CEACAM1a as a functional MHV receptor. Resistant SJ/L mice carry the Ceacam1b allele, which encodes a protein with a 27 amino acid substitution in the ﬁrst Ig domain of CEACAM1b. Extensive mutational and biochemical analysis showed that this region mediates CEACAM1

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binding to the spike protein of the MHV envelope. Three mouse lines with genetically engineered mutations in Ceacam1a were produced (247, 248). Mice with complete abrogation of the CEACAM1a protein exhibited resistance to MHV infection via the intranasal and intracerebral route with no clinical signs of disease even at high doses, which demonstrates the critical role of a virus receptor in resistance to infection. Human CEACAM1a proteins do not recognize mouse or human coronaviruses but instead bind Gram-negative bacteria in the vicinity of the MHV binding site. In addition to CEACAM1, CEACAM3, CEACAM5, and CEACAM6 proteins allow bacterial anchoring or invasion of the host cell (246). Infection with Neisseria gonorrhoeae promotes CEACAM1-mediated inhibition of CD4+ T cell activation, which indicates an immunoregulatory role for the cytoplasmic ITIM-containing tail of CEACAM1. In stark contrast, the neutrophil-restricted CEACAM3 receptor possesses an immunotyrosine activating motif (ITAM) in its intracellular domain, which promotes ITAMdependent internalization of the CEACAM3 bacterial complex and neutrophil bactericidal activity via a Syk kinase–dependent pathway (249–251). Thus, particular species of bacteria seem to exploit adhesion to CEACAM1 to promote immunosuppression.

THE FLV LOCUS: SPECIFIC INHIBITION OF WEST NILE VIRUS REPLICATION BY 2 ,5 -OLIGOADENYLATE SYNTHASE 1B West Nile virus (WNV) is a re-emerging pathogen that cycles primarily between mosquitoes and birds; humans represent one of several incidental hosts (252). The virus is widely distributed throughout Africa, the Middle East, and India but was not detected in the Western Hemisphere before 1999, when it caused an outbreak of viral encephalitis in New York (252). Since then, the virus

has propagated rapidly throughout all North America, resulting in considerable acute morbidity and mortality. Epidemiologic studies indicate that although 80% of infections remain subclinical, 20% progress to a febrile illness. Of these, more than 30% of cases in the U.S. progress to neuroinvasive disease, causing meningitis, encephalitis, and/or ﬂaccid paralysis (253). WNV is a member of the genus Flavivirus (family Flaviviridae), which also contains the Yellow Fever, Dengue, and Japanese encephalitis viruses. Flaviviruses contain a positive-sense RNA genome of 10 kb. Viral replication in host cells generates negativestrand RNA intermediates that serve as templates for nascent positive-strand RNA synthesis. WNV replicates initially in Langerhans cells, a resident DC of the skin. Following peripheral replication, WNV spreads to the CNS, possibly through TNF-mediated changes in blood-brain barrier permeability. Neurons are the primary target of WNV in the brain and spinal cord. Although WNV can directly antagonize IFN-induced responses after infection, type I IFN is still required to restrict WNV replication and spread (254). The characterization of innate resistance to ﬂavivirus-induced morbidity and mortality started as early as 1929 (255). Selective breeding in mice led to the development of resistant and susceptible lines, followed by the demonstration that a single autosomal dominant locus, designated Flv, is responsible for the differences in the host response against several types of ﬂaviviruses (256). Following intracerebral infection, viral titers in the brain of resistant mice with the Flvr resistance allele are several orders of magnitude lower than in Flvs susceptible mice. Similarly, virus titer yields in cells cultured from resistant or susceptible animals are also dramatically different, which indicates that resistance to ﬂavivirus acts intracellularly on ﬂavivirus replication. Most common laboratory-inbred strains of mice are susceptible to experimental infection, whereas wild-derived strains (e.g., PR1, MBT/Pas, MAI/Pas) are not (257). The resistance www.annualreviews.org • Immunity to Infection in the Mouse

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allele from donor PR1 mice was introduced onto the susceptible C3H/He background to produce the congenic inbred C3H.PR1-Flvr . Using this strain in crosses with the susceptible C3H/He or BALB/c mouse strains, and following the load of Murray Valley encephalitis virus in brains of infected mice, Flv was mapped to chromosome 5 (258). Additional high-resolution linkage mapping experiments delineated a minimal interval for Flv of 0.45 cM (259). In a parallel study, an identical interval was deﬁned for resistance in terms of survival to a low intraperitoneal inoculum of the recent WNV variant ISr98/NY99 (from New York) and of a different set of wildderived mouse strains (257). Physical mapping in BAC clones, exon trapping, and cDNA selection techniques were used to identify positional candidates. A cluster of genes encoding the 2 ,5 -oligoadenylate synthetases (2 ,5 OAS) contained excellent candidates on the basis of their known (IFN-inducible) antiviral activity in response to double-stranded RNA (dsRNA). Of the ﬁve functional Oas genes found in mice (Oas1g, Oas1b, Oas1a, Oas2, Oas3), only Oas1b is important for the host response to WNV and plays a key role in WNV pathogenesis. All susceptible mice tested so far have a T-to-C transition that replaces an arginine residue with a premature stop codon, which probably results in a defective enzyme without nucleotidyl transferase activity (257, 259). This assumption was reinforced by the observation that replication of WNV is less efﬁcient in neuroblastoma cell clones that overexpress the normal copy of Oas1b than in those expressing the mutant variant (259). Moreover, upregulation of OAS1B (under the control of the Tet-Off expression system) in ﬁbroblasts efﬁciently inhibited WNV replication at the early stages of the virus life cycle by dramatically reducing the levels of positivestranded viral RNA. It remains unclear what provides speciﬁcity to OAS1B in this system (259). OAS proteins are part of the OAS/RNase L system of RNA decay stimulated by the type I IFN antiviral response. Transcrip-

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tion of OAS genes is upregulated by type I IFN, and expressed OAS proteins are activated by dsRNA. Activated OAS catalyzes the synthesis of 2 ,5 -oligoadenylates, which in turn activate RNase L, a latent endoribonuclease that degrades viral and cellular RNAs. RNase L−/− murine embryonic ﬁbroblasts and PKR−/− × RNase L−/− bone marrow–derived macrophages supported increased WNV replication in vitro (recently reviewed in 214). Moreover, mice deﬁcient in both PKR and RNase L showed increased lethality following WNV infection, with greater viral loads in peripheral tissues at early time points after infection. However, the mechanisms by which Oas gene alleles affect ﬂavivirus pathogenesis remain uncertain; recent reports suggest that Oas1b gene effects on WNV replication are independent of RNase L (253). A recent study of 33 WNV-infected patients showed a positive association between the nucleotide change T210C and susceptibility to WNV disease in humans (260). Their analysis predicted that the T210C nucleotide change creates a new OAS splice enhancer site that may result in production of a dominant-negative protein (260).

THE CMV1 LOCUS AND ITS RELATIVES: RECOGNITION OF THE CYTOMEGALOVIRUSINFECTED CELL BY NATURAL KILLER CELL RECEPTORS Most humans are infected by human cytomegalovirus (HCMV), a virus of the Herpesviridae family, characterized by a large double-stranded DNA genome (200 kb), wide tissue tropism, and the ability to establish latency in immunocompetent individuals. Primary infections are mostly subclinical or they may be associated with a selflimited mononucleosis-like syndrome. However, HCMV is the most common cause of virus-induced mental retardation and congenital deafness in the Western world. In addition, HCMV infections can cause serious

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morbidity and mortality among immunocompromised subjects (recently reviewed in 261). Patients with congenital defects in NK cells are also particularly vulnerable to infection by herpesvirus (262–265). A central aspect of the pathogenesis of CMV is that many genes serve to evade or subvert mechanisms of host defense (266– 268). Infection of mice with the closely related mouse CMV (MCMV) has proven exceptionally useful for the study of the complex host:pathogen interactions (recently reviewed in 269). In mice, genetically determined resistance against MCMV is under multigenic control, with contributions from both H2 and non-H2 genes (270, 271). Inbred strains carrying the H2k haplotype, such as CBA, C3H/HeJ, or congenic BALB.K, are up to 10 times more resistant (measured as survival time) to MCMV than strains with H2b or H2d haplotypes (271). However, in strains with a C57BL background and the H2b haplotype, non-H2-linked effects override H2determined susceptibility. The Cmv1 locus is one of the best characterized loci in the C57BL/6 mouse strain that is responsible for non-H2 host resistance against MCMV infection (272). Through genetic analysis of progeny from MCMVresistant and MCMV-susceptible parents, Scalzo and coworkers (272) identiﬁed a single locus, Cmv1, as the major determinant of MCMV-resistance in the C57BL/6 mouse strain. Cmv1 is an autosomal (chromosome 6) dominant trait that restricts viral replication at the level of the spleen and other target organs such as liver and lung (272). Cmv1 function is mediated by NK cells (273). Researchers cloned the Cmv1 locus using a combination of high-resolution mapping, physical mapping, and detailed immunological characterization of C57BL/6 mice. The cloning was complicated by the fact that Cmv1 resides within an interval that contains 14 highly related genes of the Ly49 family of MHC class I receptors (277). Ly49h, a gene that encodes an activating NK cell receptor, was identiﬁed as the gene underlying the Cmv1 locus

(274–276). Ly49h is present in the MCMVresistant strain C57BL/6 but is absent in susceptible strains such as BALB/c, DBA/2, and 129Sv/J (278). In fact, a clonal expansion of Ly49H+ NK cells occurs following MCMV infection of C57BL/6 mice (279). The crucial role of Ly49H-bearing NK cells in host defense against viral infection was validated by restoring MCMV-resistance in genetically susceptible mice through transgenic expression of Ly49h (280). Ly49 molecules constitute an extended family of activating and inhibitory C-lectintype receptors that recognize MHC class I (277, 281). Activating LY49H, contrary to inhibitory ITIM-bearing Ly49 receptors, associates with the ITAM-containing DNAXactivating protein of 12 kD (DAP12) adapter protein, also known as killer cell activating receptor–associated protein (KARAP). The importance of the Ly49H/DAP12 receptor complex in NK cell–mediated resistance to MCMV infection is supported by the observation that DAP12 mutant mice present a considerable increase in MCMV titers in the spleen and liver following infection (282). Ly49H speciﬁcally recognizes MCMVinfected cells via a direct interaction with the m157 MCMV protein, which has structural homology to MHC class I molecules (283, 284). Deletion of the m157 gene is associated with gain of virulence in Ly49H+ mouse strains but not in Ly49H− strains, which indicates that m157 is the only MCMV-encoded protein that activates Ly49H+ NK cells (285). Arase et al. (283) demonstrated that m157 also binds to an inhibitory receptor, Ly49I, expressed on NK cells in 129 mice, which suggests that m157 may have evolved as a mechanism to escape NK cell killing by targeting inhibitory receptors in certain susceptible mice. These observations predicted a dynamic interaction between Ly49 receptors and MCMV evasion genes, which was recently conﬁrmed. In fact, several MCMV strains isolated from wild mice had variants of the m157 gene, many of which disrupted the open reading frame and inactivated the www.annualreviews.org • Immunity to Infection in the Mouse

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gene (286). In addition, sequential passage of the commonly used Smith strain of MCMV in Ly49H+ C57BL/6 mice (287) or in SCID mice (288) resulted in loss-of-function mutations in the m157 gene. This result demonstrates the ability of Ly49H+ NK cells to exert enough selective pressure to permit the speciﬁc outgrowth of MCMV escape mutants with alterations in m157. Further genetic analysis of MCMVresistant inbred strains demonstrated the presence of alternative NK cell–encoded resistance mechanisms, Cmv3 (289) and Cmv4 (290), which operate individually or in combination with speciﬁc H2 haplotypes. The Cmv4 locus is also tightly linked to the Ly49 cluster and presents a second example of a major locus effect in MCMV resistance present in a wild-derived inbred mouse strain, PWK/Pas. Although the Cmv4 effect is independent of Ly49H/m157, the most likely candidate remains another activating NK cell receptor that speciﬁcally recognizes MCMV-infected cells (290). Support for alternate activating Ly49 receptors determining MCMV resistance also comes from the study of the Cmv3 locus present in a resistant mouse strain, MA/My (289). This strain carries a Ly49 haplotype similar to that of the MCMV-susceptible 129 strain, which lacks Ly49h. MA/My mice also contain the protective H2k haplotype, whereas 129 (H2b ) present with susceptible haplotypes, which suggests a possible role of H2 in MA/My resistance. Linkage analyses suggested that MCMV resistance in MA/My is linked to the Ly49 gene cluster. However, only the speciﬁc combination of Ly49 (Ly49em ) with MHC (H2k ) alleles is associated with virus resistance. A model including contributions of Ly49 genes, H2-loci, and their interaction explained 40% of the phenotypic variance in this cross. A cell reporter assay, in which cells expressing the activating receptor Ly49P were responsive to challenge only with H2k target cells infected with MCMV, conﬁrmed a physical interaction between the Ly49 and H2 gene products. Blocking anti-

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bodies to class I H2k gene product, H2-Dk , directly implicated this molecule in stimulation of Ly49P (289). Thus, Ly49P recognition of MCMV infection in the context of H2Dk points to a novel mechanism of NK cell function, in which an activating Ly49P receptor mediates host resistance against MCMV through speciﬁc recognition of a virally altered MHC class I molecule. The discovery of Ly49H- and Ly49P-mediated recognition of MCMV-infected cells supports the hypothesis that activating receptors have evolved under evolutionary pressures from pathogens and has revealed an exceptional speciﬁcity of this arm of the innate immune system for unique viral determinants (Figure 3). Human killer cell Ig-like receptors (KIR) are the functional counterparts of murine Ly49s (291). Rigorous epidemiological studies based on very large patient cohorts demonstrated that combinations of KIR and HLA are associated with protection against infections by several viruses, including HIV and HCV [recently reviewed by Carrington & Martin (292)]. Remarkably, the ﬁndings suggest that these genetic interactions may manifest different mechanisms of protection. During HIV infection, speciﬁc receptor-ligand pairs may convey protection by directly increasing NK cell activation (293). In the case of protection against low inoculums of HCV, speciﬁc weakly inhibitory KIR/HLA pairs may afford protection by lowering the threshold of NK cell activation (294). By analogy with mouse models of MCMV resistance, direct NK cell killing of infected cells may reduce the risk or severity of infection with human viruses.

THE OBLIVIOUS, CPG1, LPS2, AND TRIPLE D PHENOTYPES: TOLL-LIKE RECEPTOR PATHWAYS AND THE CONTROL OF VIRAL PROLIFERATION New models of host susceptibility to infection have recently been identiﬁed in largescale mouse mutagenesis projects via the use

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Figure 3 Genetically identiﬁed molecular pathways activated in response to viral infections. Schematic representation of several major biochemical, physiological, and signaling pathways relevant to the response of cells of the immune system (CD4+ and CD8+ T cells, NK cells, and macrophages) against viral pathogens. Genes and proteins shown play a key role in these pathways; mutations identiﬁed by forward genetics as causing alterations in resistance to viruses are identiﬁed in red. (This ﬁgure is inspired by Reference 9.) See text for details. [Abbreviations: CpG DNA, DNA containing guanine–phosphate diester–cytosine dinucleotides; TRIF, Toll/IL-1 receptor (TIR) domain–containing adapter-inducing IFN-β; MyD88, myeloid differentiation primary response gene 88; STAT, signal transducer and activator of transcription; Mx, myxovirus resistance; 2 -5 OAS, 2 -5 -oligoadenylate synthetases; Ly49, killer cell lectin-like receptor; Jak1, Janus kinase 1; Tyk2, tyrosine kinase 2; Unc93b, unc-93 homolog B.]

of the chemical agent ENU (recently reviewed in 295). The technique can theoretically cause a mutation in any gene and is limited only by the ingenuity of the screening approach (recently reviewed in 296–298). In a typical whole-genome mutagenesis screen, inbred male mice are treated with ENU to

induce several hundred germ line mutations per mouse. The males are crossed to wildtype females of the same strain, and large cohorts of offspring are tested to identify the phenotypically distinct mice most likely to bear a large-effect mutation. ENU mutagenesis is a powerful tool to map novel www.annualreviews.org • Immunity to Infection in the Mouse

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biochemical and physiological pathways that play critical roles in host defenses against infections and aids in the establishment of new mouse models of human diseases. To explore TLR pathways, Beutler and coworkers (reviewed in 295) designed a genomewide screen for dominant and recessive ENUinduced mutations in C57BL/6 mice. In this experiment, thioglycolate-elicited peritoneal macrophages were collected from male mice, and researchers measured the TNF bioactivity of these cells in response to TLR agonists. Secondary screens of mice with unresponsive macrophages produced several new models of susceptibility to infection, including the Oblivious (299), Lps2 (300), CpG1 (301), and Triple D (3d ) (302) mutants that have been well characterized during in vivo infection. The Oblivious (Obl ) mutation causes a recessive immunodeﬁciency phenotype in which macrophages are insensitive to some [e.g., lipoteichoic acid, the diacylated bacterial macrophage-activating lipopeptide 2 (MALP2)] but not all (e.g., zymosan) TLR2/6-dependent microbial stimuli. Perhaps for this reason, Obl homozygous mutants are highly susceptible to Gram-positive S. aureus infections. Positional cloning of the Obl locus revealed a premature stop codon in the Cd36 gene (299). Macrophages from Obl mice have an identical phenotype to that of Cd36−/− mice, and macrophage function can be rescued by transfection of a wild-type version of Cd36, which provides convincing evidence that Cd36 is the gene responsible for the Obl phenotype (299). Unexpectedly, Obl mice permit MCMV to grow to high titers in vivo. CD36 is a member of the scavenger receptor type B family (303), previously implicated in the recognition of oxidized lowdensity lipoprotein (LDL) particles and the uptake of fatty acids (304), but unrelated to infection or TLR signaling. However, these experiments suggested that the TLR2/TLR6 complex uses CD36 as a coreceptor for some of its bacterial ligands and for a yet undeﬁned molecule derived or elicited from MCMV.

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The Lps2 mutant phenotype causes macrophages to lose their responsiveness to TLR4 and TLR3 ligands, which indicates that Lps2 may be a common intermediary of the TLR4 and TLR3 intracellular pathways (300). Of particular importance to viral pathogenesis, Lps2 macrophages also cannot generate an LPS- or dsRNA-induced type I IFN response and are highly susceptible to infection with VSV or Vaccinia virus (300). In correlation, the Lps2 mutation abolishes the activation of the gene transcription factor IRF3. As a result, Lps2 homozygotes are markedly resistant to the lethal effect of challenge with LPS, and their immune response to MCMV is compromised, which allows high viral titers in the spleen (305). The enhanced pathogenicity to MCMV is associated with a failure to produce adequate amounts of type 1 IFN, as measured in the serum early after infection. Following positional cloning, the Lps2 mutation was found to correspond to a single base pair deletion in the Trif gene (306, 307). In vitro rescue experiments with Trif again compensated for the Lps2 phenotype, and the knockout of Trif yielded a phenotype similar to that of the Lps2 allele. The identiﬁcation of Lps2 revealed the bipartite nature of LPS (which signals through MyD88 and Trif) and the role of this intracellular pathway in host resistance to virus infection (308). In parallel studies, the mouse phenotype CpG1 was ﬁrst characterized by the unresponsiveness of mutant macrophages to stimulation with a synthetic analog of CpG-DNA. This PAMP is recognized by TLR9. Accordingly, sequencing of the Tlr9 coding region indicated that the genetic defect was a nucleotide substitution that resulted in a nonconservative amino acid replacement (L499P) in a well-conserved region of the protein (301). The CpG1 mutation is associated with severe susceptibility to MCMV (301), as is a targeted mutation in the TLR9 receptor (309). These and other data suggest that the signaling pathways of both TLR9, via the adapter MyD88, and TLR3, via the adapter TRIF, are activated by viral infection and

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contribute to innate defense in an additive and nonredundant manner by stimulating the production of type I IFN for activation of effector response by NK cells (295). Finally, the Triple D (3d ) mutant phenotype abrogates macrophage signaling via the RNA and DNA agonists of the intracellular receptors TLR3, TLR7, and TLR9 (302). 3d mice succumb early to a challenge with MCMV (most likely because nucleic acids are an important by-product of virus infection); however, these mice are also susceptible to other organisms such as S. aureus and L. monocytogenes (295). 3d mice are also deﬁcient in APC activity despite normal levels of MHC class I and MHC class II antigen presenting molecules (302). Using positional cloning, researchers found that the 3d mutation introduces a single H412R substitution in the polytopic ER-resident membrane protein UNC93B. Transfection of the wild-type allele into 3d mutant mice rescues TNF production, which shows that the speciﬁc H412R mutation is responsible for the signaling defect in DCs from homozygous Unc93b mutant mice (302). The precise function of the UNC93B protein remains unknown. The mutation does not affect the stability or localization of the protein but rather abolishes the interaction between UNC93B and the transmembrane domain of TLR3 and TLR9 (310), which suggests that this physical association is crucial for TLR signaling. A notable human study by Casanova and colleagues (311) linked a mutation in UNC93B to the etiology of herpes simplex encephalitis (HSE). Cells isolated from an HSE patient presented a selective lack of response to TLR7, -8, and -9 agonists, similar to the response of cells obtained from 3d mutant mice. In addition, the production of type 1 IFNs was selectively impaired in the patient’s cells in response to stimulation with several viruses. Pedigree analysis and candidate gene sequencing associated the phenotype with a homozygous four-nucleotide deletion that introduces a premature stop codon in the UNC93B1 cDNA. Moreover, transfection

of wild-type cDNA into these cells complements cytokine secretion in the patient’s cells, which indicates that UNC93B deﬁciency is the predisposing factor to HSE in this family. Remarkably, the addition of recombinant IFN prior to viral infection fully complements the cellular phenotype of UNC93B mutant cells, which suggests that IFN could be a possible treatment in certain HSE patients. These studies provided the ﬁrst description of the genetic etiology of HSE and validated the applicability of a chemical-mutagenesis approach to identify mechanisms of human susceptibility to infection.

DOMINO AND JINX: IN VIVO DISSECTION OF HOST RESISTANCE AGAINST MOUSE CYTOMEGALOVIRUS Beutler and colleagues (312) initiated a genome-wide screen of mice derived from ENU-progenitors for host susceptibility to infection against MCMV. In this experiment, mice were challenged with MCMV and observed for severe levels of morbidity or high viral counts in the spleen. Out of approximately 11,300 mutant mice derived from ENU-progenitors, they found 11 transmissible mutations (312), of which the Domino (313) and Jinx (314) mutant phenotypes were recently characterized. The Domino mutation manifests as a severe recessive phenotype that is associated with high MCMV load and mortality by day four postinfection (313). In addition, the in vivo response to L. monocytogenes and macrophage growth of VSV are also impaired (315). This latter phenotype is not corrected by administration of type I IFN. Candidate gene sequencing and in vivo crosses with Stat−/− mice demonstrated that Domino corresponds to a point mutation that alters the binding domain of STAT1, abolishing its activation (313). The study of the Jinx phenotype led to new insights into the pathogenesis of CMV infection (314). Despite high viral load in the spleen, Jinx mutants show robust cytokine www.annualreviews.org • Immunity to Infection in the Mouse

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production, which indicates adequate sensing of the infection. Detailed phenotypic characterization of Jinx mice showed defects in NK cell and cytotoxic T lymphocyte (CTL) granule exocytosis, which are speciﬁc to viral infection. Through mapping and sequencing, investigators attributed viral susceptibility to the creation of a novel donor splice site in Unc13d. This change is predicted to introduce a premature stop codon, thus eliminating one of the two Ca2+ binding domains of the 1085 amino acid protein (314). The precise function of the UNC13-D protein remains undeﬁned, although it is required for the fusion competence of cytoplasmic vesicles only in certain immune cells (316). UNC13D is conserved throughout evolution from C. elegans to humans (317). Remarkably, mutations in the human ortholog MUNC13-4 cause type 3 familial hemophagocytic lymphohistiocytosis (FHL3), a severe disease characterized by hepatosplenomegaly, anemia, and thrombocytopenia (318). However, FHL3 may require either a virus or bacteria as an infectious trigger for expression of the phenotype (319). Likewise, Jinx mice do not present hepatosplenomegaly in the absence of infection. However, hemophagocytic lymphohistiocytosis (HLH) can be induced by at least one virus, the arenavirus lymphocytic choriomeningitis virus (LCMV), which conﬁrms that UNC13-D is responsible for the Jinx phenotype (314). MUNC134/UNC13-D belongs to a large family of paralogous proteins, of which the closest relatives (MUNC13-1 to MUNC13-4) are expressed in the brain (320), where they serve partially redundant functions in the priming of synaptic vesicles (321, 322). This is clearly not the case for UNC13-D, whose role in NK function seems unique, and can now be explored in the Jinx model.

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many aspects of host defenses against infections, which has led to numerous seminal discoveries. These discoveries include the major histocompatibility locus (and the concept of self-recognition), the presence of discrete cell populations such as macrophages, DCs, and lymphocytes responsible for early antigen recognition, the orchestration of the adaptive immune response, and the resulting long-term protection against reinfecting pathogens. With the advent of laboratoryinbred mouse strains some 50 years ago, scientists began readily to report reproducible differences in the responses of different mouse stocks to different types of infections, and the ﬁeld of immunogenetics was born. Early segregation studies revealed that some of these strain-speciﬁc differences in susceptibility are inherited as monogenic traits, and scientists were prompt to realize that identiﬁcation of the gene involved may yield important insight into the mechanisms by which the host recognizes, interacts with, and ultimately eliminates infectious agents. However, the absence of adequate mapping tools, including a paucity of genetic markers, the resulting frail genetic map, and the lack of DNA analysis methods rendered the task of going from a chromosomal localization to identiﬁcation of the gene and protein involved largely impossible. The advent of interspeciﬁc mouse crosses, simple sequence length polymorphisms (dinucleotide repeats), ﬂuorescence in situ hybridization, long-range physical mapping by pulsed-ﬁeld gel electrophoresis, large insert libraries in artiﬁcial yeast and bacterial chromosomes, exon ampliﬁcation, and efﬁcient DNA sequencing technologies gave birth to the ﬁeld of genomics 20 years ago. The years that followed saw the positional cloning of some of the oldest and best known so-called host-resistance loci, such as Mx (p47GTPase), Ity/Lsh/Bcg (Nramp1), Lps (Tlr4), Cmv1 (Ly49h) and several others covered in this review. Some of these discoveries had a dramatic impact on our understanding of host:pathogen interactions and shed considerable light on the critical early steps of

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this interaction. In particular, the discovery of LRRs as modular structures for the recognition of PAMPs and the critical role of LRRcontaining proteins (including the TLR and NLR families) uncovered a previously unknown pathway for extracellular and intracellular antigen recognition by macrophages and other cells. The next wave of innovation came in the form of a complete draft of the whole genome, an annotated transcript map together with powerful technologies such as high-throughput DNA sequencing and genotyping, and microarrays for whole-genome transcript proﬁling. These technologies have greatly facilitated the process of positional cloning, which has now become a fairly routine, albeit still laborious, endeavor, and have led to the discoveries of many other genes and

proteins important for the antimicrobial arsenal of the mammalian host. What is next? These new high-throughput technologies have opened the door to a more systematic sampling of the genetic diversity of inbred mouse stocks for differential response to infectious stimuli. Expanded genetic diversity may come not only from commercially available inbred mouse strains (Mouse Phenome Project), but also more importantly from ENU-mutagenized mouse stocks with the long-term potential to test the contribution of every gene in the genome to host response to infections. Although this is an enormous endeavor, several such large screens are currently underway. These screens will no doubt generate a wealth of additional information in this key area of immunology.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

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307. Yamamoto M, Sato S, Mori K, Hoshino K, Takeuchi O, et al. 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-β promoter in the Toll-like receptor signaling. J. Immunol. 169:6668–72 308. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, et al. 2003. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301:640– 43 309. Krug A, French AR, Barchet W, Fischer JA, Dzionek A, et al. 2004. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21:107–19 310. Brinkmann MM, Spooner E, Hoebe K, Beutler B, Ploegh HL, Kim YM. 2007. The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLR signaling. J. Cell Biol. 177:265–75 311. Casrouge A, Zhang SY, Eidenschenk C, Jouanguy E, Puel A, et al. 2006. Herpes simplex virus encephalitis in human UNC-93B deﬁciency. Science 314:308–12 312. Beutler B, Crozat K, Koziol JA, Georgel P. 2005. Genetic dissection of innate immunity to infection: the mouse cytomegalovirus model. Curr. Opin. Immunol. 17:36–43 313. Crozat K, Georgel P, Rutschmann S, Mann N, Du X, et al. 2006. Analysis of the MCMV resistome by ENU mutagenesis. Mamm. Genome 17:398–406 314. Crozat K, Hoebe K, Ugolini S, Hong NA, Janssen E, et al. 2007. Jinx, an MCMV susceptibility phenotype caused by disruption of Unc13d: a mouse model of type 3 familial hemophagocytic lymphohistiocytosis. J. Exp. Med. 204:853–63 315. Beutler B, Georgel P, Rutschmann S, Jiang Z, Croker B, Crozat K. 2005. Genetic analysis of innate resistance to mouse cytomegalovirus (MCMV). Brief. Funct. Genomic Proteomics 4:203–13 316. Neeft M, Wieffer M, de Jong AS, Negroiu G, Metz CH, et al. 2005. Munc13–4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells. Mol. Biol. Cell 16:731–41 317. Brose N, Hofmann K, Hata Y, Sudhof TC. 1995. Mammalian homologues of Caenorhabditis elegans unc-13 gene deﬁne novel family of C2-domain proteins. J. Biol. Chem. 270:25273–80 318. Feldmann J, Callebaut I, Raposo G, Certain S, Bacq D, et al. 2003. Munc13–4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3). Cell 115:461–73 319. Fisman DN. 2000. Hemophagocytic syndromes and infection. Emerg. Infect. Dis. 6:601– 8 320. Brose N, Rosenmund C, Rettig J. 2000. Regulation of transmitter release by Unc-13 and its homologues. Curr. Opin. Neurobiol. 10:303–11 321. Verhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, et al. 2000. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287:864–69 322. Varoqueaux F, Sons MS, Plomp JJ, Brose N. 2005. Aberrant morphology and residual transmitter release at the Munc13-deﬁcient mouse neuromuscular synapse. Mol. Cell Biol. 25:5973–84

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:81-132. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislˆaine Martins and Kathryn Calame Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032; email: [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:133–69

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

transcription, repressor, terminal differentiation, cytokines

This article’s doi: 10.1146/annurev.immunol.26.021607.090241

Abstract

c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0133$20.00

B lymphocyte–induced maturation protein-1 (Blimp-1), discovered 16 years ago as a transcriptional repressor of the IFNβ promoter, plays fundamentally important roles in many cell lineages and in early development. This review focuses on Blimp-1 in lymphocytes. In the B cell lineage, Blimp-1 is required for development of immunoglobulin-secreting cells and for maintenance of long-lived plasma cells (LLPCs). Direct targets of Blimp-1 and the transcriptional cascades Blimp-1 initiates to trigger plasmacytic differentiation are described. Blimp-1 also affects the homeostasis and function of CD4+ , CD8+ , and regulatory CD4+ T cells, and Blimp-1 levels are highest in antigen-experienced T cells. Blimp-1 attenuates T cell proliferation and survival and modulates differentiation. Roles for Blimp-1 in Th1/Th2 speciﬁcation, regulatory T cell function, and CD8 differentiation and function are under investigation. Signals that induce Blimp-1 in B cells include Toll-like receptor ligands and cytokines; in T cells, T cell receptors and cytokines induce Blimp-1. In spite of some commonalities, different targets and regulators of Blimp-1 in B and T cells suggest intriguing evolutionary divergence of this regulatory machinery.

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INTRODUCTION

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Zinc finger: the structural domain of a protein, composed of ∼30 amino acids binding a zinc ion, that mediates sequence-speciﬁc binding to DNA

Although transcription factors containing zinc ﬁnger DNA-binding domains constitute the largest family of transcription factors (1), a single zinc ﬁnger–containing transcriptional repressor, B lymphocyte–induced maturation protein-1 (Blimp-1, also called PRDIBF1) plays critical and nonredundant roles in both B and T lymphocytes. In addition, Blimp-1 plays important roles in certain nonlymphoid lineages in adults and in various aspects of embryonic development in many organisms. In this article, we review the role of Blimp-1 in B and T lymphocytes in detail, with an emphasis on recent ﬁndings. Other reviews including this topic have appeared recently (2–7).

BLIMP-1 BASICS Although there is nothing unusual about its structure, an unusual role for Blimp-1 in B cells was indicated in early work.

Discovery Blimp-1 cDNA was ﬁrst cloned by Maniatis and colleagues (8), who used expression cloning to identify a human cDNA encoding a zinc ﬁnger–containing protein that bound to the positive regulatory domain I (PRDI) of the human IFNβ promoter. They named the protein PRDIBF1 (positive regulatory domain I-binding factor 1) and, in addition to verifying its binding speciﬁcity, demonstrated that the protein was a transcriptional repressor, which was induced upon virus infection of the human osteosarcoma line U20S. Three years later, Davis and colleagues (9) isolated a murine cDNA from a subtractive screen of BCL1 lymphoma cells compared before and after induction of differentiation to Ig-secreting plasma cells by treatment with IL-2+IL-5. Because the message was induced following cytokine-dependent differentiation of the cells, they called it B lymphocyteinduced maturation protein-1, or Blimp-1 (9). Although they did not show directly that the zinc ﬁnger protein encoded by their cDNA 134

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was a transcription factor or deﬁne a binding site, they made the critical observation that ectopic expression of the protein was sufﬁcient to drive the BCL1 cells to mature into Igsecreting plasmacytoid cells. Later that year, Huang (10) brought these two lines of study together by showing that Blimp-1 was the murine homolog of PRDIBF1, although the mouse protein had a slightly different N terminus from the human, containing 67 additional amino acids (10). Indeed, the human and mouse proteins are highly homologous and are interchangeable in functional assays. (For simplicity, we use the name Blimp-1 for both the human and mouse proteins in this review.) Huang’s recognition that PRDIBF1 and Blimp-1 were homologs identiﬁed this protein as a transcriptional repressor, with a deﬁned binding speciﬁcity, that was capable of driving BCL1 lymphoma cells to differentiate into Ig-secreting plasma cells. Subsequently the gene structure of prdm1 (11), encoding Blimp-1, as well as its location on human chromosome 6q21, and the syntenic region of mouse chromosome 10 (12) were deﬁned. The mouse gene extends over ∼23 kb and contains 8 exons. Exons 6, 7, and 8 encode the zinc ﬁnger domains (Figure 1a,b).

Protein Domains and Biochemical Mechanism of Action Murine Blimp-1 contains 856 amino acids and is predicted to be a 95,835-Da protein. Human Blimp-1 has 789 amino acids and a predicted molecular weight of 87,990 Da. The ﬁve C2 H2 zinc ﬁnger motifs in the C terminus of Blimp-1 were clearly implicated as the DNA-binding domain; however, further study showed that only the ﬁrst two ﬁnger motifs are necessary for recognition of the PRDI region in the IFNβ promoter (13). The consensus-binding site for Blimp-1 was determined and, consistent with the Maniatis papers (8, 13), was very similar to that of IFN regulatory factor (IRF)1 and IRF2 (14). In fact, Blimp-1 and IRF1/2 compete for binding to the site in the IFNβ promoter (14).

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prdm flox Figure 1 Blimp-1 mRNA and the prdm1 gene. (a) Murine Blimp-1 mRNA showing which portions are encoded by each exon. 5 and 3 noncoding regions are white. Coding regions are colored, with acidic regions light blue, PR region orange, proline-rich region dark blue, and zinc ﬁngers dark green (11). (b–d ) Murine prdm1 gene with exons shown as raised boxes. Coding regions are blue, and noncoding are white. The approximate location of clustered transcription start sites is indicated by an arrow. The gene covers about 23 kb; in the ﬁgure, the scale for introns is 2 times less than the scale for exons. (c) Alterations in prdm1 to make the prdm1gfp knockin allele. A region containing a splice acceptor site followed by stop codons in all three frames ( yellow hexagon), an internal ribosome entry site (IRES), and cDNA encoding e-GFP (green ﬂuorescent protein) followed by a SV40 polyadenylation site (pA) was inserted into intron 6 (24). (d ) Structure of the prdm1ﬂox allele. LoxP sites (red bolts) were inserted in intron 5 and 3 to exon 8. Upon Cre-dependent deletion, exons 6–8, encoding the zinc ﬁnger domains, are deleted (34).

Other domains of the protein (Figure 1a) include a proline-rich region N-terminal to the zinc ﬁngers and a PR domain conserved between Blimp-1 and the Rb-binding protein RIZ1 (encoded by prdm2) (15, 16). The proline-rich region along with the zinc ﬁngers is required for transcriptional repression and has been shown to mediate association of Blimp-1 with transcriptional corepressor hGroucho (17) and histone deacetylases 1 and 2 (18). Deacetylation of histone lysine residues is associated with a repressive chromatin structure. The PR domain was named for the ﬁrst two proteins where it was discovered, PRDIBF1 and RIZ. PR domains in Blimp-1 and RIZ have similarity to SET domains found

in histone methyl transferases (HMT) (19), and the PR domain of RIZ1 does have HMT activity (20). Although the PR domain of Blimp-1 does not have demonstrable HMT activity, Blimp-1 recruits the G9a HMT to the IFNβ promoter (21). G9a methylates lysine 9 on histone 3, a repressive histone modiﬁcation. H3K9 methylation occurs in the IFN-β promoter upon ectopic expression of Blimp-1 (21). Finally, in primordial germ cells, Blimp-1 complexes with prmt5, an arginine HMT that catalyzes symmetrical dimethylation of arginine 3 on H2A and H4 (22). Thus, Blimp-1 appears to repress transcription by recruiting proteins or corepressor complexes that modify histones (by deacetylation, H3K9 methylation, and www.annualreviews.org • Blimp-1 in T and B Lymphocytes

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IHC: immunohistochemistry

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GC: germinal center Cre and “floxed” alleles: bacteriophage P1’s Cre recombinase excises DNA between two similarly oriented 34 bp loxP recombination sites, leaving a single loxP site. DNA ﬂanked by loxP sites is said to be “ﬂoxed” and is a target for Cre-dependent deletion

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arginine methylation) to create a more closed or repressive chromatin structure. However, further work is necessary to learn exactly how chromatin is modiﬁed at speciﬁc Blimp1 target genes when Blimp-1 represses them and to learn if chromatin modiﬁcation is the only mechanism by which Blimp-1 represses transcription.

BLIMP-1 IN B CELL BIOLOGY Based on the demonstration by Davis and colleagues (9) that Blimp-1 was sufﬁcient to drive plasmacytic differentiation, much work has focused on the role of Blimp-1 in the B cell lineage. This work provides a paradigm for exploring its roles in other cell lineages.

Expression Pattern of Blimp-1 Blimp-1 expression in the B cell lineage has been studied by immunohistochemistry (IHC) in mice and humans and using a green ﬂuorescent protein (GFP) reporter gene knocked into one allele of the prdm1 locus in mice (Figure 1c). In mice, both IHC (23) and GFP (24) studies show that some plasmablasts and all plasma cells express Blimp-1. GFP analyses provide evidence that increasing levels of Blimp-1 correspond to stages of plasma cell differentiation from plasmablasts to long-lived plasma cells (LLPCs) in the bone marrow (24). Consistent with this idea, plasma cells resulting from a secondary response had higher levels of Blimp-1 compared with those formed in a primary response (25). No Blimp-1 expression has been observed in memory B cells in mouse (26) or human (27). Although peritoneal B-1 cells express low levels of Blimp-1 mRNA (28) and protein, indicated by the GFP reporter (29), Blimp1 mRNA is induced by lipopolysaccharide (LPS) upon Toll-like receptor (TLR) 4 signaling in B-1 cells (30), and the kinetics of induction, measured using the GFP reporter, are more rapid than that for B2 cells (29).

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Although IHC is not quantitative, it is probably more sensitive than the GFP reporter. Furthermore, half-lives of Blimp-1 and GFP mRNA and protein are probably not exactly the same, introducing some doubt into using GFP as an absolute indicator of endogenous Blimp-1 expression. IHC for endogenous Blimp-1 has revealed that a small fraction (∼5%–15%) of cells in germinal centers (GCs) also express Blimp-1. (Blimp-1 has not been detected in B cells at earlier stages.) Blimp-1+ cells in GCs do not express Bcl-6 but do contain cytoplasmic Ig and probably represent centrocytes that are fated to leave the GC as plasmablasts (23). Blimp-1 appears in these GC cells before Syndecan-1 (CD138) (31). Blimp-1 expression patterns in humans are similar to those in mice (32, 33). The GC B cells that express Blimp-1 are Pax5+ Bcl-6− ; outside the GC, human plasma cells do not express Pax5 (32, 33). In an in vitro system in which human centrocytes were induced to become Ig-secreting plasma cells, high levels of Blimp-1 mRNA were not achieved until levels of Bcl-6, Pax5, and Bach2 had fallen signiﬁcantly (27).

Blimp-1 Is Required for Ig Secretion Mice with a conditional deletion of prdm1 in the B lineage were created by crossing mice with “ﬂoxed” prdm1 alleles to CD19Cre transgenic mice (34). In these mice, exons 6– 8, which encode all the zinc ﬁnger domains, are deleted in mature B cells in the presence of Cre recombinase (Figure 1d ). Consistent with Blimp-1’s expression pattern, these mice revealed that Blimp-1 is required for plasma cell formation and for normal Ig secretion in response to both T-independent (TI) and Tdependent (TD) antigens. In the B cell conditional knockout (CKO) mice, peripheral B cell subsets were normal, and GCs formed in response to the TD antigen. Class switch recombination (CSR) occurred normally, as evidenced by the appearance of switch circles (M. Shapiro-Shelef & K. Calame, unpublished),

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but secretion of all isotypes was severely reduced. In addition, GCs were enlarged in CKO mice, suggesting a developmental block at the late/post-GC stage. Subsequent studies on the CD19Cre CKO mice revealed that, although B1 B cells were present in normal numbers and were capable of self-renewal, they failed to secrete Ig normally (30). Thus, Blimp-1 is required for normal Ig secretion in all B cell subsets. A recent report (110) has investigated in more detail the low Ig secretion observed in mice lacking Blimp-1 in the B cell lineage (34). Using reconstitution of Rag−/− mice with fetal liver cells from prdm1gfp/gpf mice, these researchers showed that there is an early phase in plasmacytic differentiation, which they call a “preplasmablast,” during which Pax5 activity is inhibited and genes repressed by Pax5 and other plasma cell genes, but not Blimp-1, are expressed. These cells secrete low amounts of Ig and provide evidence for an early phase of plasmacytic differentiation that is independent of Blimp-1 but requires inhibition of Pax5. Thus, these authors conclude that, although Blimp-1 is required for full plasmacytic differentiation and normal levels of Ig secretion, B cells can enter an initial phase of plasmacytic differentiation without Blimp1. One caveat regarding this work, however, is the question of whether the prdm1gfp allele used in this study is a true null allele or a hypomorphic allele, owing to the potential, via differential splicing, to form Blimp-1 mRNA (Figure 1c ). Embryos homozygous for the prdm1gfp allele live until at least E13 or E14, allowing transfer of fetal liver cells for reconstitution studies (35). In contrast, embryos homozygous for either of two different deletion alleles, including one generated from the prdm1ﬂox allele (Figure 1d ), die signiﬁcantly earlier at E10.5 (36). Blimp-1 is not required for formation of memory B cells because in the CKO mice there is an exaggerated GC response upon a secondary challenge even though these cells are unable to differentiate into plasma cells (34). This is consistent with the ﬁnding that

Blimp-1 mRNA is not present in human memory B cells formed in in vitro cultures (27) or in ex vivo puriﬁed human memory cells (26). Preplasma memory cells in the bone marrow, however, were dramatically reduced in the CKO mice, suggesting a requirement for Blimp-1 to form this interesting but controversial (26, 37, 38) subset. Using an inducible gene deletion system, investigators showed that LLPCs (see the Long-Lived Plasma Cells sidebar) in the bone marrow, formed in the presence of Blimp-1, require continued expression of Blimp-1 for their maintenance (39). LLPCs in the bone marrow provide a second form of humoral memory by providing continuous immunity to pathogens that have been previously encountered. They survive in the bone marrow without proliferation or antigen stimulation for long periods of time (40). The continued requirement for Blimp-1 shows that the repressive changes in chromatin that Blimp-1 facilitates must be labile on at least an important subset of its target genes, requiring continued activity of Blimp-1 to maintain repression. These studies showed that when prdm1 was deleted, CD138+ , Ig-secreting cells, resident in the bone marrow disappeared; however, whether they died or dedifferentiated was not established and requires further study. Thus, Blimp-1 is uniquely required for formation and maintenance of all Ig-secreting B cells, and interfering with its activity might be a way to target LLPC in autoimmunity or other pathological conditions.

Plasmacytic differentiation: terminal differentiation of an activated B cell to become an Ig-secreting plasma cell

Targets of Blimp-1 Not only is Blimp-1 necessary for plasma cell formation and function, it is also sufﬁcient. The striking ability of ectopically expressed Blimp-1 to drive plasmacytic development, originally demonstrated in 1994 by Davis and colleagues (9), was subsequently extended beyond BCL1 cells to normal splenic B cells (47, 48). Importantly, however, when Blimp-1 expression is forced in B cells at earlier developmental stages, it causes cell death (49). www.annualreviews.org • Blimp-1 in T and B Lymphocytes

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LONG-LIVED PLASMA CELLS In a primary response, most plasma cells formed after a T cell–dependent GC reaction secrete antibodies with high afﬁnity and switched isotypes. Because of changes in chemokine receptors such as CXCR5, they exit the follicles, and some migrate to niches in the bone marrow. In the bone marrow niches, they receive survival signals that include IL-6, made by bone marrow stromal cells (41), and TNF family members BAFF (B cell-activating factor) or APRIL (a proliferation-inducing ligand) that signal through BCMA (B cell maturation antigen) (42). LLPCs have been shown to survive for months to years, sometimes for the lifetime of the organism (43) in the absence of an antigen (44) or cell division (45). Interestingly, when an organism mounts a new primary response, some previously formed LLPCs, resident in the bone marrow, are mobilized to leave the bone marrow survival niches, presumably providing space for newly formed plasma cells and ensuring a dynamic repertoire of LLPCs, reﬂecting the immunological experience of the organism (46). Because they provide constant immunological vigilance in the form of secreted antibodies, LLPCs are considered one form of humoral immunity. For more details on LLPCs, see a recent review (40).

XBP-1: X-box binding protein-1 Gene expression profile: the unique, global pattern of genes expressed as steady-state mRNA in a given cell type

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Ig-secreting plasma cells differ signiﬁcantly, both morphologically and functionally, from activated B cells; they express neither BCR nor MHC class II on their surface, and they are postmitotic (6). The ability of a single transcription factor to trigger such a complex and dramatic developmental decision is unusual and was a driving force behind efforts to identify targets of Blimp-1-dependent repression in B cells. Gene expression analyses comparing Burkitt lymphoma lines with or without forced expression of Blimp-1 revealed more than 250 genes whose expression was altered by Blimp-1 (50). Obviously such experiments reveal both direct and indirect Blimp-1 targets (Figure 2). Three main programs of gene expression were altered by Blimp-1. (a) A proliferative program including cMYC, E2F1, and other genes required for entry into cycle and cell division was repressed. Myc had Martins

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previously been shown to be a direct target of Blimp-1-dependent repression (51, 52). (b) A program involved in Ig secretion, including J chain, X-box binding protein-1 (XBP-1), as well as Ig heavy and Ig light chain genes, was induced. (c) An extensive program of gene expression characteristic of activated or GC B cells was repressed, including genes encoding the critical transcription factors Pax5 and Bcl-6. Pax5 was previously shown to be a direct target of Blimp-1 (53), and recent studies show that bcl6 is also directly repressed by Blimp-1 (L. Cimmino & K. Calame, unpublished). The “B cell program” repressed by Blimp-1 includes repression of genes involved in response to signals from the BCR; genes, notably AICDA, involved in CSR and somatic hypermutation (SHM); genes encoding costimulatory molecules for T cells; and genes for chemokine receptors (Figure 2). Direct targets among this group also include class II transactivator (CIITA) via promoter III, required for MHC class II expression in B cells (48, 54), ID3 and SPIB (50) (Figure 3, Table 1). Another gene expression study identiﬁed genes regulated by Blimp-1 in BCL1 and M12 cells (55). This study conﬁrmed much of the earlier work but also identiﬁed other interesting genes regulated by Blimp-1 in B cells, including induction of irf4 and repression of taci. Using mutant forms of Blimp-1, investigators also identiﬁed genes whose regulation depended on the PR domain of Blimp-1, including ell2 (55). XBP-1 is a transcriptional activator that is required for plasma cell development and function (56). When the gene expression proﬁles of splenic B cells lacking Blimp1 and XBP-1 were compared (57), it was clear that Blimp-1 expression is necessary for expression of XBP-1 and that XBP-1 is the proximate activator of multiple genes necessary for expanded endoplasmic reticulum and protein secretion as well as of genes causing increased cell size, lysosome content, mitochondrial mass and function, ribosome numbers, and total protein synthesis. In the

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a

Blimp-1 myc

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dhrf odc1 ldha cdc2a

gadd45a ddit3 cdkn1a cdkn2c

id3

pax5

aicda blnk cd19 cd79a ciita syk

igh igl igj xbp1

igl

Direct repression spi-b

ciita

bcl-6

btk

h2 cd74

cdkn1a trp53 prdm1

Secretory genes

Blimp-1 Direct?

b

Indirect?

e2f1

mybl1/ mybl2

ebf1

pou2f2/ pou2af1

tcfe2a

myc mcm7 dhfr cdc2a pcna myb2

sox-4 cdc2a

pax5 cd79a

cd79a h2 blr1

aicda

Figure 2 Transcription factors that are targets of Blimp-1 in B cells. Panel (a) indicates genes shown to be directly repressed by Blimp-1, and panel (b) indicates genes Blimp-1 represses by a currently unknown mechanism. In both cases, target genes that are known to be regulated by the indicated transcription factors and that had their expression altered by Blimp-1 (50) are shown. Green indicates genes involved in cell proliferation, purple, genes involved in B cell phenotype and function, and blue, genes involved in Ig secretion. All genes are given murine designations for clarity and consistency.

absence of Blimp-1, neither XBP-1 nor its target genes are expressed normally (57). The mechanism responsible for Blimp-1dependent induction of XBP-1 may be via repression of pax5 since Pax5 has been reported to repress xbp1 (59). However, a detailed gene expression study in B cells lacking Pax5 did not show that XBP-1 was elevated (60), suggesting another, currently unknown, mechanism may be responsible for Blimp-1-dependent expression of XBP-1. In chicken DT40 cells lacking Pax5, both Blimp-1 and XBP-1 were elevated (61), providing evidence that Pax5 does repress xbp1 in this setting. Thus, although it is established that Blimp-1 is necessary for XBP1 induction in B cells, the exact mechanism

by which Blimp-1 regulates XBP-1 remains uncertain.

Regulation of Blimp-1 and Plasmacytic Differentiation Plasmacytic differentiation must be strictly regulated. Failure to mount a humoral response in a timely fashion would clearly jeopardize the organism. However, if plasmacytic differentiation occurred too soon during a TD response, before germinal center reactions were completed, afﬁnity maturation and class switching would be compromised, and the strength and quality of both primary and secondary responses would be www.annualreviews.org • Blimp-1 in T and B Lymphocytes

Germinal center (GC) reaction: In response to T cell–dependent antigens and T cell help, B cells form GCs, where they undergo rapid proliferation, afﬁnity maturation, and CSR. Memory cells and plasmablasts result from the GC reaction

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MITF

Bach2

irf4

Naive B cell

Pax5

prdm1

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Plasma cell

xbp1

GC B cell

Pax5

Bach2 Bcl-6

Figure 3 Inhibition of genes required for plasma cell differentiation in earlier naive follicular B cells and by germinal center (GC) B cells. Red bars indicate transcriptional repression, and the blue arrow indicates induction. Factors in naive B cells are shown in the yellow box; factors in GC B cells are shown in the blue box (59–61, 90, 95–97, 100, 103, 106).

diminished. In addition, peripheral tolerance requires B cells to selectively respond to signals from self-antigens by dying or becoming anergic rather than undergoing plasmacytic differentiation. Finally, it is important that memory B cells be poised to differentiate to Table 1

plasma cells rapidly in response to secondary antigenic challenge but also be regulated so that spontaneous differentiation does not occur in the absence of a stimulus. Expression of Blimp-1 sets plasmacytic differentiation into effect in what is normally an irreversible process. Thus, understanding the regulation of Blimp-1 expression is critical for understanding how appropriate primary humoral responses, peripheral B cell tolerance, and B cell memory are regulated. The data detailed below support a general model in which (a) the B cell developmental stage and (b) the nature, strength, and duration of signals that B cells receive from antigen, TLRs, and cytokines combine to determine Blimp-1 expression and plasmacytic development. In B cells, Blimp-1 expression is regulated primarily at the level of transcription initiation (11). Multiple, clustered transcription initiation sites have been mapped in the mouse gene that give rise to a full-length protein (11). An alternative transcription start site, located 5 of exon 4 in the human gene,

Direct targets of Blimp-1 repression in lymphocytes

Gene

Transcriptional regulator?

B cell target

T cell target

References

bcl6

Yes, repressor

Yes

Yes

L. Cimmino & K. Calame, unpublished

myc

Yes, activator and repressor

Yes

No

51; G. Martins, L. Cimmino & K. Calame, unpublished

ciita (promoter III)

Yes, coactivator

Yes

Not expressed

48, 54

fos

Yes, activator

n.d.

Yes

162a; E. Magnusdottis, G. Martins & K. Calame, unpublished

Id3

Yes, inhibitor of E proteins

Yes

n.d.

50

ifng

No

Not expressed

Yes

L. Cimmino & K. Calame, unpublished

il2

No

n.d.

Yes

62; G. Martins & K. Calame, unpublished

pax5

Yes, activator and repressor

Yes

Not expressed

53

spib

Yes, activator

Yes

n.d.

50

tbet

Yes, activator

n.d.

Yes

L. Cimmino & K. Calame, unpublished

n.d., not determined.

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gives rise to a shorter protein known as the beta form, which lacks 101 N-terminal amino acids, contains only a partial PR domain, and has reduced transcription repression activity (62). This form is usually present in submolar amounts relative to the full-length form. Blimp-1 mRNA and protein are labile, with half-lives of less than 2 h (C. Tunyaplin & K. Calame, unpublished). Although phosphorylation of the protein has been observed ( J. Noronha & K. Calame, unpublished), there is no evidence that levels of phosphorylation are regulated, and the effect of phosphorylation on function has not been assessed. IHC shows Blimp-1 is localized entirely in the nucleus in mouse (23) and human (32) B cells. Activators of prdm1 transcription. Pattern-recognition receptors (63), including TLRs, RIG-I-like receptors, and possibly NOD-like receptors, induce Blimp-1 in many settings. This was ﬁrst demonstrated by induction of Blimp-1 by Sendai virus (double-stranded RNA) infection of U20S cells (8). In B cells treated in vitro, LPS, which activates TLR4, is a strong inducer of Blimp-1 mRNA for murine splenic B cells and for B-1 cells from the peritoneal cavity (30, 47). CpG, which activates TLR9, induces Blimp-1 in human tonsillar B cell cultures (T. Kuo & K. Calame, unpublished). Reishi polysaccharides, which activate TLR4/TLR2, also induce Blimp-1, apparently using different signaling pathways in human and murine B cells (64). These data are consistent with an analysis of TLR4−/− and MyD88−/− mice (65). [MyD88 is an obligate signal transducer for all TLRs except TLR3 and TLR4, which have both MyD88-dependent and MyD88independent pathways (63, 66).] B cells from these mice do not secrete IgM or IgG antibodies normally. Further analysis showed that for a TI-2 response to ﬂagellin, which activates TLR5, MyD88−/− B cells had a defective IgM and IgG1, but not IgG3, response. In a TD GC reaction, both TLR4−/− and MyD88−/− B cells were defective in formation of GC

B cells. GC B cells lacking MyD88 had decreased Blimp-1 mRNA and increased Bcl-6 mRNA. These data are consistent with the idea that TLR signals induce Blimp-1 in both TI and TD responses. However, a more recent paper (67) calls into question the role of TLR signals for B cell responses as it showed that mice lacking all TLR-dependent signaling mounted normal responses to TD antigens delivered in various adjuvants. Thus, the role of TLRs in B cell responses is unclear at present, although their ability to induce Blimp-1 is unquestioned. Pattern recognition receptors activate NF-κB (63), and NF-κB appears to be a direct activator of prdm1 transcription (Table 3), as evidenced by the failure of p65−/− p50−/− 3T3 cells to induce Blimp-1 in response to Sendai virus infection, failure of splenic B cells to induce Blimp-1 in response to LPS in the presence of NF-κB inhibitors, and the binding of p65 in vivo to multiple κB sites in the region 5 to transcription initiation on prdm1 (T. Kuo, E. Magnusdottir & K. Calame, unpublished). TLR or RIG-I activation of Blimp-1 mRNA is sufﬁcient to induce plasmacytic differentiation in activated B cells. However, activated NF-κB is apparently not sufﬁcient to induce Blimp-1 in all stages of B cell development because it plays important roles earlier in B cell development (68), yet Blimp1 is not expressed in these earlier B cells. Furthermore, BCR, CD40 ligation, and B cell–activating factor (BAFF)-dependent signaling also activate NF-κB but do not induce Blimp-1 mRNA (69). In fact, BCR, CD40, and IL-4 signals block LPS-dependent induction of Blimp-1 in murine splenic B cells (69), although in human B cells BCR and CD40 activation enhance IL-21’s ability to induce plasmacytic differentiation and Blimp-1 expression (70). There has, however, been a report that a stress response in macrophage and B cell lines induces Blimp-1 in a way that depends on NF-κB (71). Other transcription factors, including NF-IL6 and IRF1, IRF3, and IRF8 are also induced by TLR signaling (72) and may also induce Blimp-1, especially www.annualreviews.org • Blimp-1 in T and B Lymphocytes

BAFF: B cell activating factor

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Table 2

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Signals that regulate Blimp-1 expression

Function

Signal

B cells

References

T cells

References

IL-2

Yes (BCL1 line)

9

Yes

74, 76

IL-4

n.d.

—

Yes

132

IL-5

Yes

9

n.d.

—

IL-6

Yes (lymphoma lines, bone marrow)

K.L. Lin & K. Calame, unpublished

n.d.

—

IL-10

Yes

27

n.d.

—

IL-21

Yes

77

n.d.

—

B/TCR

Indirect via Bcl-6 or others

78

Yes

76, 35

LPS

Yes

47

n.d.

—

CpG

Yes

29

n.d.

—

Cytokine

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Clonotypic receptor TLR ligand

n.d., not determined.

because IRF8 induces Blimp-1 in myeloid cells (73). Several cytokines, including IL-2, IL5, IL-6, IL-10, and IL-21, induce Blimp1 mRNA, as ﬁrst noted by Davis and colleagues (9) who showed that Blimp-1 mRNA was induced by IL-2+IL-5 in BCL1 cells (Table 2). Indeed, in T cells as well as B cells, IL-2 is a strong inducer of Blimp-1 mRNA (74). IL-6 induces plasmacytic differentiation of certain human B cell lines (75), and this is accompanied by induction of Blimp-1 mRNA (K.-I. Lin & K. Calame, unpublished). Although there is little evidence that IL-6 is important for driving plasmacytic differentiation during a primary response in vivo, IL-6, secreted by stromal cells, is critical for maintenance of LLPCs in the bone marrow (41). One of its important functions in this setting may be to induce Blimp-1, which is continuously required for the maintenance of these plasma cells (39), although this has not been directly demonstrated. Plasmacytic differentiation of human B cells can be induced by IL-10 following activation by BCR and CD40 signals (79–81). Indeed, if IL-10 is added to human memory cell cultures, rapid plasmacytic differentiation

STAT: signal transducer and activator of transcription

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ensues (82) and is accompanied by induction of Blimp-1 mRNA (27). IL-21 is necessary for normal Ig secretion (83, 84) and induces differentiation of B cells to plasma cells in mice (77). Interestingly, IL21 induces both Blimp-1 and Bcl-6 mRNA (77). Similarly, when human B cells are stimulated via BCR and CD40, IL-21 is a strong inducer of both plasmacytic differentiation and Blimp-1 mRNA, and the effect of IL-21 is enhanced in combination with IL-2 (70). IL-2, IL-6, IL-10, and IL-21 all activate signal transducer and activator of transcription 3 (STAT3), strongly implicating STAT3 as a direct activator of prdm1 transcription. Although this has not been deﬁnitely demonstrated, forced expression of a dominantnegative form of STAT3 inhibited Blimp-1 mRNA induction in BCL1 cells (85), providing evidence that STAT3 does activate prdm1 transcription. In addition, and consistent with early observations in BCL1 cells, IL-5, which primarily activates STAT5 (86), induces Blimp-1 mRNA and, like IL-21, also induces genes involved in GC B cells such as bcl6 and aicda (87). IL-2 also activates STAT5 (88), and STAT5 may be important in the IL-2-dependent activation of Blimp-1. However, one study (89) has shown that activated

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Table 3

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Transcription factors that regulate prdm1 transcriptiona

Transcription factor

Activity

B cell

References

T cell

References

AP-1 (fos-Jun)

Activator

Yes

90

(Yes)

—

Bach2

Repressor

Yes

95

Not expressed

96

Bcl-6

Repressor

Yes

97

Yes

L. Cimmino & K. Calame, unpublished

Blimp-1

Autorepressor

n.d.

—

Yes

L. Cimmino & K. Calame, unpublished; 162a

IRF4

Activator

Yes

93

n.d.

—

Foxp3

Activator

Not expressed

—

Yes

98

NFAT

Activator

Yes

99

(Yes)

—

NF-κB

Activator

Yes

T. Kuo and K. Calame, unpublished

(Yes)

—

Pax5

Repressor

Yes

102a

(No)

—

STAT3

Activator

Yes

85

(Yes)

—

STAT5

Activator

n.d.

—

Yes

D. Gong & T. Malek, personal communication

a Parentheses indicate activities implied but not experimentally demonstrated. n.d., not determined.

STAT5 blocks plasmacytic differentiation, so the role of STAT5 in Blimp-1 regulation in B cells is currently unclear. In addition to Jak/STAT pathways, these cytokines also activate other signaling pathways, including the Ras-Raf-ERK, the phosphatidylinositol 3kinase (PI3K), the JNK/SAPK, and the p38 signaling pathways, and these may play a role in induction of Blimp-1 as well, although most of these possibilities remain unexplored. Two other transcriptional regulators, IRF4 and AP-1, bind prdm1 and directly activate its transcription (Table 3). AP-1 was ﬁrst implicated as an activator of the human gene when it was found that Bcl-6 repressed Blimp-1 transcription by associating with c-Jun and inhibiting the ability of AP-1 to activate PRDM1 (90). Evidence that AP-1 also induces murine prdm1 transcription comes from studies using c-Fos transgenic mice. Splenic B cells from these mice undergo more signiﬁcant plasmacytic differentiation in response to CD40 and IL-4 than do controls. Further studies on these mice also demonstrated binding of AP-1 to the prdm1 gene by chromatin

immunoprecipitation (ChIP) (91). Because Blimp-1 can be induced in mice lacking c-Fos, it seems that, although AP-1 activates Blimp-1 transcription, AP-1 is not essential (91). IRF4 is necessary for plasma cell formation (92) and more recently has also been shown as a requirement for CSR (93, 94). Mice lacking IRF4 in their B cells have been analyzed to determine if IRF4 might be upstream of Blimp-1 in a regulatory cascade. Two groups obtained different answers to this question. Klein et al. (94) created mice with a conditional deletion of irf4 in GC B cells. Upon LPS stimulation of splenic B cells in vitro, plasmacytic differentiation was blocked, but Blimp-1 mRNA was similarly induced in both wild-type and CKO, providing evidence that IRF4 does not act upstream of Blimp-1 and is not required for Blimp-1 induction. However, when Sciammas et al. (93) studied B cells from the original knockout mice (92), they also observed a failure in plasmacytic development after LPS stimulation, but in their studies, Blimp-1 mRNA failed to be induced. They went on to perform ChIP studies, which www.annualreviews.org • Blimp-1 in T and B Lymphocytes

ChIP: chromatin immunoprecipitation

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identiﬁed a site between exon 5 and 6 of prdm1 that was occupied by IRF4 in vivo in stimulated, but not in naive, B cells. They concluded that IRF4 is a direct transcriptional activator of prdm1. Although the discrepancy between the two studies remains unexplained, it may be that minor differences in activation conditions or the developmental stage of the cells studied altered the availability of IRF4 partners or other regulators of prdm1 in the two studies, accounting for different results. The ChIP studies (93), however, strongly suggest that IRF4 does indeed induce prdm1 transcription in at least some conditions.

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Repressors of prdm1 transcription. It is becoming increasingly clear that repression of Blimp-1 is important for its regulated expression in B cells. Bcl-6 was the ﬁrst repressor of Blimp-1 to be identiﬁed, based on microarray analyses of Burkitt cell lines expressing Bcl-6 or a dominant-negative Bcl-6 (97). Forced expression of Bcl-6 also inhibited plasmacytic differentiation and Blimp-1 expression in murine splenic B cells following activation (85). Bcl-6 represses prdm1 transcription by interfering with the AP-1 activator (90) and by binding directly to a conserved site in exon 5 of the murine gene (100). MTA3, a cell type-speciﬁc subunit of the corepressor complex Mi-2/NuRD, acts as a corepressor with Bcl-6, and when MTA3 and Bcl-6 expression is forced in myeloma cell lines, Ig secretion and Blimp-1 mRNA expression are inhibited, essentially causing dedifferentiation of these transformed plasma cells (101). Signals that lead to strong activation of PI3K in B cells favor plasmacytic differentiation and inhibit CSR (102). Part of this effect appears to depend on Blimp-1. The mechanism involves PI3K-dependent activation of Akt, which inhibits FoxO-dependent activation of Bcl-6, thus decreasing Bcl-6 and allowing derepression of prdm1 when PI3K is strongly activated (102). Loss of Pax5 in mature murine B cells leads to increased expression of Blimp-1 (60), 144

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and similar results are observed in chicken DT40 cells (61). However, whether this effect is due to direct repression of prdm1 by Pax5 or to Pax5-dependent induction of Bcl-6, which then represses prdm1, or both was not clear in those studies. In mice, loss of Pax5 did not alter Bcl-6 mRNA levels, but in DT40 cells, Bcl-6 was diminished in the absence of Pax5, and induction of Blimp-1 in the absence of Pax5 could be blocked by Bcl-6 (61). A very recent report shows that Pax5 binds to prdm1 and directly represses it (102a). Another repressor of prdm1 transcription is Bach2, a repressor that interacts with small Maf proteins (95). Bach2 is speciﬁcally expressed in B cells and required for normal CSR and SHM. Bach2−/− mice have a hyperIgM syndrome and spontaneous plasmacytic differentiation of IgM-secreting cells (95). LPS treatment of Bach2−/− splenic B cells led to abnormally elevated Blimp-1 and XBP-1 mRNA and to repression of AID mRNA (95). Subsequently, a binding site for the Bach2MalK heterodimer in the prdm1 promoter region was identiﬁed, and binding of MalK in vivo was demonstrated by ChIP (103), providing additional evidence that Bach2 directly represses prdm1. Repression of plasmacytic differentiation and Blimp-1 expression. There are several mechanisms that prevent premature plasmacytic differentiation by repressing transcription factors required by plasma cells, and most of these mechanisms involve Blimp-1 (Figure 3). Of the three prdm1 repressors discussed above, Bach2 and Pax5 are expressed in preB and naive B cells (96), as well as in GC B cells. Additionally, Pax5 has been reported to repress xbp1 (59, 61), although there are data to the contrary (60). Finally, microphthalmiaassociated transcription factor MITF, which is most abundant in naive B cells, represses IRF4 mRNA by an undetermined mechanism. B cells lacking MITF have spontaneous plasmacytic differentiation (106), providing direct evidence that MITF is important for normal control of terminal differentiation.

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Because IRF4 is a direct activator of prdm1 (93), its repression indirectly represses prdm1. Expression of Bcl-6 protein is highly restricted to GC B cells (33, 104, 105). Thus, Bcl-6 appears to add a second layer of prdm1 repression speciﬁcally at the GC stage. Thus, Bach2, MITF, and Pax5 repress plasmacytic differentiation in naive B cells and at developmental stages before and during the GC reaction; in the GC, Bcl-6 is present to further ensure that plasmacytic differentiation is inhibited. Interestingly, when human memory B cells develop in vitro from tonsillar centrocytes, Bach2, Pax5, and Bcl-6 mRNA levels decrease signiﬁcantly, suggesting that one reason memory cells can differentiate into plasma cells quickly is because they lack these repressors (27). Induction and establishment of plasmacytic differentiation. Current understanding of Blimp-1 regulators, as detailed above, shows that Blimp-1 expression and the plasmacytic differentiation that ensues require a combination of two kinds of events: (a) removal of inhibitors Bcl-6, Pax5, MITF, and Bach2 and (b) induction of activators including IRF4, AP-1, NF-κB, and STAT3 (Figure 4). Interestingly, all three general signals necessary for Ig secretion—antigen, TLR ligands, and cytokines—provide one or more of these signals. The requirement both to remove inhibitors and to supply activators may help explain why LPS is such a potent immunogen because TLR4 activates both MyD88-dependent and -independent pathways (107). The requirement may also explain why activated NF-κB present in B cells at earlier developmental stages or BCR signals alone are unable to induce Blimp-1 mRNA. How are the inhibitors removed? This is a critical question that relates directly to the question of how exit of plasmablasts from the GC is regulated. Unfortunately, information is far from complete, but dissecting the regulation of Blimp-1 in GC and post-GC B cells provides one way to ap-

B cell

Plasma cell

BCR PI3K

MITF

NFAT

irf4

IL-21 FoxO Mad1

Bcl-6

STAT3

prdm1

NF-κB TLR

Pax5

Bach2

xbp1

Provide activation

Remove inhibition Figure 4 Two signals necessary for plasma cell differentiation. Arrows and bars indicate positive or negative regulation, and the orange star indicates protein degradation (27, 29, 30, 47, 78, 93, 99, 100, 108–112).

proach the question. Recent studies establish that high-afﬁnity BCRs direct GC B cells to a plasmacytic fate (108, 109), so models must incorporate BCR signals. Studies using in vitro cultures of human tonsillar centrocytes that develop into plasma cells in response to IL-10 (27) show that levels of Bcl6, Pax5, and Bach2 mRNA drop signiﬁcantly before large increases in Blimp-1 mRNA occur. There is little information available concerning the mechanisms responsible for loss of Pax5 and Bach2 expression, although decreased Pax5 was observed in preplasmablasts prior to Blimp-1 induction (110). Several mechanisms remove Bcl-6 from GC B cells. BCR-dependent activation of mitogen-activated protein kinase (MAPK) leads to phosphorylation of Bcl-6, which targets Bcl-6 for ubiquitin-dependent degradation (78). Because Bcl-6 is abundant in GC B cells, this mechanism may only be operative in response to strong BCR signals or in combination with other signals, and it may initiate exit of B cells from the GC, as a decrease in Bcl-6 activity is required for both post-GC plasma cell and memory B cell differentiation www.annualreviews.org • Blimp-1 in T and B Lymphocytes

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(27). Acetylation inactivates the ability of Bcl6 to repress transcription, and Bcl-6 is acetylated in GC B cells, although there is no knowledge of if or how acetylation of Bcl-6 may be regulated in GC B cells (111). The E box factor Mad1 is elevated in plasma cells and directly represses Bcl-6 (112). Finally, because FoxO3A activates Bcl-6 (113) and Akt inactivates FoxO transcription factors (114), strong BCR signals, activating PI3K and Akt and inactivating FoxO, lead to a reduction in Bcl-6 and an increase in Blimp-1 in B cells (102). Interestingly, Bcl-6 is not abundant in marginal zone B cells or B1 B cells, and neither Bach2, Pax5, nor Bcl-6 mRNA are found in human memory B cells formed in vitro (27). Thus, in these settings, induction of Blimp-1 can occur more rapidly when appropriate activators are induced. Mice lacking OBF-1 have impaired plasmacytic differentiation in response to certain kinds of stimulation (115). Without OBF-1, bcl6 and pax5 are not repressed, and prdm1 is not induced when B cells are activated in vitro with CD40L and IL-4. However, stimulation by LPS is normal in the absence of OBF-1 (115). The mechanism requiring OBF-1 for induction of Blimp-1 is not clear, although ChIP assays provided no evidence for direct binding of octamer-OBF-1 complexes to prdm1. OBF-1 may indeed be needed to remove one or more repressors such as Bcl-6 in response to certain signals. Many mechanisms are likely to induce or activate activators of prdm1. TLR signals, activating NF-κB, induce Blimp-1 in GC B cells (27) and B-1 B cells (29, 30) and probably in naive and marginal zone B cells present in splenic B cell preparations following treatment with LPS (47). Vav signaling is required downstream of TLR signals to induce Blimp-1 mRNA and to form Igsecreting plasma cells (116). Mature marginal zone B cells, stimulated with TLR ligands, proliferate and express IRF4 but do not express Blimp-1 when they lack Vav proteins, demonstrating that IRF4 alone is not sufﬁ-

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cient to induce Blimp-1. NFAT (nuclear factor of activated T cells) factors are important in B cells, and calcineurin/NFAT-dependent induction of IRF4 is important for post-GC plasmacytic development (99). Because IRF4 is probably a direct activator of prdm1 (93), it follows that NFAT signaling indirectly induces Blimp-1 mRNA in post-GC plasma cells. The role of BCR in regulating prdm1 is interesting and, given rather fragmentary data, certainly deserves further study. There is no evidence that BCR signals alone are sufﬁcient to induce prdm1, and in combination with TLR signals, BCR signals actually block plasma cell formation in vitro (117) and Blimp-1 expression (47). However, NFAT, an indirect activator of prdm1 (99), and AP-1, a direct activator (90), are activated by BCR signals (118). Furthermore, in anergic B cells that do not induce Blimp-1 or develop into plasma cells, NFAT signaling downstream of BCR is uncoupled, whereas extracellular signal regulated kinase (ERK) signaling downstream of BCR is maintained (119). BCR signaling via Ras and MAPK kinase (MEK) to ERK integrates BCR and cytokine signals. ERK activation, via continuous BCR signaling, inhibits Blimp-1 induction, and IL-2 and IL-5 inactivate ERK via induction of the DUSP 5 phosphatase (120). The mechanism for ERK-dependent inhibition of Blimp-1 was not identiﬁed, but it might involve Bcl-6 or its cofactors. It is also interesting that in mice lacking Bruton’s tyrosine kinase, TI-2 activation of B cells initially induces Blimp-1, but this is not sustained, and the cells do not progress to become plasmablasts (121). Once Blimp-1 is expressed, it is sufﬁcient to cause plasmacytic differentiation. Blimp-1 also speciﬁcally represses genes encoding two critical transcription factors required for GC B cells—pax5 (53) and bcl6 (90, 100). Thus, the plasma cell program is enforced, and earlier stages of B cell development are inhibited by Blimp-1.

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Blimp-1 in B Cell Malignancies

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Recent studies provide evidence that abnormal regulation or abnormal activity of Blimp1 may be one causal event in some B cell malignancies. If Blimp-1 acts as a tumor suppressor, lack of its activity could be important in some forms of lymphoma. Alternatively, its ability to enforce a plasma cell phenotype may be important in plasma cell tumors.

Diffuse large B cell lymphoma. When an early study identiﬁed myc as a direct target of Blimp-1-dependent transcriptional repression (51), it was logical to speculate that Blimp-1 might function as a tumor suppressor, especially given the prominent role of dysregulated c-Myc expression in B cell tumors (122). Interestingly, however, aged mice with conditional deletion of prdm1 in the B cell lineage do not develop B cell tumors spontaneously (M. Shapiro-Shelef & K. Calame, unpublished), indicating that loss of Blimp-1 alone is not sufﬁcient for B lymphomagenesis. Nevertheless, there is growing evidence that deletion or mutation of PRDM1 is frequently found in a subset of diffuse large B cell lymphoma (DLBCL), providing evidence that it may indeed function as a tumor suppressor (123–125). Similar PRDM1 mutations were not found in B or T or myeloid leukemias or in the 467 common carcinomas examined (126). In the most thorough study on DLBCL, PRDM1 was inactivated by structural alterations in 24% (8 out of 34) activated B cell–like diffuse large cell lymphomas but not in GC B cell–like (n = 0/37) or unclassiﬁed (n = 0/21) DLBCLs (125). However, a subset of DLBCL, which expressed Blimp-1, lacked detectable plasmablastic or immunoblastic changes and displayed more aggressive behavior, with a shorter failure-free survival (127). Thus, further studies are warranted to determine how expression and lack of expression of Blimp-1 affect the formation and properties of different subsets of DLBCL.

Murine plasmacytoma and human multiple myeloma. Malignant plasma cells in both mice (plasmacytoma) and humans (multiple myeloma) express abundant levels of Blimp-1, which likely reﬂects their differentiated state as plasma cells and the fact that they usually secrete Ig. Blimp-1 is also present in a subset of DLBCL but not in marginal zone lymphomas or chronic lymphocytic leukemia (32), and abortive plasmacytic differentiation in some Hodgkin and Reed Sternberg cells is indicated by the presence of Blimp-1 (128). Apparently, the ability of Blimp-1 to repress myc is overcome in these tumors by oncogenic changes, such as chromosomal translocation, that activate and dysregulate myc transcription or the dominant activity of other oncoproteins. [However, Blimp-1 is apparently able to repress myc in other tumors, such as myeloid tumors, where it is induced by IRF8 (73).] There is active interest in determining how forcing expression of Blimp-1 or, alternatively, blocking its activity or knocking down its expression might affect the growth and phenotypic properties of myeloma or other B cell tumors. In a recent study, treatment of myeloma cell lines with 2-methoxyestradiol, which suppresses their growth and induces apoptosis, also led to an elevation of Blimp1 and XBP-1 and repressed MYC and PAX5 (129). In some myeloma cell lines, the proportion of the beta form of Blimp-1 is elevated in comparison with normal plasma cells (62, 130), which might decrease the overall activity of Blimp-1, but the functional consequences of elevated beta form have not been elucidated clearly. Assuming Blimp-1 is necessary for the Ig secretion program in myeloma, as it is in normal plasma cells, one reasonable prediction is that blocking Blimp-1 in the tumors might block Ig secretion. Whether this would cause apoptosis owing to endoplasmic reticulum stress as observed upon administration of protease inhibitors (131) or to failure of other survival mechanisms (39) or dedifferentiation (101) remains to be determined. www.annualreviews.org • Blimp-1 in T and B Lymphocytes

DLBCL: diffuse large B cell lymphoma

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BLIMP-1 IN T CELL BIOLOGY

LCMV: lymphocytic choriomeningitis virus

The understanding that Blimp-1 is also important in the T lymphocyte lineage emerged only within the past 2–3 years. Thus, this work is at an earlier stage. Although the overall effect of lacking Blimp-1 T cells is fatal, owing to spontaneous inﬂammatory disease, the cellular and molecular effects are more subtle and complex than the effects in B cells.

Expression of Blimp-1 in T Cells Blimp-1 protein is expressed in human (32, 132) and mouse (35, 74) T cells, and in both species Blimp-1 levels are signiﬁcantly higher in antigen-experienced cells. Indeed, the levels of Blimp-1 mRNA and protein in antigen-experienced murine and human T

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Regulatory T (Treg) cells: those CD4+ CD25+ regulatory T cells, both constitutive and inducible, that express FoxP3 and suppress T cell responses

Activation and differentiation status Figure 5 Blimp-1 expression in T cells. Blimp-1 mRNA (represented by arbitrary units on the y-axis) is present in low levels in naive CD4+ and CD8+ T cells but is abundant in ex vivo isolated antigen-experienced (CD62LLo CD44Hi and CD62LHi CD44Hi ) cells. Memory CD8+ T cells and some of the memory CD4+ T cells are contained in the CD62LHi CD44Hi subpopulation, which expresses slightly lower levels of Blimp-1 mRNA than the CD62LLo CD44Hi cells. In vitro stimulation (not depicted) of the naive T cells results in a slow increase in Blimp-1 expression, reaching levels similar to that observed in the ex vivo isolated antigen-experienced cells. Ex vivo isolated CD4+ CD25+ naturally occurring, regulatory T (Treg) cells express Blimp-1 transcripts at the same levels as effector nonregulatory CD4+ T cells (35, 76, 132). 148

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cells are similar to those observed in cultures where plasma cells are generated in vitro by LPS treatment of splenic B cells (76, 132). Although steady-state Blimp-1 mRNA is present in low levels in mouse thymocytes (76, 133) and naive CD4+ and CD8+ T cells (76, 132), levels increase 20–40 times upon stimulation with α-CD3, α-CD28, and IL-2 (35, 76, 132). Induction of Blimp-1 mRNA and protein in vitro is slow, with maximum levels achieved 3–5 days poststimulation (76, 132). T cell receptor (TCR) restimulation in the presence of α-CD28 leads to a further increase in Blimp-1 mRNA expression (132; L. Cimmino & K. Calame unpublished). Blimp-1 mRNA is also found in high levels in ex vivo puriﬁed CD4+ or CD8+ effector/memory (CD62LLo /CD44Hi ) and memory phenotype (CD62LHi /CD44Hi ) T cells, with the ﬁrst subpopulation showing slightly more abundant Blimp-1 transcripts (Figure 5) (76). Finally, Blimp-1 transcripts are observed in high levels in ex vivo puriﬁed CD4+ CD25+ CD62LHi , which are mostly composed of Foxp3+ cells, known as naturally occurring, regulatory T (Treg) cells (35, 76). Prdm1-GFP knockin (Figure 1c) reporter mice (24) have been used to monitor Blimp-1 expression in T cells (24). GFP expression is not detected in the thymus or in naive peripheral T cells, but it was observed in in vitro– activated T cells, in vivo antigen-experienced CD4+ and CD8+ peripheral T cells, and in CD4+ CD25+ CD62LHi cells, which are primarily Foxp3+ Treg cells (24). Thus, despite the limitations of using GFP as a reporter for Blimp-1 expression, these data are consistent with observations described above showing that within the T cell lineage Blimp-1 is found in highest levels in antigen-experienced cells and in naturally occurring Treg cells. In agreement with these patterns, Blimp-1 is upregulated in vivo in CD8+ antigen-speciﬁc T cells upon infection with herpes simplex virus (HSV) (35) and with lymphocytic choriomeningitis virus (LCMV) (R. Rutishauser & S. Kaech, unpublished).

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Although Blimp-1 expression is consistently elevated in antigen-experienced CD4+ T cells, levels vary: Naive CD4+ T cells stimulated under Th2 conditions (IL-2, IL-4, and anti-IFN-γ) express higher levels of Blimp1 steady-state mRNA than do cells stimulated under Th1 (IL-2, IL-12, and anti-IL4) or nonpolarizing conditions (IL-2) (132; L. Cimmino & K. Calame, unpublished). Indeed, Blimp-1 mRNA expression seems to be repressed as cells differentiate under Th1 conditions because at the end of a 6-day culture, Th1 cells express less Blimp-1 steadystate mRNA than do cells stimulated under nonpolarizing conditions (L. Cimmino & K. Calame, unpublished). However, differential expression of Blimp-1 in Th subsets was not observed in the Blimp-1-GFP knockin reporter mice (35). Nonetheless, immunoblotting reveals that Blimp-1 protein is higher in Th2 than in Th1 cells after two rounds of polarization in vitro (L. Cimmino & K. Calame, unpublished). Importantly, these differences are observed before any restimulation at the end of the differentiation rounds, suggesting that the differential levels of Blimp-1 expression are maintained in resting Th1 and Th2 cells and might be related to the maintenance of the respective T helper phenotype.

Blimp-1 and T Cell Development The pattern of Blimp-1 mRNA expression in T cells—low in thymocytes and naive T cells, high in antigen-experienced T cells—is consistent with the idea that Blimp-1 is more important for T cell function than development; however, conditional deletion of Blimp1 in T cells (prdm1F/F Lck-Cre mice) resulted in signiﬁcant alterations in the thymus (76). The prdm1F/F Lck-Cre mice have a threefold decrease in the numbers of total thymocytes (76), which can be attributed to a marked reduction in the absolute numbers of CD4+ and CD8+ double-positive (DP) as well as singlepositive (SP) CD4+ or CD8+ thymocytes. This defect is observed as early as 4 weeks after birth and progresses with age. There

is no signiﬁcant alteration in the number of double-negative (DN) and γδ thymocytes and no signiﬁcant differences in the distribution of the various DN thymocyte subsets between control and prdm1F/F Lck-Cre mice, as determined by CD25 and CD44 expression. Also, developmental stage-speciﬁc surface markers (CD5, TCRβ, HSA, CD24, and CD69) and short-term BrdU incorporation are indistinguishable between control and prdm1F/F LckCre mice, suggesting that the maturation and proliferation of each subset are preserved in the absence of Blimp-1. However, DP thymocytes are more susceptible to cell death, suggesting that Blimp-1 may regulate survival during negative selection of DP thymocytes (76). Nevertheless, the possibility that decreased survival of the prdm1F/F Lck-Cre mice DP thymocytes is secondary to the immune activation in the periphery cannot be excluded, and more work is required to clarify the role of Blimp-1 in thymocyte development.

Functions of Blimp-1 in T Cells Mice lacking Blimp-1 in their T cells have provided valuable information about the role(s) of Blimp-1 in the T lineage. Although these CKO mice were created in different ways in two laboratories, most of the observations from the two groups were consistent. However, one key difference is the possible role Blimp-1 may play in CD4+ Treg cells. Spontaneous inflammatory disease in mice with T cells lacking Blimp-1. Two strategies were used to generate mice with Blimp1-deﬁcient T cells. One group crossed the prdm1F/F mice (Figure 1d ) with proximal Lck-Cre or CD4-Cre transgenic mice, generating mice in which Blimp-1 deletion was speciﬁcally restricted to T lymphocytes (76; G. Martins & K. Calame, unpublished). The other group used fetal liver cells from prdm1gfp/gfp (Figure 1c) embryos to reconstitute the hematopoietic compartment of Rag-1-deﬁcient mice, resulting in the www.annualreviews.org • Blimp-1 in T and B Lymphocytes

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generation of chimeric mice in which cells from the myeloid and lymphoid lineages were prdm1gfp/gfp and Blimp-1 deﬁcient (35). Despite the differences in the two approaches, both studies revealed that lack of Blimp-1 results in profound alterations of T cell function and homeostasis, culminating with the spontaneous development of inﬂammatory disease (35, 76). Whereas in the ﬁrst model (T cell–speciﬁc deletion of Blimp-1) the inﬂammatory disease was concentrated in the colon (35, 76), mice reconstituted with prdm1gfp/gfp fetal liver cells also had inﬂammation in other organs, including lungs and liver (35). Although the nature of this difference has not yet been systematically addressed, it may simply reﬂect the different approaches used (deletion of Blimp-1 in T cells only versus deletion of Blimp-1 in the lymphoid and myeloid compartments). If so, one would predict that Blimp-1 might be important for regulating the function of hematopoietic cells other than T and B lymphocytes, a possibility that remains to be investigated. It is also possible that the difference in severity of the inﬂammatory disease observed in the two models is related to differences in the genetic background: The prdm1F/F LckCre mice were of mixed 129xC57BL/6 background, whereas the prdm1gfp/gfp mice where backcrossed into C57BL/6. In other models of spontaneous development of inﬂammatory diseases (134) the severity and target organs might vary in different genetic backgrounds. Finally, differences in the animal facilities used to house the different Blimp-1 CKOs could also explain differences in the inﬂammatory phenotype. The cellular and molecular mechanisms associated with development of colitis in mice with Blimp-1-deﬁcient T cells are not completely understood. T cell dysfunction is associated with colitis in many murine models, which are generally characterized by deregulated activation of effector T cells with excessive production of IFN-γ and decreased production of IL-10 and/or defective devel-

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opment/function of Tregs (135, 136). Current data indicate several probable causes of colitis development in the prdm1F/F Lck-Cre mice. Blimp-1-deﬁcient CD4+ T cells produce increased amounts of IFN-γ, and their production of IL-10 is signiﬁcantly impaired (see below), indicating deregulated T cell activation and/or biased Th1 differentiation. There is also partial impairment of Treg function in the absence of Blimp-1, discussed below. Interestingly, a small population of CD4+ T cells with colitogenic potential can be found in normal mice (137). Apparently, these cells can be driven into the antigen-experienced pool by the presence of commensal bacteria but are normally kept under control by Treg cells in an IL-10-dependent manner (137). Hence, it would be interesting to know if the antigen-experienced cells that accumulate in mice with T cell–speciﬁc deletion of Blimp-1 contain these potentially colitogenic cells. If that is indeed the case, the defective production of IL-10 by Blimp-1-deﬁcient Treg cells (see below) could provide a mechanistic explanation for their accumulation. Nonetheless, direct evidence linking the accumulation of the antigen-experienced cells with the spontaneous development of colitis in the T cell– speciﬁc Blimp-deﬁcient mice is still lacking, and it is not known if the defect in IL-10 production is associated with colitis in these mice. T cell homeostasis and attenuation of IL2 production. The elevated expression of Blimp-1 in peripheral antigen-experienced T cells and the substantial accumulation of these cells in both prdm1F/F Lck-Cre and prdm1gfp/gfp mice, despite the different genetic backgrounds of the mice, provide strong evidence that Blimp-1 is important in T cell homeostasis. Evidence from studies in which Blimp-1 is ectopically expressed (74, 132) or deleted from T cells (35, 76) indicates that Blimp1 regulates responsiveness and homeostasis of peripheral T cells by attenuating both proliferation and survival. Blimp-1-deﬁcient antigen-experienced CD4+ T cells are less susceptible to apoptosis upon restimulation

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in vitro (35; G. Martins & K. Calame, unpublished), and in vitro–differentiated Blimp1-deﬁcient Th1 cells are more resistant to cell death caused by cytokine withdrawal (35), suggesting that Blimp-1 normally promotes apoptosis of effector T cells. In addition, it appears that Blimp-1 attenuates proliferation induced upon TCR stimulation of naive T cells. Ectopic expression of Blimp-1 resulted in decreased proliferation of CD4+ T cells (132), and naive CD4+ T cells from the prdm1F/F Lck-Cre mice proliferate better than wild-type cells when stimulated in suboptimal conditions (76). Stimulation in optimal conditions, including CD28-mediated costimulation and addition of exogenous IL-2, abrogates the proliferation differences between Blimp-1-sufﬁcient and -deﬁcient cells (35, 76), suggesting that regulation of proliferation by Blimp-1 is limited to conditions in which costimulation is absent or weak, such as presentation of a self-antigen in the periphery where the normal result is anergy of the responder T cell (138, 139). Thus, lack of Blimp-1 may cause increased responsiveness to self-antigens. Furthermore, one may speculate that Blimp-1 normally attenuates tonic signaling through the TCR (140). Both possibilities remain to be experimentally evaluated. Blimp-1-deﬁcient CD8+ T cells seem to respond abnormally to exogenous antigens, as indicated by the increased accumulation of antigen-speciﬁc CD8+ T cells after infection of prdm1gfp/gfp mice with HSV (35). Also, in a system where P14 TCR transgenic, prdm1deﬁcient or control naive CD8+ T cells were transferred to wild-type recipient mice and the recipients infected with LCMV, there was signiﬁcantly more accumulation of Blimp-1deﬁcient cells (R. Rutishauser & S. Kaech, unpublished). One key mechanism by which Blimp-1 modulates T cell responsiveness is likely to be through repression of IL-2. IL-2, acting in both autocrine and paracrine manners, regulates the initial expansion of naive T cells upon TCR stimulation in vitro (141, 142) and

possibly in vivo. Paracrine IL-2 is also important for the in vivo survival of Tregs, which in turn regulate the proliferation of peripheral T cells (142, 143). Multiple observations support the idea that Blimp-1 represses IL-2. Production of IL-2 is elevated in naive Blimp1-deﬁcient CD4+ T cells following activation in vitro (76), and IL-2 steady-state mRNA levels in naive Blimp-1-deﬁcient CD4+ T cells are elevated relative to controls both before and following activation in vitro (G. Martins & K. Calame, unpublished). Ectopic expression of Blimp-1 signiﬁcantly decreases IL-2 production in wild-type CD4+ and CD8+ T cells, and IL-2 production declines when Blimp-1 levels are highest after TCR stimulation (74, 132). Moreover, ectopic expression of Blimp-1 inhibited the expression of an IL-2 promoter-GFP reporter (74) in vitro. However, the relationship between IL-2 and Blimp-1 is additionally complicated and interesting. As detailed below, IL-2 signaling strongly induces prdm1 transcription (74). Thus, T cell activation induces il2 transcription, IL-2 signaling induces prdm1 transcription, and Blimp-1 feeds back to repress il2 transcription (Figure 6). Blimp-1 therefore appears to be a key component of the pathway by which IL-2 downregulates its own expression during later phases of T cell activation (144) (Figure 6). Together these data suggest a model (Figure 7) in which Blimp-1 controls T cell function in two different stages: At the initial activation stage it may regulate responsiveness by attenuating IL-2 production and proliferation, and, subsequently, it may enhance the elimination of effector cells via apoptosis. Additionally, Blimp-1 might be required for the proper function of Treg cells, another pathway by which Blimp-1 regulates T cell responses. Thus, Blimp-1-deﬁcient T cells would proliferate more when differentiating into effectors, and the effectors would survive better. This could help to explain the abnormal accumulation of antigen-experienced cells observed in the Blimp-1-deﬁcient mice. www.annualreviews.org • Blimp-1 in T and B Lymphocytes

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Figure 6 The regulatory feedback loop involving Blimp-1 and IL-2 in T cells. (1) Antigen and costimulation induce il2 (pink rectangle) transcription via NFAT, AP-1, and NF-κB. (2) IL-2 secreted from the cell, in either an autocrine or paracrine manner, binds the IL-2 receptor. (3) IL-2 signaling, probably via activated STAT3 or STAT5, induces prdm1 transcription and Blimp-1 protein (74). Blimp-1 represses il2 directly and also represses fos, a component of AP-1 which activates il2 (4) (G. Martins & K. Calame, unpublished).

T helper differentiation. Blimp-1 appears to play a role in Th2 cells by repressing Th1 genes (Figure 8). Deletion of Blimp1 in T cells causes increased production of IFN-γ and decreased production of IL-10 (35, 76). Production of IL-4 was decreased in one model of T cell deletion of Blimp-1 (35) but not signiﬁcantly altered in another (76). The increased production of IFN-γ by Blimp-1-deﬁcient T cells suggests that Blimp1 may attenuate Th1 differentiation. Consistent with that, Blimp-1 transcripts and protein are more abundant in Th2 than in Th1 cells differentiated in vitro (132; L. Cimmino & K. Calame, unpublished). Despite these observations, Blimp-1-deﬁcient CD4+ T cells can be polarized into Th1 or Th2 cells in vitro, and IL-4 production seems unaffected, although IFN-γ production is slightly increased in both Th1 and Th2 cells derived from Blimp-1deﬁcient mice (L. Cimmino & K. Calame, unpublished). It will be important to determine if Blimp-1-deﬁcient Th cells can still 152

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polarize normally in an in vivo immune response, where the factors directing Th polarization, such as strength of TCR stimulation and concentration of different cytokines, are more complex than in the in vitro cultures. Consistent with increased IFN-γ production, Blimp-1-deﬁcient T cells show increased levels of IFN-γ and Tbet steady-state mRNA (L. Cimmino & K. Calame, unpublished). Another important player in the regulation of Th differentiation by Blimp-1 appears to be Bcl-6. Blimp-1-deﬁcient T cells have increased Bcl-6 mRNA levels (L. Cimmino & K. Calame, unpublished), and Bcl-6 proteins can repress Th2 differentiation by interfering with the function of GATA-3 (145), a master regulator of Th2 differentiation (reviewed in 146), and by direct repression of il5 transcription (147). Thus, alleviating bcl6 repression could be one mechanism responsible for increased production of Th1 cytokines in Blimp-1-deﬁcient T cells. The idea that Blimp-1 might be required to inhibit Th1

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Proliferation and survival Figure 7 A model for Blimp-1 attenuation of proliferation and survival in CD4+ T cells. TCR stimulation of naive T cells induces IL-2 production and Blimp-1 expression. Blimp-1 attenuates T cell proliferation and survival following primary activation in several ways. (1) Blimp-1 attenuates induction of the il2 gene indirectly by repressing fos and directly by repressing il2. (2) Blimp-1 also represses transcription of bcl2a1 (although it is not known if this occurs directly or indirectly). By interfering with pathways 1 and 2, Blimp-1 might regulate proliferation and survival upon primary TCR stimulation. Pathway 2 could also remain operative in antigen-experienced cells at later stages of differentiation because Blimp-1-deﬁcient Th1 effector cells seem more resistant to cytokine deprivation cell death (35). Additionally, Blimp-1 might repress genes related to activation-induced cell death (AICD) induction (not shown). This latter pathway is more likely to be operative in antigen-experienced cells, at later stages of differentiation, as indicated by the phenotype of mice with Blimp-1-deﬁcient T cells (35, 76).

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Figure 8 A model for Blimp-1 attenuation of Th1 cell differentiation. Blimp-1 represses ifng directly and indirectly via repression of tbet. It also represses bcl6 and tbet directly and indirectly via repression of ifng (L. Cimmino and K. Calame, unpublished).

differentiation also ﬁts with the observation that ablation of Blimp-1 in T cells results in spontaneous development of colitis, which, in most of the cases, is mediated by IFNγ-producing cells (135, 136). However, to date there is no direct evidence that a biased Th1 response is a cause of the spontaneous colitis/inﬂammatory disease observed in mice with Blimp-1-deﬁcient T cells. Colitis development in the prdm1F/F LckCre mice might also be associated with the impaired production of IL-10. The decreased production of IL-10 by the Blimp1-deﬁcient cells is most likely a cell-intrinsic defect because it is observed in conditions in which IFN-γ is minimal or nonexistent, such as when puriﬁed CD4+ CD25+ Foxp3+ Treg cells are stimulated in vitro (G. Martins & K. Calame, unpublished). This ﬁnding also www.annualreviews.org • Blimp-1 in T and B Lymphocytes

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indicates that regulation of IL-10 production by Blimp-1 extends to the Treg cells, and this defect could be one of the triggers of the inﬂammatory disease observed in these mice, as discussed below.

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Differentiation of CD8+ T cells. The expression pattern of Blimp-1 is similar in CD4+ and CD8+ cells (35, 74, 76, 132), but little is known about the role of Blimp-1 in CD8 cells. As mentioned above, prdm1gfp/gfp mice are able to mount an efﬁcient CD8+ -mediated response to HSV infection, and in this model, Blimp-1-deﬁcient CD8+ T cells performed immediate effector functions, such as IFN-γ production and cytotoxicity, normally. Interestingly, there was signiﬁcant accumulation of antigen-speciﬁc CD8+ T cells by day 55 postinfection in the prdm1gfp/gfp mice. Kallies et al. (35) suggested that these were memory cells, but neither their phenotype nor function was evaluated. If these cells are indeed memory cells, these results would indicate that Blimp-1 attenuates the formation of memory cells or enhances their elimination. This would be consistent with the idea that Blimp-1 is important for the elimination of antigen-experienced cells and, in the CD8+ lineage, may limit the transition from effectors to memory cells. Blimp-1-deﬁcient antigen-speciﬁc cells also accumulate in vivo after infection with LCMV, in a transfer system where only the antigen-speciﬁc CD8+ T cells were Blimp-1 deﬁcient (R. Rutishauser & S. Kaech, personal communication). In this system, the accumulated cells have a memory precursor effectorlike phenotype (CD127Hi KLRG1Lo ) characterized by high levels of expression of the IL-7 receptor. Taken together, these results suggest that Blimp-1 might play an important role in the differentiation of CD8+ T cells, but this requires further investigation. It will be important to know if the memory cells that accumulate in the absence of Blimp-1 in both systems can actually provide protection upon infection when transferred to a naive recipient. Also, identiﬁcation of Blimp-1 154

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target genes in these systems could provide important insights into the molecular mechanisms regulating the differentiation of memory cells in the CD8+ lineage. In this context, an important player could be Bcl-6, which is a target of Blimp-1 in B and T cells and is known to support the survival of memory CD8+ T cells (148, 149). Treg cell function in vivo. The expression of high levels of Blimp-1 in Treg cells and the inﬂammatory disease associated with Blimp-1 ablation in T cells suggest a role for Blimp-1 in Treg cell differentiation and/or function. However, CD4+ CD25+ Foxp3+ Treg cells develop normally in the absence of Blimp-1 (35, 76). Indeed, prdm1gfp/gfp mice show increased frequency of splenic CD4+ /FoxP3+ cells (35), and peripheral CD4+ /Foxp3+ cells numbers increase with age in the prdm1F/F Lck-Cre mice (G. Martins & K. Calame, unpublished observations). It is important to determine if this expansion is due to increased IL-2 in the mice. Blimp-1-deﬁcient Treg cells perform normally in in vitro suppression assays and are also able to suppress colitis caused by homeostatic expansion of naive CD4+ T cells in Rag1-deﬁcient mice (35, 76). Nevertheless, Blimp-1-deﬁcient Treg cells cannot suppress acute colitis induced by dextran sodium sulfate (DSS) administration (76) and do not prevent inﬂammatory disease in Blimp-1-deﬁcient mice (35, 76), demonstrating a functional impairment. The reason that Blimp-1-deﬁcient Tregs control colitis in the RAG reconstitution system but not in the DSS system is not clear but may be related to the differential requirement for IL-10. IL-10 is associated with resistance to DSS-induced colitis (150, 151) but may not be as critical for preventing colitis induced by homeostatic proliferation of naive CD4+ cells (137). Available data indicate that Blimp-1 is required for IL-10 production by Treg cells (G. Martins & K. Calame, unpublished observations) as well as for nonregulatory CD4+ T cells (35, 76). Because Blimp-1 is a repressor,

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it is likely that Blimp-1 represses a gene that negatively regulates IL-10 expression, but this remains to be determined.

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Targets of Blimp-1 in T Cells RNA microarray analyses and gene expression studies using a quantitative reverse transcriptase PCR (qRT-PCR) have begun to reveal genes that are targets of Blimp-1 repression in T cells. This list includes bcl2a1, bcl6, il2, erg2, ifng, prdm1, tbet, and fos. Some of these targets (bcl2a1 and bcl6 ) are in common with B cells; others seem to be unique to T cells (Table 1). qRT-PCR studies, showing higher levels of mRNA in Blimp-1-deﬁcient T cells compared with controls, combined with ChIP assays, demonstrating binding of endogenous Blimp-1 in primary T cells, have shown that il2, fos, ifng, tbet, and bcl6 are direct targets of Blimp-1-dependent repression in T cells (G. Martins, L. Cimmino & K. Calame, unpublished). The evidence that Blimp-1 represses IL-2 production was discussed above, and it makes sense that il2 is a direct target of Blimp-1 repression. It seems likely that, in addition to direct repression of il2, repression of fos is important for the Blimp-1-dependent attenuation of T cell activation and IL-2 production. mRNA encoding Fos, a member of the AP1 family of transcription factors, is elevated in Blimp-1-deﬁcient CD4+ T cells following TCR stimulation. Fos is an important activator of IL-2 production upon TCR stimulation in T cells (152, 153). Thus, repression of il2 transcription by Blimp-1 in T cells seems to occur both directly and indirectly. Although not yet directly tested, Blimp-1 may repress il2 in Treg as well as in nonregulatory CD4+ T cells (Figure 9). Repression of ifng, tbet, and bcl6 is very likely to be important for Blimp-1-dependent repression of Th1 differentiation. Ifng is not only directly repressed by Blimp-1 but may also be indirectly repressed by suppression of tbet. In turn, decreased IFN-γ production would decrease expression of both tbet

and bcl6. In yet another example of redundant regulation, Blimp-1 also directly represses both of these genes. Thus, Blimp-1 is part of a complicated network of regulation that inhibits Th1 differentiation (Figure 8). However, the functional importance of this aspect of Blimp-1’s activity remains largely untested. An interesting common target gene for Blimp-1 in both B and T cells is bcl2a1, encoding A1, an antiapoptotic member from the Bcl-2 family of proteins (reviewed in Reference 154). A1 (a and b) are highly expressed by Blimp-1-deﬁcient cells in microarray studies comparing in vitro stimulated CD4+ T cells from control and prdm1F/F Lck-Cre mice (G. Martins & K. Calame, unpublished). Accordingly, A1 transcripts, measured by qRTPCR, are more abundant in Blimp-1-deﬁcient CD4+ T cells stimulated in vitro. In B cells, bcl2a1 is repressed by Blimp-1 (50), and enforced expression of A1 rescued Blimp-1induced cell death (155). In T cells, A1 is expressed upon TCR stimulation (156), and it may play a role in protecting cells from cytokine deprivation–induced cell death (157). A CD2-driven bcl2a1 transgenic mouse showed an increased number of peripheral T cells, but their activation status was not evaluated. T cells from these transgenic mice also show decreased death upon stimulation with T cell mitogens (157). Similar to other Bcl-2 family members, A1 does not seem to play any nonredundant role in protecting T cells from activation-induced cell death (AICD) (157). This is consistent with the fact that AICD, as assayed following in vitro activation of naive CD4+ T cells, was unaltered in CD4+ T cells from the prdm1f /f mice (76). Nonetheless, Blimp-1-deﬁcient in vitro–generated CD4+ effector T cells are more resistant to cytokine withdrawal–induced cell death (76; G. Martins & K. Calame, unpublished). Thus, derepression of bcl2a1 in the Blimp-1deﬁcient T cells might contribute to the accumulation of the antigen-experienced cells in the periphery of the prdm1f /f LckCre mice. Sequence inspections reveal the www.annualreviews.org • Blimp-1 in T and B Lymphocytes

AICD: activation-induced cell death

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IL-2 IL-2 IL-2 IL-2R

Foxp3

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NFAT AP-1 (Fos)

Blimp-1

STAT3 and STAT5

pdrm1

il2 il10 Figure 9

A model for Blimp-1 in Treg cells. Blimp-1 is expressed in high levels in Treg cells, induced by Foxp3 directly and probably also by IL-2, to which Treg cells are highly responsive. Blimp-1 could promote IL-10 expression by repressing a factor that normally inhibits its expression. IL-2 production is normally repressed in Treg cells by Foxp3, which interferes with NFAT induction of the il2 gene. Direct or indirect repression of il2 by Blimp-1 may also occur, as discussed in the text. Dotted lines represent regulation that is predicted based on ﬁndings in nonregulatory T cells but not yet conﬁrmed in Treg cells.

presence of Blimp-1 putative-binding sites in the promoter region of the murine bcl2a1 gene (G. Martins & K. Calame, unpublished observations). ChIP studies are needed to clarify if A1 is a direct target of Blimp-1 in T cells.

Regulation of Blimp-1 Expression in T Cells While several aspects of transcriptional regulation of prdm1 appear to be conserved in T cells and B cells, the overall regulatory pathways in T cells are distinct, and in T cells there is a clear role for antigen receptor stimulation in Blimp-1 induction. TCR and IL-2. Blimp-1 mRNA and proteins are induced during T cell activation. In vitro stimulation of naive CD4+ T cells with α-CD3, or α-CD3 and α-CD28, results in Blimp-1 mRNA induction, but maximum levels are induced when exogenous IL2 is administered together with α-CD3 and α-CD28 (132; L. Cimmino & K. Calame unpublished), suggesting that IL-2 greatly po156

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tentiates prdm1 induction upon TCR stimulation. Indeed, T cells lacking the β-chain of the IL-2 receptor (IL-2Rβ−/− ) express signiﬁcantly less Blimp-1 than wild-type cells upon TCR stimulation, and TCR stimulation in the presence of IL-2-neutralizing antibodies resulted in very low/undetectable Blimp1 protein (74). Therefore, a large portion, but not all, of prdm1 induction upon TCR stimulation seems to be secondary to induction of IL-2. Furthermore, IL-2 is required to maintain Blimp-1 expression after the initial in vitro activation of CD8+ T cells, a function that IL-15, another common gamma chain cytokine, was unable to perform (74). The importance of IL-2 for Blimp-1 expression is consistent with the late expression of Blimp-1 during T cell activation in vitro, and it is likely to have important regulatory functions because Blimp-1 represses il2, as discussed above. The components downstream of IL-2 signaling required for Blimp-1 induction remain to be identiﬁed, but STAT5 is probably an important element in this pathway.

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Other regulators. High levels of Blimp-1 in naturally occurring Foxp3+ Treg cells are likely to be caused by IL-2 signaling, which is critical for maintenance and proliferation of Treg cells in the periphery (158) (Figure 9). In addition, a genome-wide analysis of Foxp3 targets showed that prdm1 is directly activated by Foxp3 (159), adding further support to the idea that Blimp-1 is required for proper function of Treg cells. Extrapolating from the data in nonregulatory cells, we can surmise that Blimp-1 likely represses il2 transcription in Treg cells. This would provide another mechanism, in addition to competition with NFAT (160), by which Foxp3 represses il2 in Treg cells (Figure 9). Comparative analysis of gene expression proﬁles in Blimp-1-sufﬁcient and -deﬁcient Treg cells will be necessary to further elucidate Blimp-1’s mechanisms of action in Treg cells. The fact that Blimp-1 transcripts can be induced, albeit to low levels, in the IL-2Rβ−/− T cells upon TCR stimulation, indicates the existence of IL-2-independent pathways for Blimp-1 induction. Blimp-1 protein levels were increased in these cells upon TCR stimulation in the presence of IL-4 or IL-12, suggesting that these cytokines can also induce Blimp-1 expression (74). Interestingly, in these studies IL-4 seems to be a stronger inducer of Blimp-1 protein than IL-12 (74). In wild-type naive CD4+ T cells, Blimp-1 mRNA is also induced more strongly by IL4 than by IL-12, and in this same system, administration of exogenous IFN-γ reduces the induction of Blimp-1 transcripts upon TCR stimulation (L. Cimmino & K. Calame, unpublished). Thus, consistent with the expression patterns discussed above, TCR stimulation in Th2-promoting conditions (IL4) results in strong induction of Blimp-1, whereas Th1-promoting conditions (IL-12 and IFN-γ) result in weak Blimp-1 induction. IL-21 is expressed in T cells as well as B cells (160a), and although IL-21 induces prdm1 in B cells, there is currently no information as to whether IL-21 induces prdm1 in T cells. IL21 is expressed at high levels in inﬂammatory

conditions (161, 162), and if it induces Blimp1 in T cells, this could be a mechanism for downregulation of T cell function and containment of inﬂammation. In addition to being regulated by TCR stimulation and cytokines, preliminary data indicate that prdm1 is autoregulated by Blimp1. This was ﬁrst discovered in epidermal keratinocytes, where a Blimp-1 deﬁciency results in elevated amounts of transcripts from the deleted allele relative to transcripts from the wild-type allele. In this same system, ChIP assays show that Blimp-1 directly binds the prdm1 gene (162a), thus indicating that lack of Blimp-1 results in increased transcription of Blimp-1 mRNA. Similarly, in vitro– stimulated CD4+ T cells from prdm1 CKO mice show an increase in Blimp-1 mRNA as detected by real-time PCR using primers for the region upstream of the deleted region (L. Cimmino & K. Calame, unpublished). CD4+ CD25+ T cells from prdm1gfp/gfp mice also seem to have increased amounts of prdm1 mRNA, as demonstrated by semiquantitative RT-PCR (35). The physiological relevance of Blimp-1 autoregulation in T lymphocytes still remains to be addressed, but given the general action of Blimp-1 in attenuating T cell responses, it might be that autoregulation was selected to avoid inappropriate termination of immune responses. In this context, it would be interesting to learn if Blimp-1 autoregulation only occurs when Blimp-1 is expressed in very high levels or, in other words, if prdm1 itself is among the set of Blimp-1 targets that are susceptible to regulation only when Blimp-1 is available at very high levels.

OVERVIEW Interestingly, although mice lacking Blimp1 in their B cells are deﬁcient in antibodies, they survive well in pathogen-free conditions. In contrast, mice lacking Blimp-1 in T cells die of inﬂammatory disease within a few months. In B cells, Blimp-1 is clearly required and sufﬁcient for terminal differentiation of plasma cells. In T cells, its roles www.annualreviews.org • Blimp-1 in T and B Lymphocytes

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appear more complicated, probably reﬂecting the more complicated pathways of activation, effector and memory differentiation, and homeostatic maintenance in this lineage. Whereas a common role for Blimp-1 in both lineages may involve terminally differentiated functions, the apparent roles of Blimp-1 in memory T cells and in attenuating responses to activation appear to be unique and not found in the B lineage. While it is too early to understand fully how the molecular mechanisms of Blimp-1 action and regulation may differ in T and B cells, there appear to be interesting commonalities between the two lineages.

ﬁne-tuning of these regulatory networks. A ﬁnal common theme is that Blimp-1 is often involved in feedback loops. In both B and T cells, Blimp-1 represses bcl6, and Bcl-6 represses prdm1. Such mutual repression loops can help establish mutually exclusive states—a GC B cell versus a plasma cell in the B lineage and Th1 versus Th2 in the CD4+ T cell lineage. A different feedback loop is present in naive T cells where activation induces IL-2 (Figure 6), IL-2 in turn induces Blimp-1, and Blimp-1 then represses il2 itself as well as fos, and Fos is a mediator of activation. In this setting, Blimp-1 is apparently important in the attenuation of the immune response.

Common Themes

Remaining Questions

In spite of apparently serving quite different regulatory roles in the life of B and T lymphocytes, there are some aspects of Blimp-1-dependent regulation that are strikingly similar. First, the majority (8 of the 10) identiﬁed direct targets of Blimp-1 repression (Table 1) are themselves transcriptional regulators with their own, often extensive and important, sets of gene targets. Thus, Blimp-1 acts early in speciﬁc transcriptional regulatory cascades. For example, direct target Pax5 both activates genes required for B cell commitment (163) and function (164) and represses genes expressed in other hematopoietic lineages and genes expressed in plasma cells (60). A second common theme is the redundancy in how Blimp-1 regulates some genes. For example, in B cells, myc is repressed by Blimp-1 both directly and indirectly by repression of an important activator, E2F (50). AID mRNA is repressed indirectly by Blimp-1-dependent repression of two activators, Pax5 and E2A (50). In T cells, Blimp-1 represses il2 directly and indirectly by repressing an activator of IL2 transcription, fos. Tbet is repressed both directly and indirectly by repression of its strong inducer, IFN-γ. It seems reasonable to suggest that this redundancy reﬂects the importance of repressing given target genes and/or provides the possibility for multiple ways of

Important and interesting questions remain to be answered regarding Blimp-1’s role in lymphocytes in both a short-term and a longterm sense. In the short-term, a complete understanding of Blimp-1’s roles in various T cell subsets and in lymphoid malignancies remains to be established. It will be of interest to identify additional direct and indirect targets to understand more fully the regulatory pathways dependent on Blimp-1, especially in Treg cells and CD8+ T cells, which have not yet been studied in detail. Many groups are currently identifying various signals and transcriptional mechanisms that regulate Blimp-1, and given the importance of Blimp-1 action, understanding its regulation is a key issue. The functional importance of alternate promoter usage to generate the beta form needs further study, as does the possibility that Blimp-1 mRNA or protein may be regulated posttranscriptionally by degradation, covalent modiﬁcation or other mechanisms. The mechanism of action of Blimp-1 requires more study to identify corepressors that are required for its activity. Structural determination of a repressorcorepressor complex would provide molecular information that might aid in designing small-molecule inhibitors of Blimp-1. The possibility of competition between Blimp-1

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and IRF1 or IRF2 also deserves additional study. Finally, it is intriguing to speculate that, although Blimp-1 appears normal in some forms of common variable immune deﬁciency (CVID) (165), there could be mutations in prdm1 in other forms of CVID. In a long-term sense, as more information is obtained on the role, regulation, and mechanism of action of Blimp-1 in both normal and abnormal or malignant lymphocytes, it can be compared with the growing understanding of the roles of Blimp-1 in early embryogenesis in many species including Drosophila

(166), Xenopus (167), Zebraﬁsh (168–172), Fugu (173), and mouse (22, 36, 174) as well as with results of studies showing roles for Blimp-1 in other adult murine cell lineages such as sebocytes (175) and epidermal keratinocytes (162a). Hopefully, when more data are available, such comparisons will reveal information about how transcriptional regulatory pathways involving Blimp-1 have evolved and may provide insight into the currently unanswered question of why this single transcription factor plays such important and varied roles in multiple cell lineages.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We are grateful to members of the Calame laboratory and many colleagues for helpful discussions. This work was supported by RO1AI 50569 and RO1AI43576 to K.C.

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9. Describes cloning of murine Blimp-1 cDNA. Shows that forced expression of Blimp-1 is sufficient to drive BCL1 lymphoma cells to secrete Ig.

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10. Huang S. 1994. Blimp-1 is the murine homolog of the human transcriptional repressor PRDI-BF1. Cell 78:9 11. Tunyaplin C, Shapiro MA, Calame KL. 2000. Characterization of the B lymphocyteinduced maturation protein-1 (Blimp-1) gene, mRNA isoforms and basal promoter. Nucleic Acids Res. 28:4846–55 12. Mock BA, Liu L, LePaslier D, Huang S. 1996. The B-lymphocyte maturation promoting transcription factor BLIMP1/PRDI-BF1 maps to D6S447 on human chromosome 6q21-q22.1 and the syntenic region of mouse chromosome 10. Genomics 37:24– 28 13. Keller AD, Maniatis T. 1992. Only two of the ﬁve zinc ﬁngers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-speciﬁc DNA binding. Mol. Cell. Biol. 12:1940–49 14. Kuo TC, Calame KL. 2004. B lymphocyte-induced maturation protein (Blimp)-1, IFN regulatory factor (IRF)-1, and IRF-2 can bind to the same regulatory sites. J. Immunol. 173:5556–63 15. Canote R, Du Y, Carling T, Tian F, Peng Z, Huang S. 2002. The tumor suppressor gene RIZ in cancer gene therapy (review). Oncol. Rep. 9:57–60 16. Huang S, Shao G, Liu L. 1998. The PR domain of the Rb-binding zinc ﬁnger protein RIZ1 is a protein binding interface and is related to the SET domain functioning in chromatin-mediated gene expression. J. Biol. Chem. 273:15933–39 17. Ren B, Chee KJ, Kim TH, Maniatis T. 1999. PRDI-BF1/Blimp-1 repression is mediated by corepressors of the Groucho family of proteins. Genes Dev. 13:125– 37 18. Yu J, Angelin-Duclos C, Greenwood J, Liao J, Calame K. 2000. Transcriptional repression by Blimp-1 (PRDI-BF1) involves recruitment of histone deacetylase. Mol. Cell. Biol. 20:2592–603 19. Dillon SC, Zhang X, Trievel RC, Cheng X. 2005. The SET-domain protein superfamily: protein lysine methyltransferases. Genome Biol. 6:227 20. Derunes C, Briknarova K, Geng L, Li S, Gessner CR, et al. 2005. Characterization of the PR domain of RIZ1 histone methyltransferase. Biochem. Biophys. Res. Commun. 333:925–34 21. Gyory I, Wu J, Fejer G, Seto E, Wright KL. 2004. PRDI-BF1 recruits the histone H3 methyltransferase G9a in transcriptional silencing. Nat. Immunol. 5:299– 308 22. Ancelin K, Lange UC, Hajkova P, Schneider R, Bannister AJ, et al. 2006. Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nat. Cell Biol. 8:623–30 23. Angelin-Duclos C, Cattoretti G, Lin KI, Calame K. 2000. Commitment of B lymphocytes to a plasma cell fate is associated with Blimp-1 expression in vivo. J. Immunol. 165:5462–71 24. Kallies A, Hasbold J, Tarlinton DM, Dietrich W, Corcoran LM, et al. 2004. Plasma cell ontogeny deﬁned by quantitative changes in Blimp-1 expression. J. Exp. Med. 200:967– 77 25. Gonzalez-Garcia I, Ocana E, Jimenez-Gomez G, Campos-Caro A, Brieva JA. 2006. Immunization-induced perturbation of human blood plasma cell pool: progressive maturation, IL-6 responsiveness, and high PRDI-BF1/BLIMP1 expression are critical distinctions between antigen-speciﬁc and nonspeciﬁc plasma cells. J. Immunol. 176:4042– 50

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26. Blink EJ, Light A, Kallies A, Nutt SL, Hodgkin PD, Tarlinton DM. 2005. Early appearance of germinal center-derived memory B cells and plasma cells in blood after primary immunization. J. Exp. Med. 201:545–54 27. Kuo TC, Shaffer AL, Haddad J Jr, Choi YS, Staudt LM, Calame K. 2007. Repression of BCL-6 is required for the formation of human memory B cells in vitro. J. Exp. Med. 204:819–30 28. Tumang JR, Frances R, Yeo SG, Rothstein TL. 2005. Spontaneously Ig-secreting B-1 cells violate the accepted paradigm for expression of differentiation-associated transcription factors. J. Immunol. 174:3173–77 29. Fairfax KA, Corcoran LM, Pridans C, Huntington ND, Kallies A, et al. 2007. Different kinetics of Blimp-1 induction in B cell subsets revealed by reporter gene. J. Immunol. 178:4104–11 30. Savitsky D, Calame K. 2006. B-1 B lymphocytes require Blimp-1 for immunoglobulin secretion. J. Exp. Med. 203:2305–14 31. Lampert IA, Van Noorden S, Wotherspoon AC. 2005. Centrocytoid plasma cells of the germinal center. Appl. Immunohistochem. Mol. Morphol. 13:124–31 32. Cattoretti G, Angelin-Duclos C, Shaknovich R, Zhou H, Wang D, Alobeid B. 2005. PRDM1/Blimp-1 is expressed in human B-lymphocytes committed to the plasma cell lineage. J. Pathol. 206:76–86 33. Cattoretti G, Shaknovich R, Smith PM, Jack HM, Murty VV, Alobeid B. 2006. Stages of germinal center transit are deﬁned by B cell transcription factor coexpression and relative abundance. J. Immunol. 177:6930–39 34. Shapiro-Shelef M, Lin KI, McHeyzer-Williams LJ, Liao J, McHeyzer-Williams MG, Calame K. 2003. Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and preplasma memory B cells. Immunity 19:607– 20 35. Kallies A, Hawkins ED, Belz GT, Metcalf D, Hommel M, et al. 2006. Transcriptional repressor Blimp-1 is essential for T cell homeostasis and self-tolerance. Nat. Immunol. 7:466–74 36. Vincent SD, Dunn NR, Sciammas R, Shapiro-Shalef M, Davis MM, et al. 2005. The zinc ﬁnger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for speciﬁcation of primordial germ cells in the mouse. Development 132:1315–25 37. Driver DJ, McHeyzer-Williams LJ, Cool M, Stetson DB, McHeyzer-Williams MG. 2001. Development and maintenance of a B220− memory B cell compartment. J. Immunol. 167:1393–405 38. Bell J, Gray D. 2003. Antigen-capturing cells can masquerade as memory B cells. J. Exp. Med. 197:1233–44 39. Shapiro-Shelef M, Lin KI, Savitsky D, Liao J, Calame K. 2005. Blimp-1 is required for maintenance of long-lived plasma cells in the bone marrow. J. Exp. Med. 202:1471– 76 40. Radbruch A, Muehlinghaus G, Luger EO, Inamine A, Smith KG, et al. 2006. Competence and competition: the challenge of becoming a long-lived plasma cell. Nat. Rev. Immunol. 6:741–50 41. Minges Wols HA, Underhill GH, Kansas GS, Witte PL. 2002. The role of bone marrowderived stromal cells in the maintenance of plasma cell longevity. J. Immunol. 169:4213– 21 42. O’Connor BP, Raman VS, Erickson LD, Cook WJ, Weaver LK, et al. 2004. BCMA is essential for the survival of long-lived bone marrow plasma cells. J. Exp. Med. 199:91–98 www.annualreviews.org • Blimp-1 in T and B Lymphocytes

34. Describes mice with B cells lacking prdm1 and shows that Blimp-1 is necessary for formation of plasma cells and for Ig secretion. 35. One of two independent studies that reported a nonredundant role for Blimp-1 in T cell homeostasis and function.

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51. Identified myc as the first known target of Blimp-1dependent repression in B cells.

56. Showed that XBP-1 is required for plasma cell differentiation.

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74. Shows that IL-2 induces prdm1 and shows that Blimp-1 downregulates IL-2 production upon T cell activation. 76. One of two independent studies that reported a nonredundant role for Blimp-1 in T cell homeostasis and function.

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97. This paper used microarray analyses to identify direct targets of Bcl-6 and was the first to show that Bcl-6 directly represses prdm1.

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125. The most complete study showing that PRDM1 is inactivated in one subset of DLBCL, implying a role for Blimp-1 as a tumor suppressor.

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:133-169. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Contents

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Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack,1,2,3,4 James P. Scott-Browne,2 Shaodong Dai,1,2 Laurent Gapin,2 and John W. Kappler1,2,4,5,6 1

Howard Hughes Medical Institute, 2 Integrated Department of Immunology, National Jewish Medical and Research Center, 3 Department of Biochemistry and Molecular Genetics, 4 Department of Medicine, 5 Department of Pharmacology, and 6 Program in Biomolecular Structure, University of Colorado Denver Health Science Center, Denver, Colorado 80206; email: [email protected], [email protected], [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:171–203

Key Words

First published online as a Review in Advance on January 22, 2008

T cell receptor, major histocompatibility complex, evolution, conserved interactions, tolerance, selection

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090421 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0171$20.00

Abstract The rules for the conserved reaction of αβ T cell receptors (TCRs) with major histocompatibility complex (MHC) proteins plus peptides are poorly understood, probably because thymocytes bearing TCRs with the strongest MHC reactivity are lost by negative selection. Thus, only TCRs with an attenuated ability to react with MHC appear on mature T cells. Also, the interaction sites between TCRs and MHC may be inherently ﬂexible and hence difﬁcult to spot. We reevaluated contacts between TCRs and MHC in the solved structures of their complexes with these points in mind. Relatively conserved amino acids in the TCR complementarity-determining regions (CDR) 1 and CDR2 are often used to bind exposed areas of the MHC α-helices. These areas are exposed because of small amino acids that allow somewhat ﬂexible binding of the TCRs. The TCR amino acids involved are speciﬁc to families of variable (V) regions and to some extent different rules may govern the recognition of MHCI versus MHCII.

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HISTORICAL INTRODUCTION MHC: major histocompatibility complex

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TCR: αβ T cell receptor

172

Almost 50 years have elapsed since the discovery that the thymus is involved with immune responses. Shortly thereafter, cells derived from the thymus were found to improve the ability of B cells to make antibodies. This ﬁnding was followed by the discovery of the carrier effect, the observation that the antibody response to haptens requires simultaneous recognition of both the hapten and epitopes on the attached protein carrier. These ﬁndings dovetailed unexpectedly well to give rise to the idea that T cells created in the thymus react with one portion of the antigen and then help B cells produce antibody against a different determinant on the same molecule (1–4). This satisfying explanation for cell cooperation in immune responses left one key problem unresolved for immunologists: the major histocompatibility complex (MHC). Researchers studying the rejection of grafts and tumors (5, 6) recognized that the MHC represents a special case for immune responses. They found that differences at the MHC were recognized by the immune system extraordinarily rapidly. This phenomenon led Niels Jerne (7) to propose that lymphocyte receptors (by which he meant immunoglobulin proteins) had evolved to react with alleles of the MHC. In a groundbreaking theoretical paper, he suggested that lymphocytes developing in the thymus somatically mutate their evolutionarily generated receptors such that the receptors no longer react with the MHC of their host, but retain the ability to react well with the MHC of others. Meanwhile, others were beginning to realize that T and B cells do not react with the same determinants on antigens. Senyk and coworkers (8), for example, showed that after immunization with bovine glucagons, rabbits and guinea pigs make antibodies against a glucagon N-terminal peptide, whereas their lymphocytes divide in response to the Cterminal part of the protein. This seemed to be due to different reactivities of T and B cells because we showed (9) that mouse B and

Marrack et al.

T cells cross-react differently with red blood cells of different species. Furthermore, after immunization with sheep red blood cells, B cells can bind these cells directly and form rosettes but T cells cannot, even though the T cells are clearly able to respond to the antigen (9, 10). At approximately the same time, a number of experiments showed that T cells were geared toward recognition of antigens that were cell-associated rather than soluble. These kinds of experiments suggested that T and B cells recognize antigen in radically different ways and led to a lengthy search for the antigen receptor on T cells. The search was confused by reports that T cells secrete soluble factors that could bind antigen, by intermittent reports that T cells expressed immunoglobulin molecules, and by the idea that genes encoded by the MHC might be part of T cell antigen receptors. Conversely, a giant clue came from immune response (Ir) genes, which control the immune response against certain antigens and map to the MHC (reviewed in Reference 11). A shift in perspective led to the concept that resolved all these apparent contradictions, starting with the discovery by Zinkernagel & Doherty (12) that T cells must recognize not only antigen, but also products of the MHC. The issue of whether these were properties of a single receptor or two receptors, one speciﬁc for antigen and the other speciﬁc for MHC, was cleared up by the results of a dual TCR T cell hybrid experiment. This experiment showed that fused T cell hybrids able to react with two combinations of antigen and MHC (antigen a + MHC A and antigen b + MHC B) could not react with the mixed combinations (antigen a + MHC B and antigen b + MHC A) (13). Hence, a single receptor must react with a speciﬁc combination of antigen and MHC. This conclusion was conﬁrmed by the discovery of the polypeptides and genes of the αβ T cell receptor (TCR) (14–20). TCRs are now known to comprise two chains, α and β, each composed of variable (Vα, Nα, Jα; Vβ, Dβ, Nβ, Jβ) and constant elements with all but the N regions

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encoded by the germ line (21). Biochemical, cellular immunological, and later, crystallography experiments showed that TCRs react usually with a complicated ligand composed of the α helices of MHC proteins and peptides derived from antigen, bound to specially designed grooves of MHC molecules (22–26). Investigators have reported a number of structures of TCRs bound to their MHC/peptide ligands (27–46). TCRs bind MHC/peptide via their complementaritydetermining region (CDR) loops: CDR1α, CDR2α, CDR1β, and CDR2β, encoded in the germ line, and CDR3α and CDR3β, made up at least partially of non–germ line encoded residues and the C-terminal and N-terminal ends of Vα or Vβ and Jα or Dβ/Jβ, respectively. In these structures, the TCRs often lie on a diagonal above the face of MHC/peptide (Figure 1). The six CDR loops of TCRs contact this face, to varying degrees, usually with CDR1α and CDR2α over the α2 helix of MHC class I (MHCI) or the β helix of MHC class II (MHCII), and CDR1β and CDR2β over the α1 helix of MHCI or the α helix of MHCII. Conversely, the interactions of CDR3α and CDR3β usually focus on amino acids of the peptide. We now know that in addition to αβ T cells an entirely different set of T cells exists, which bear receptors made up of γ and δ chains. Adams and coworkers (47) solved the structure of a γδ TCR bound to its ligand. Although the interactions of such receptors with their ligands are of great interest, these receptors are not the subject of this review and are not discussed further.

EVIDENCE THAT TCRs MAY NOT HAVE BEEN SELECTED EVOLUTIONARILY TO REACT WITH MHC All these wonderful discoveries left unresolved Jerne’s (7) original hypothesis: the idea that what are now known to be TCRs have evolved to react with MHC proteins. In fact the notion fell out of favor after

a

β2

MHC α1

Peptide α1

β1

α3

β3

MHC β1 or α2

α2 β3

b β2

α2

β1 α3 α1 Peptide

MHC α1

MHC β1 or α2

Figure 1 The TCR usually contacts MHC/peptide on a diagonal, via the loops of its CDRs. (a) A schematic of the contacts between the CDR1–3 loops of the α and β chains of a TCR and a space-ﬁlling surface of MHC and peptide. The example shown here is a mouse TCR bound to the MHCII protein, IAb , engaged by the peptide, 3K (FEAQKAKANKAVD) [Protein Data Bank (pdb) XXX]. Shown are the α1/α region of MHC (cyan), the α2/β region of MHC (magenta), the peptide ( yellow), as well as the CDR loops, color coded and labeled in their corresponding colors. (b) An elevation perspective of the interactions shown in a.

the discovery of positive selection, the phenomenon by which developing thymocytes are picked out to survive on the basis of the reaction between their receptors and self MHC + self peptides in the thymus (48, 49). The existence of positive selection allowed

www.annualreviews.org • The Interaction Between αβ TCRs and MHC

CDR: complementarity determining region

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immunologists to account for the obsession of T cells with MHC without proposing that evolution was responsible. Nevertheless, some still hoped that structures of TCRs bound to their MHC/peptide ligands would shore up the evolutionary hypothesis. They proposed that conserved amino acids in the MHC helices and TCR CDR loops would serve as lynchpins for the interaction and that other interactions among variable amino acids would determine speciﬁcity and afﬁnity. However, the ﬁrst few reported structures of TCRs bound to MHC/peptide failed to reveal any obvious rules governing their interaction, at least not at the ﬁne structure level. There was no consistent pattern to the TCR amino acids that bound MHC or vice versa, thus suggesting that evolution had not selected TCRs that reacted in some predictable way with MHC. Other evidence weakens the evolutionary hypothesis. For example, TCR genes and MHC genes lie on different chromosomes; thus, there is no obvious genetic mechanism that will maintain coexpression of particular TCR variable (V) region alleles and particular versions of the rapidly evolving MHC genes. Although there are a few examples of TCR V region alleles or family members with a bias toward a particular MHC allele or class (50–52), in general most Vα and Vβ elements can be found in TCRs that recognize any of the extremely polymorphic alleles and isotypes of MHCI and MHCII. Worse still, in special cases, apparently quite conventional TCR α and β chains react with MHC-like proteins [such as the cluster of differentiation (CD1) proteins, for example CD1d] and their ligands, which are quite different stereochemically from classical MHCI and MHCII (53–60). How could evolution select for TCR V regions that could cope with all these differences? It is simplest to conclude that the somatic process of positive selection in the thymus must choose receptors that can react with MHC from an immense collection of receptors with random speciﬁcities.

V region: TCR variable region

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CD1: cluster of differentiation

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The CDR sequences of TCR V regions do not offer many clues to help us understand this problem. TCR genes do not usually mutate (61), a fact that weakens Jerne’s idea that somatic mutation might help solve the problem. If TCR CDR regions evolved to react with MHC, then one might expect their sequences to offer some clue as to how that has happened. Clearly the evolutionary secret cannot lie in the CDR3 regions of TCRs, because CDR3 regions are created somatically and are only partly encoded in the genome. The secret, if there is one, probably lies in the CDR1 and CDR2 sequences of the V regions of the α and β chains of the TCR. However, inspection of these sequences does not offer much hope. There are many different sequences for TCRα and β CDR1s and CDR2s, and although they can be, and have been, assigned to families on the basis of their amino acid contents and predicted structures, they are not particularly well conserved between mouse and human (62–64).

EVIDENCE FOR EVOLUTIONARY SELECTION FOR TCRs TO REACT WITH MHC Despite the lack of evidence for the evolutionary hypothesis based on the sequences of the TCR V genes or the initial structural data, several results suggest that Jerne might have been correct, in principle. First, in the solved structures TCRs usually bind MHC/peptide in approximately the same orientation (as mentioned above): angled across the MHC α-helices with the TCR α chain over the α2 helix of MHCI or the β helix of MHCII, and the TCR β chain CDR regions over the MHC helices α1/α (reviewed in References 65 and 66) (Figure 1). If MHC recognition is simply a matter of positive selection, why would the reverse orientation never occur? Mazza & Malissen (67) suggest that the accessory proteins CD4, CD8, or perhaps CD3 lock the TCR/MHC/peptide complex into the observed positions by binding

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simultaneously to both TCR and MHC; this remains an untested possibility. Second, the pivot point of the TCR on MHC is usually in approximately the same location: centered over peptide amino acids 4–6 in MHCI and peptide amino acid 5 (P5) in MHCII complexes. If TCR obsession with MHC is simply a matter of positive selection, why wouldn’t the TCR slide drastically to one end or the other of MHC, or even interact with the side of MHC? Actually a dramatic shift of this nature is seen in the reaction of a natural killer T cell (NKT) TCR with CD1d (see below) (68) but was observed only once for the reaction of TCR with classical MHC (38). Again, we can invoke the effects of CD4 and CD8 (see above) as an explanation. Third, although TCR CDR regions vary between species, they are better conserved evolutionarily in length and sequence than their counterparts in immunoglobulin (69, 70), a fact that suggests some required function. Finally, there is a small amount of direct experimental evidence that suggests evolution has played a role in the obsession of TCRs with MHC. For example, we showed that random combinations of TCR α and β chains react with MHC more frequently than expected (71), and others demonstrated that even before positive selection has had a chance to have an impact, TCRs react with surprising frequency with MHC (72, 73).

NEGATIVE SELECTION OBSCURES THE BIASES OF TCRs TO REACT WITH MHC The speciﬁcity of the TCRs on developing thymocytes is tested via both positive and negative selection. Positive selection allows the maturation of thymocytes bearing TCRs that react with self MHC + self peptides (48, 49), but negative selection deletes all thymocytes bearing TCRs that react too well (74, 75). We reasoned that if TCR V elements have been selected through evolution to react with MHC, this reactivity must be at-

tenuated in the thymus to allow escape from negative selection. Thus, whereas the CDR1s and CDR2s of most Vα/Vβ combinations can produce inherently MHC-reactive TCRs, CDR3s generated via somatic recombination will sterically interfere to varying degrees with this reactivity, producing a repertoire of TCRs with a wide range of afﬁnities for MHC. Only T cells with TCRs that exhibit reactivity for self MHC/peptides in a narrow afﬁnity window will pass the tests of both positive and negative selection. Thus, in the resulting mature TCR repertoire only a few of the germ line–encoded MHC interactions of the CDR1s and CDR2s may still be evident, although these interactions still contribute to the overall interaction of the TCRs for MHC, and their maintenance in the germ line is critical to provide a starting point for selection. The consequence of this line of reasoning is that the germ line–encoded MHC interactions may be difﬁcult to identify structurally in T cells that have experienced normal negative selection. Thus, the best place to look for these interactions is in the window between positive and negative selection. We therefore examined the properties of TCRs in mice in which positive selection is relatively normal but negative selection is limited. Although several available mice ﬁt the bill, we chose animals we had described previously, in which the MHCII protein has been replaced with an MHCII that is covalently bound to a single peptide (76). These animals are not ideal for the purposes of this experiment because they still express normal levels of MHCI, bound to the many different host peptides with which it normally engages, and they express one MHCII/peptide combination. Therefore, any thymocytes with TCRs that could react well with any of these combinations would still be deleted. However, these animals have two advantages for the planned studies: Previous characterization showed that they contain high frequencies of T cells that can react very well with MHC, and in fact the T cells cross-react with MHC of many different alleles (76), properties

www.annualreviews.org • The Interaction Between αβ TCRs and MHC

NKT cell: natural killer T cell

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Table 1 MHC

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T cells that have experienced only limited negative selection react frequently with

Mouse donor

Immunogen

C57BL/6 B6→MHCI+

MHCII−

Ii− IAb /Eα+a

MHCI+ MHCII− Ii− IAb /Eα+b a

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b

IAb /3K

5

IAb /3K

2

0

IAb /3K

19

40.4

4.4

The bone marrow–derived cells are B6, but the thymus epithelial cells are MHCI+ MHCII− Ii− IAb /Eα. Mice express a single MHCII protein, IAb , bound to the Eα peptide.

expected of TCRs manifesting their evolutionarily selected abilities to bind MHC. Secondly, the animals contain mature CD4+ T cells that could be primed with MHC/peptide, and therefore we could identify at least one target MHC/peptide combination for these cells. Mice that express a single class II protein, IAb , bound to a single peptide from Eα (MHCI+or− MHCII− Ii− IAb /Eα mice) and normal C57BL/6 (B6) animals were primed with IAb bound to another peptide, called 3K because it contains three lysine residues that point straight up out of the IAb groove. Thus, Table 2 The number of deleted MHC ligands controls T cell alloreactivity

Number of TCRs tested

Mouse donor

% of target MHC tested that stimulated hybridomas bearing the TCRs

C57BL/6

20

3.1

MHCI+ MHCII− Ii− IAb /Eα+a

19

44.1

MHCI+ MHCII− Ii− IEk /99A+b

8

52.2

MHCI+ MHCII− Ii−c

3

18.2

MHCI− MHCII− Ii− IAb /Eα+d

45

41.1

MHCI− MHCII− Ii−e

28

67.1

a

Mice express a single MHCII protein, IAb , bound to the Eα peptide. Mice express wild-type MHCI of the k allele and IEk bound to the 99A peptide (83). c Mice express MHCI of the b allele but no MHCII. d Mice express no MHCI and a single MHCII protein, IAb , bound to the Eα peptide. e Mice lack almost all MHC proteins. b

176

Number of TCRs tested

% of target MHC tested that stimulated hybridomas bearing the TCRs

Marrack et al.

these three lysines should bind to TCRs that reach with IAb /3K. We prepared T cell hybridomas speciﬁc for IAb /3K from the two sets of animals. Only one of the IAb /3K speciﬁc TCRs from B6 mice showed any alloreactivity, whereas many of the TCRs speciﬁc for the same MHC/peptide combination from MHCI+or− MHCII− Ii− IAb /Eα animals were extravagantly alloreactive (Table 1) (77). We tested two of these TCRs in detail and found that they react with both MHCI and MHCII. This phenomenon is not a special feature of TCRs from the MHCI+or− MHCII− Ii− IAb /Eα mice, because we obtained similar results with TCRs from other mice in which negative selection is limited (78–83). Also, the degree of MHC reactivity of the TCRs appeared to correlate with the degree to which negative selection was absent (Table 2). The few T cells in mice lacking almost all MHC proteins (MHCI− MHCII− Ii− in Table 2) reacted most frequently with allogeneic MHC proteins. To conﬁrm that the ability to produce very MHC-reactive TCRs on mature T cells was due to lowered opportunities for negative selection in the MHCI+ MHCII− Ii− IAb /Eα mice, chimeras were examined in which the bone marrow–derived cells were B6 but the thymus epithelial cells were MHCI+ MHCII− Ii− IAb /Eα. In these animals, positive selection of most conventional T cells occurs on thymus epithelium, whereas negative selection occurs on both the epithelial and bone marrow–derived cells. IAb /3K-speciﬁc

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T cells from these chimeric mice were not MHC cross-reactive (Table 1) (77). Therefore, mature T cells bearing very MHCcross-reactive TCRs are allowed to appear in MHCI+ MHCII− Ii− IAb /Eα mice because of the limited opportunities for negative selection in these animals. Further studies showed that the MHCcross-reactive TCRs display several unexpected properties. Although the MHC-crossreactive TCRs react with their immunogen (IAb /3K) with the same kinetics, afﬁnities, and footprints as normal TCRs do, they show less speciﬁcity for particular side chains of the amino acids of their ligand (77, 84). That is, the MHC-cross-reactive TCRs are more accepting of amino acid substitutions in both IAb and the 3K peptide than are normal TCRs. This result provides additional evidence that these TCRs are more cross-reactive. If the TCRs can accept amino acid substitutions, they are more likely to recognize different MHC alleles and classes than their wild-type counterparts. These results support the view that the T cell repertoire is intrinsically MHC reactive and that this reactivity is partially masked by negative selection in the thymus. To improve our understanding of the results, we recently solved the crystal structures of two TCRs, both bound to the IAb /3K complex (84a). One TCR (B3K506) came from a T cell developing in normal mice and was extremely peptide and MHC speciﬁc. The other TCR (YAe62) came from one of the very cross-reactive T cells described above. Both TCRs displayed approximately the same afﬁnity for the IAb /3K ligand (84). The cross-reactive TCR showed a very concentrated footprint on IAb /3K with very strong contributions from several amino acids in Vα CDR1(Y31) and Vβ CDR2 (Y48 and E54). These amino acids also contributed to the binding of the speciﬁc TCR. However, in the peptide- and MHC-speciﬁc TCR these amino acids were less important in the overall footprint, which was distributed over a much wider area. These results further supported our view that germ line–encoded interactions

may best be revealed by these cross-reactive T cells that have avoided negative selection and that normally developing T cells may have attenuated forms of these interactions.

TCR VARIABLE REGIONS HAVE BUILT-IN BIASES FOR REACTION WITH MHC Investigators have inspected the increasing numbers of solved structures of TCRs bound to MHC/peptide many times in the hope that these structures will reveal the rules that govern the reactions between TCRs and MHC (27–46). Apart from the usual diagonal mode of interaction and the usual placement of the TCR V region loops over the α-helices of the MHC, a set of general rules is not apparent. However, some researchers noticed that particular TCR amino acids often make contact with MHC, and in the case of structures involving the same TCR Vβ region, bound to different ligands, some of the V region amino acids that bind MHC are identical and bind MHC in the same positions (42, 85). This observation gave hope that rules were present, although they may be difﬁcult to make out. There are now more than 20 published structures of TCR/MHC complexes. This is both an advantage and a disadvantage for people who are searching for the rules that underlie the reactions. On the one hand, the increasing numbers of structures provide more data for analysis and a greater likelihood of spotting patterns. On the other hand, some of the new structures were chosen deliberately to illustrate unusual interactions, such as those with self antigens or with very large peptides. Therefore, these structures may not illustrate the general rules that guide the recognition of MHC by TCRs. Bearing these issues in mind, we revisited the published TCR/MHC structural information with new eyes and more ﬂexible criteria for conserved interactions. We were guided by several thoughts: 1. Almost all the structures published so far involve TCRs that have undergone

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normal negative selection. Therefore, these TCRs are unlikely to demonstrate all their built-in abilities to react with MHC. Had the TCRs been able to react strongly with MHC, the cells that bear them would have been deleted in the thymus. Perhaps each TCR/MHC/peptide combination will manifest only a few of the evolutionarily selected interactions, and other interactions are prevented by the TCR CDR3 regions or the peptide. Thus, different TCR/MHC/peptide combinations will use different evolutionarily determined interactions, and some of the combinations may not use any at all. 2. Given the variability in TCR V region CDR1 and CDR2 sequences, the evolutionarily selected interactions may be distinct for different TCR V regions. 3. The angle and pitch with which TCRs settle onto MHC varies because of differences in peptide and CDR3 sequences. The evolutionarily selected interactions may therefore need a builtin ﬂexibility to accommodate shifts in the relative positions of the amino acids involved. 4. The evolutionarily selected interactions may not be the same for MHCI and MHCII because some TCR V regions are used preferentially to react with one or the other MHC class (50–52). With these thoughts in mind, we have reexamined the contacts between the CDR1 and CDR2s of Vα and Vβ regions and MHC in

Annu. Rev. Immunol. 2008.26:171-203. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

ANRV338-IY26-06

all the published structures of TCRs bound to MHC/peptide ligands. The analyses also included data from two structures we solved recently, mentioned above. The only omissions from the analyses are TCR/MHC/peptide structures that are duplicates of particular TCR/MHC combinations and those well analyzed in this context in a recent publication (85). The results of these analyses are shown in Figure 2. The analyses are inherently incomplete because only a few structures of TCRs bound to their ligands are known, in comparison with the many existing TCR V region genes in human and mouse. The results are also distorted by the fact that TCRs containing some V regions are better behaved in crystallographic studies than others, perhaps because the V regions in question allow the TCRs to fold in a more stable fashion. Whatever the reason, the TCR/ligand structures reported so far are heavily biased in favor of TCRs using Vβ8 in the mouse or Vβ13 (a related Vβ) in human, and a few other Vβ regions related to these two. The Vα regions that have been crystallized show less bias. These caveats limit our ability to make predictions about the universe of human and mouse TCR V regions. However, the structures may help us see patterns of reactivity for those V regions that are overrepresented in the solved structures. Some patterns are evident from the limited numbers of structures available. As previously reported and mentioned above, Vα CDR amino acids usually contact amino acids

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 2 Contacts between TCR CDR1 and CDR2 and MHC. (a) The cells contain the sequences of CDR1α of TCRs whose structure, bound to the MHC/peptide ligands, is known. In the same cell as each CDR amino acid are the MHC amino acids bound by that CDR residue, color coded ( pink or blue) to indicate the MHC region on which they lie. Amino acids from the α1/α regions of MHCI/MHCII are shaded blue, and those from the α2/β regions are shaded pink. Dashes indicate a gap in the CDR region, introduced according to References 63 and 64 to best align TCR sequences. TCR and MHC amino acids are numbered according to References 63 and 64. Structures are from References 27–46, 84a. (b-d ) As in a, except the sequences of TCR CDRα2, CDRβ1, and CDRβ2 and their contacts are displayed. PDB, Protein Data Bank; Pep, peptide. 178

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α1/α region of MHCI/II α2/β region of MHCI/II

Human TCRs binding MHCI

a

VαCDR1

Vα

PDB

TCR

MHC

Pep

10GA

JM-22

A2

MP

hVα10.1 S

–

–

V

1BD2

B7

A2

TAX

hVα21.1 N

S R170 –

M

S

A

2BNQ

1G4

A2

1AO7

A6

A2

1MI5

LC13

B8

2AK4

SB27

B35

2NX5

ELS4

B35

26

ESO-9C hVα23.1 D TAX

EBV

hVα2.1

hVα4.1

27

27a

W167

–

29

30

S Q155 S

F

D T163 Y Q155

I

Y Q155 N

G W167 S

Q Y159 S

E58 Y59

Q155

W167

T163 A150 R151

Q155

T R62 I

R

31

E154

F

K66

W167

D E58 R R170 –

13mer hVα12.1 T

28

S R62 G A158 T Y159 D

Y V152

D

Y

T

E154 Q155

T A158 Y L163

A158

EPLP hVα7.2

T

S E166 –

G E166

KK50.4 HLA-E Leader hVα4.2

T

I

S

G D162 N T163 E

–

G162 T163 Y E166 W167

F L163 N L163 G E154

2ESV

Y H155 A158

Mouse TCRs binding MHCI

1LP9

AHIII

A2

p1049 mVα8.5

S

T

Pkb1 mVα2.7

1FO0

BM3.3

Kb

Pbmi

mVα17.3 T

Q R62 D

S T163 S A158 Y

F R155

2OL3

BM3.3 Kbm8 pBM8 mVα17.3 T

Q R62 D

S T163 S

Y

F R155 Y R155

Kb

dEV8 mVα3.1

2OI9

2C

Ld

QL9

E58

E58

T W167 F

A158

Kb

2C

–

F Q155

1KJ2 KB5-C20

2CKB

D R62 S

E154

A158

S Y159 P

N T163 Y

Y R62 S R62 –

A

R62

T E166 P

Y

A E163

T Y159 P

A158

mVα3.1

S

–

Y155

Y A158

Human TCRs binding MHCII

E163

Mouse TCRs binding MHCII

Annu. Rev. Immunol. 2008.26:171-203. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

26 February 2008

1FYT

HA1.7

2IAM

1ZGL

DR1

flu HA hVα1.2

S

–

V

E8

DR1 mut TPI hVα13.1 D H81 –

–

S

3A6

DR2

T V85 –

G

MBP

S

hVα22.1 A

H81

H81

P

T77 H81

V

H81

Y159

P

Y T77

N T77 N D76 T77

Y R80 P

S

H81 D76

1YMM Ob.1A12 DR2

MBP

hVα3.1

Conalb mVα2.5

T

–

–

D

T77 S H81 –

S

I

T

T77 H81

F

N T77 N T77 H81 R70 D A73 Y R70 T77

1D9K

D10

IAk

1U3H

172.10

IAu

MBP

mVα2.3

N

S Y81 –

A

Y81

F

XXX

B3K506

IAb

3K

mVα4.1

A

S H81 –

G

T77 H81

Y T77 P

T77

D T77 Y D76

A

H81 D76

XXX

YAe62

IAb

3K

mVα4.6

T

T

–

G

Y T77 P

T

H81

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α1/α region of MHCI/II α2/β region of MHCI /II

b PDB 10GA

TCR JM-22

MHC A2

Pep MP

VαCDR2

Vα

48

S T hVα10.1

49

50

51

H151 V E154 T

V

E154

1BD2

B7

A2

TAX

hVα21.1 S

I

G A158

S Q155 S T163

Human TCRs binding MHCI

A158

Annu. Rev. Immunol. 2008.26:171-203. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

52

1G4

A2

ESO-9C hVα23.1 N

L

G

E

D

A6

A2

TAX

hVα2.1

S

1MI5

LC13

B8

EBV

hVα4.1

H R151 G

2AK4

SB27

B35

H151

Q155 Q155 E154 Y Q155 S A158 N E166 G A158

I

D

E154

A150

hVα12.1 13mer Y Y R

H151

Q H151 S Q155 S E154 Q E154 R

I

1AO7

54

R157 A158 I E161 K G162

A150

2BNQ

53

L Q155 T

S E154 N

V R151

A158

R

E154 R157

N E154 S

F A158 D

E

E161

2NX5 2ESV

ELS4

B35

EPLP hVα7.2 G

KK50.4 HLA-E Leader hVα4.2

R151

Y E154 N

V A158 L R157 D

H

G

L R157 K

S

F E154 T E154 D

G

L

N

N

E

N

K

R

I Q155 R E161 S E166 V G162 S

D

E154

H151

1LP9

Mouse TCRs binding MHCI

1KJ2 KB5-C20

p1049 mVα8.5

H151

Kb

Pkb1 mVα2.7

E154 Y R155 K E154 K A158 E154 Y R155 K E154 K A158

mVα17.3 R

Q

D

S

2OL3

BM3.3 Kbm8 pBM8 mVα17.3 R

Q

D

S

K

Y

Y A158 S G162 G

2C

Kb

Kb

2OI9

2C

Ld

1FYT

HA1.7

DR1

Pbmi

dEV8 mVα3.1

QL9

mVα3.1

flu HA hVα1.2

K A150 Y

K

I

P

S D76 G

T

K

K

A

D

K

G

L E69 I

R

S D76 N E69 E

R

3A6

DR2

MBP

hVα22.1 T

1YMM Ob.1A12 DR2

MBP

hVα3.1

D10

IAk

1U3H

172.10

IAu

Conalb mVα2.5 MBP

mVα2.3

P

S T77

D66 E69 Q70

E69 Q70 A73

A73

D

A73 T77

A

I

S

E69

L

S

I

L

E69 A73

S T77

E69 73 T77

E69 R72 A73 D76

XXX

B3K506

IAb

3K

mVα4.1

R E66 A

S

XXX

YAe62

IAb

3K

mVα4.6

Q

T

V

A E69 A

P

E69 A73 T77

1ZGL

1D9K

D

T163 A150 G151 E154 Y E154 S R157 G R155 D Y155 A158 Y159 E69

DR1 mut TPI hVα13.1 Y

Y

A158

R155

T

E8

Marrack et al.

E166

BM3.3

2IAM

(Continued )

A158

S

1FO0

2CKB

Mouse TCRs binding MHCII

Human TCRs binding MHCII

A2

Q155

Figure 2

180

AHIII

R

T

V

E69 R72 A73

T

S

N

V A73 S

D

D

K E69 E

A

N

N

E69

ANRV338-IY26-06

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α1/α region of MHCI/II α2/β region of MHCI/II

Human Vβ binding MHCI

c

Annu. Rev. Immunol. 2008.26:171-203. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

20:33

TCR

MHC

Pep

Vβ

10GA

JM-22

A2

MP

hVβ17.1

N

L

N

H

D

1BD2

B7

A2

TAX

hVβ13.1 D

M

N

H

E

2BNQ

1G4

A2

1AO7

A6

A2

TAX

1MI5

LC13

B8

EBV

2AK4

SB27

B35

2NX5 2ESV

Human V β binding MHCII

Mouse V β binding MHCI

1LP9

Mouse Vβ binding MHCII

Vβ CD R 1

PDB

ELS4

26

28

27

Q72 T73

29a

29

A

–

Y

–

Y

–

Y

–

D

M

N V76

H

E

hVβ13.1

D

M

N

H

E

hVβ6.2

I

S

G

H

V

N80

S

D

M

N

H

N

T69

S

T69 T73

13mer hVβ13.3

E76

– –

Q65 T69

T

E

N

H

R

KK50.4 HLA-E Leader hVβ16.1

I

S

G

H

D K146 N

T

N

N

H

D P

W

–

P

W V76

–

P

W

–

Q149 N K146 N A150

–

A2

EPLP

25

hVβ12.1

AHIII

B35

ESO-9C hVβ13.1

24

p1049 mVβ8.1

1KJ2

KB5-C20

Kb

Pkb1

mVβ2

N

S

K146 Q Q149 Y A150 A150

1FO0

BM3.3

Kb

Pbmi

mVβ2

N

S

Q

Kbm8 pBM8

Y

Q72

Y

Y

– –

T73

–

2OL3

BM3.3

mVβ2

N

S

Q K146 Y

2CKB

2C

Kb

dEV8

mVβ8.2

T

N

N K146 H

2OI9

2C

Ld

QL9

mVβ8.2

T

N

N

H

N

V76 N77

N

–

1FYT

HA1.7

DR1

D

M

D

H

E

A64 V65 A68

N

–

2IAM

E8

DR1 mut TPI hVβ13.6

D

M

N

H

E

A61 V65

Y

1ZGL

3A6

DR2

MBP

hVβ5.1

I

S

G

H

R W61 S

1YMM Ob.1A12 DR2

MBP

hVβ2.1

L

D

F

Q

A

T

E55 Q57

T

Conalb mVβ8.2

T

N

N

H

N

N Q61

–

flu HA hVβ3.1

G58

Y60

–

–

1D9K

D10

IAk

1U3H

172.10

IAu

MBP

mVβ8.2

T

N

N

H

N H68 N Q61

–

XXX

B3K506

IAb

3K

mVβ8.1

T

N

N

H

D

Y Q61

–

XXX

YAe62

IAb

3K

mVβ8.2

T

N

N

H

N H68 N Q61

–

E55

Figure 2 (Continued ) www.annualreviews.org • The Interaction Between αβ TCRs and MHC

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α1/α region of MHCI /II α2/β region of MHCI /II

d

VβCDR2

Human Vβ binding MHCI Mouse Vβ binding MHCI Human Vβ binding MHCII

TCR

MHC

Pep

Vβ

46

10GA

JM-22

A2

MP

hVβ17.1

1BD2

B7

A2

TAX

hVβ13.1 Y

2BNQ

ESO-9C hVβ13.1

Y

1G4

A2

1AO7

A6

A2

TAX

hVβ13.1

Y

1MI5

LC13

B8

EBV

hVβ6.2

Y

2AK4

SB27

B35

2NX5

ELS4

B35

2ESV

Mouse Vβ binding MHCII

Annu. Rev. Immunol. 2008.26:171-203. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

PDB

13mer hVβ13.3 EPLP

hVβ12.1

KK50.4 HLA-E Leader hVβ16.1

S

V

S

V

S

V

F

Q

S

A

Y Q65 S

Y

H

V

Q72 E76

Y

F

G

T

V

K

D K68

Q65

Q

D

K

T

D

K

T

D

K

Q72

V76 T80

E

R79

S R75 K

Q

V76

D69 Q72 I73

D

R65

E

R65

K V

mVβ2

T

L

R

S

V76 R79

P

G

D

K

E

V

mVβ2

T

L

R

S

R79

P

G

D

K

E

V

E

K

E

K

mVβ8.2

Y

S

DR1 mut TPI hVβ13.6

Y Q57 S

DR2

E

mVβ8.2

hVβ5.1

hVβ2.1

Conalb mVβ8.2

Y

Y

Y

F

Q57

Y

T

V76 R79 T30 V76 T80 S73 V76 G69 Q72 V76

G

R79

A

R79 R75 R79

G R79 S

V76 R79

T

G R75 S

Q72 R75

T

Q72

G

V76

A

Q57 G58 A61 A64

D

A64 K67

V

K

M L60

K

K39

E

K39 Q57

K

V

Q57 G58 A61

G

A

G

I

A61

T

Q57

D Q57

K

F

A61 V65

S

E

T A61 Q Q57

R

Q57 G58 A61

N

E55 Q57

K

S

N

Y Q57 S

Y

D10

IAk

1U3H

172.10

IAu

MBP

mVβ8.2

Y Q57 S

Y

XXX

B3K506

IAb

3K

mVβ8.1

Y

Q57 Q61

S

Y

XXX

YAe62

IAb

3K

mVβ8.2

Y Q57 S

Y

Marrack et al.

E

S

D Q72 R75

E

1D9K

182

L

K

S

(Continued )

T

Q

D Q72

flu HA hVβ3.1

Figure 2

I

R79

R65

G

R65

Y

MBP

D

P

Y

S

1YMM Ob.1A12 DR2

T

Q72

Kbm8 pBM8

MBP

K68

S

BM3.3

3A6

I

Q72 V76

2OL3

1ZGL

G

Q

R

Pbmi

E8

D

Q

E

K

T

A

R79

I73 V76

Q72

G

N

G

I

K

L

Kb

2IAM

A

G

R65

T

BM3.3

DR1

G

Q72

Q

E

1FO0

HA1.7

A

F

Q72 R75

T

mVβ2

1FYT

G Q72

D

N

55

S

Pkb1

QL9

A

54

D

Kb

Ld

Q72 R75 E76 Q65 Q65 K68 T69

G

Q72 V76

53

K68 A69 Q72

A

KB5-C20

2C

R65 K68 A69 Q72

V

52

Q72

1KJ2

2OI9

I

51

V

p1049 mVβ8.1

dEV8

Q

A69 Q72 T73 V76

50

R65 K68 A69

A2

Kb

S

49

Y

AHIII

2C

R65

48

S

1LP9

2CKB

Y

R65

47

Q57 G58 L60 Q61 A64 Q57 L60 Q61 A64 Q57 L60 Q61 Q57 L60 Q61 A64

A61 A64 V65

K39

L60 A64

E

G

S

K

A

G

A

G

S

T

K39

E

K39 Q57 L60

K

G

A

G

S

T

K39

E

K39 Q57 L60

K

V

A

D

S

T

E

Q57

K

G

A

G

S

T

E

K39 Q57 L60

K

K67

T

K39

Q57

Y

Q57

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in the α2 helix of MHCI or the β helix of MHCII. Conversely, Vβ CDR amino acids bind residues in the α1- or α-helices of MHCI or MHCII, respectively (Figures 2a–d ). However, the results also suggest that the contacts between TCRs and MHC might follow more rules than this one. For example, as previously noted, TCRs often bind MHC via the use of amino acids in positions 28, 29, and 31 of CDR1α; positions 50, 51, and 52 of CDR2α; positions 28 and 29 of CDR1β; and position 48 of CDR2β. (TCR V regions and their amino acids are numbered as listed in References 63 and 64. In all ﬁgures, MHC amino acids are from the MHC α chain unless speciﬁcally marked “β”.) This usage is to some extent independent of whether the target is MHCI or MHCII. We, among others, have discussed a number of these positions (αY29, βY46, and βY48) at length in previous publications, so in the discussion below we concentrate on positions that have not been covered as thoroughly.

Amino Acids Often Used by Vαs to Bind MHC Figure 2a and Figure 3 show that in VαCDR1, Y/F31 is very often bound to a site on MHC that includes amino acids around Q/H/R 155 of the α2 helix of MHCI or the equivalent position on MHCII, R70 on the β helix. Approximately 40% of human and mouse TCRαs have a Y or F at position 31, at the C-terminal end of their CDR1 regions (Figure 3a). So, if this is an interaction that routinely affects TCR/MHC binding, it may apply to many TCRs. Investigators have solved the structures of 13 TCRs that contain a VαY/F31, bound to different MHC ligands. Figure 3b–m shows the relative positions of VαY/F31 and MHC/peptide in 12 of these structures. (We omitted one structure, 2OL3, because it involves a TCR that was already included, bound to a closely related MHC.) VαY/F31 does not bind MHC in three of these structures (2AK4, 1KJ2, and 1U3H; Figure 3i, j,

and m). In one of these structures (2AK4), the entire TCR is lifted away from MHC by a very pronounced bulge in the engaged peptide, which correlates with an unusual 13mer in the structure. In another case (1KJ2), the TCR has a very long CDR3β, and the total number of interactions between this TCR and MHC is fewer than normal (Figure 2). The orientation of VαY/F31 is not identical in the nine structures shown in which it binds MHC; sometimes VαY/F31 adopts a vertical conﬁguration, reacting with the C-terminal side of α2H/Q/R155 or βR70 (in 1LP9, 2ESV, 2CKB, 1FO0, and 1FYT; Figure 3c, d, f, h, and l ), and in other structures VαY/F31 reacts with the N-terminal side of the same amino acid or is oriented horizontally relative to the axis of the MHC α helix (in 1MI5, 1BD2, and 1DK9; Figure 3b, g, and k). For both MHCI and MHCII, the approach of VαCDR1 to the MHC in this region is often facilitated by the lack of a side chain on a highly conserved alanine on top of the MHC α helix (A158 for MHCI and βA73 for MHCII). This leads to the formation of a cup on the surface of the MHC α helix into which Y/F of TCR α31 can nestle in various orientations while still maintaining Van der Waals–like interactions with some portion of the exposed helix backbone and the surrounding amino acid side chains. The interaction is in a way analogous to a ball and socket structure. Almost as striking as the fact that VαY/F31 tends to bind to a particular site on MHC is the fact that other amino acids at this same position do not. Alternate amino acids in known structures at position 31α include G, A, S, N, and T. These amino acids engage MHC in only one of these structures (1YMM). In many cases, the VαY/F31 interaction seems to be replaced by binding between amino acid Vα29 or Vα30 and the ligand (Figure 2a). In human Vα22 and Vα4, VαY29 is a special case. In the three available structures, VαY29 interacts strongly with βH81 and βT77 of the MHCII β chain helix (84a). An adjacent proline at position 30

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a

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31

hVα01.2, 3, 4 hVα02 hVα04.1 hVα04.2 hVα05.1 hVα08.1 hVα08.2 hVα12.1 hVα14.1 hVα14.2.1 hVα14.2.3 hVα15.1 hVα16.1 hVα17.1 hVα20.1 hVα21.1

SS - VPPY NS - A FQY T I SGT DY T I SGN E Y NY - SPAY DS - ASNY NS - ASDY T RD T T Y Y T SESDYY T S ENDY Y T SESNYY DS - SS T Y V S - GN P Y NT - A FDY N I A T NDY NS - MF DY

mVα1.1 mVα1.2, 8 mVα1.7 mVα2.1, 5, 9 mVα2.2, 4, 7 mVα2.3, 6 mVα3.1, 3, 9 mVα3.2, 5, 6 mVα3.4, 8 mVα3.7 mVα5.1 mVα5.3 mVα6.1 mVα8.1, 6 mVα8.3 mVα8.5 mVα8.13 mVα8.14 mVα9.1 mVα12.1 mVα13.1 mVα15.1 mVα16.1P mVα17.1 mVα17.3 mVα20.1

DS - A SQY DR - N FQY DR - NVDY DS - T FDY DS - T FNY NS - A FDY YS - ATPY Y F - GT PY YG - GS I Y SS - VTPY DP - NSYY DS - A T AY T I SGN E Y T I YSNP F TTYS - PF STYS - PF TAYS - TF T I YSNA F YS - AAPY N I ATSEY NS - ASNY DT - ASSY NA - A TNY RD - S S Y F QD - S S Y F ND - MF DY

b

c

hVα α4.1 (1MI5)

mVα8.5 (1LP9)

R151 Q155

Q155

mVα3.1 (2CKB)

Y155 R155

G151

βE69

HLA-E

g

Q155

H151

A158

A158

Ld

βR70 βT77 βA73

E154

Kb

i

hVα1.2 (1FYT)

βE69

E154

E154

IAk

l

hVα21.1 (1BD2)

G151

A158

mVα17.3 (1FO0)

βR70

S151

E154

HLA-A2

f

mVα3.1 (2OI9)

mVα2.5 (1LP9)

βT77 βA73

E154

HLA-B8

h

H155 A158

E154

e

k

hVα4.2 (2ESV)

H151

A158

A158

d

hVα12.1 (2AK4)

j

HLA-A2 mVα2.7 (1KJ2)

m

HLA-DR1 mVα2.3 (1U3H) βR70

R151 R155 A158

Q155

G151

R155

K

b

βT77 βA73

A158

A158 E154

G151

E154

HLA-B35

βE69 E154

K

b

IAu

Figure 3 TCRs often bind the same site in MHC via Y/F31 in TCRα CDR1α. (a) CDR1 sequences of the human (h) and mouse (m) Vα regions that contain an F or Y residue at position 31 (highlighted in salmon pink). V regions were omitted if the sequences of both their CDR1 and CDR2 regions were identical to one already displayed. V regions and their amino acids are numbered according to References 63 and 64. The sequences were selected from 48 human Vαs and 75 mouse Vαs. (b–j ) The arrangements of MHC amino acids around Y/F31α ( green with red hydroxyl groups) in nine of the solved structures of TCRs bound to MHCI, with the α2 helix of MHCI in magenta. Also indicated are the Vα region, MHC allele, and pdb number. The structures are arranged from b to j roughly according to the predicted strength of the interaction between Y/F31α and MHCI; there is no predicted contact for Y/F31α in 2AK4 (i ) and 1KJ2 ( j ). (k–m) As in b–j, but for TCRs bound to MHCII, with the β helix of MHCII in magenta. There is no predicted contact between Y/F31α and MHCII in 1U3H (m). Structures were selected from the references in Figure 2.

of these Vαs orients VαY29 to point straight out from the tip of CDR1, making VαY29 particularly available for interaction. In the other structures in which Vα contains a P30, the amino acid at p29 (P, T, or S) also interacts with the MHC helix in the same region (Figure 2a). This Vα29 interaction may be a conserved structural feature of Vαs with this proline. 184

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In VαCDR2, a serine at position 51 is the most conserved amino acid among the published structures (63, 64). In MHCI this serine most often interacts with the MHC helix slightly C-terminal to the site engaged by VαY/F31 (Figure 4b–g ). Only occasionally does VαS51 not engage MHC (2AK4 and 1FO0; Figure 4h,i ). The approach of αCDR2 to the MHCI α helix is again

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a hVα1.1, 2.1 hVα1.2.5 hVα1.3 hVα1.4 hVα1.5 hVα2.1.1 hVα2.1.2 hVα2.2 hVα2.3 hVα3.1 hVα6.1.1 hVα6.1.2 hVα6.1.1 hVα9.1 hVα12.1 hVα13.1 hVα15.1 hVα19.1 hVα21.1 hVα23.1 hVα27.1 hVα28.1 hVα30.1 mVα1.1, 7 mVα1.2, 8 mVα1.5 mVα2.2, 3, 4 mVα2.6 mVα2.7 mVα3.1, 5, 6, 7, 9 mVα3.4, 8 mVα5.1 mVα5.3 mVα7.1 mVα10.1 mVα10.2, 3, 6, 9 mVα10.5 mVα11.1, 6 mVα11.6 mVα13.1 mVα15.1 mVα16.1 mVα17.1, 3 mVα18.1, 2, 3 mVα20.1

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51 K Y F SG D P K Y T SA A T K Y L SG S T K Y F SG D T K Y T SA A T F I Y SN G D S I Y SN G D Y T Y SSGN S VY SSGN L I R SN E R Y QG S Y D Q Y QG S Y D E D I R SN V G R H I SR E R RN S F D E Y I P SG T K Y I F SNM D M L S SG K K S I SS I KD L I Q SSQR V L L SN G A M L T SSG I T L Y SAG E S I F SN G E S I F SD G D S I F SN G N S I S SV S D S I L SV S N S I R SV S D K Y Y SG D P K Y Y SG N P K V F SS T E S V L SN V D R QT SS S T S N P SG T Y N P SG T Y S P SWA Y L A SG T Y L A SG T D I R SNM E D I R SN V D D I R SN V N R QD S Y K K L I R SN E R S V R SN V D

b

c

hVα α21.1 (1BD2)

hVα2.1 (1AO7)

Q155 T163 E154 A158 E166

E166 T163

HLA-A2

f

g

Q155 T163

A158

E154

R155

G162

hVα1.2 (1FYT)

βT77

βA73

E154

R157

k

βT77

h

L163 E166 A158

b

hVα13.1 (2IAM) βA73

Ld

i

Q155 E154

HLA-DR1

mVα17.3 (1FO0)

T163 E166

l

G162

R157

hVα3.1 (1YMM)

βD76

R157

m

βT77 βA73

Kb

mVα2.3 (1U3H)

βT77

βA73

βD76 HLA-DR1

HLA-DR2

IAk

Figure 4 S51 in TCRα CDR2α is often used in TCRs and often engages MHC at the same site. (a) The CDR2 sequences of the human (h) and mouse (m) Vα regions that contain an S at position 51 (highlighted in salmon pink). V regions were omitted and numbered as in Figure 3a. (b–i) The arrangements of MHC amino acids around αS51 (blue with red hydroxyl group) in eight of the solved structures of TCRs bound to MHCI, with the α2 helix of MHCI in magenta. Also indicated are the Vα region, MHC allele, and pdb number. The structures are arranged from b to i roughly according to the predicted strength of the interaction or alignment between S51α and MHCI; there is no predicted contact for S51α in 2AK4 (h) and 1FO0 (i ). ( j–m). As in b–i, but for TCRs bound to MHCII, with the β helix of MHCII in magenta. Structures were selected from the references in Figure 2.

facilitated by the conserved MHC A158, as well as G151. In a few cases the hydroxyl group of VαS51 hydrogen bonds to the backbone or amino acid side chains of the MHC helix; other Van der Waals interactions can involve the participation of the backbones of VαS51 and the MHC helix. On MHCII the binding site for VαS51 is similar to that of MHCI, but more ﬁxed, and VαS51 engages MHCII βT77 in various orientations (Figure 4j–m). Particularly in the case of

R155 E154

A158

HLA-B35

βD76

βD76

R157

G162

hVα12.1 (2AK4)

G162

K

mVα3.1 (2OI9)

Kb

R157

HLA-A2

j

G162

mVα2.7 (1KJ2)

e

R155 Y155 E154 E166 E163 A158 A158 E154

E166 T163

R157

E166 T163 A158

R157

G162

E154

mVα3.1 (2CKB)

HLA-A2

hVα23.1 (2BNQ)

E166

A158

G162

R157

G162

Q155

d

MHCI, VαS51 binds at a region of the MHC α helix with some ﬂexibility in its exact site and pitch on the MHC protein. Others have mentioned the involvement of VαY50 in the interaction of several TCRs with MHCI (86). Vαs with this amino acid are not abundant (present in 12%–16% of Vαs) (Figure 5b), but in the VαCDR2s of the published structures VαY50 and other amino acids at this position very often interact with MHC, using the same area of MHCI

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a

b Vα αs with a Y at aa29

Vαs with a Y at aa50

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29 hVα22.1 hVα31.1

Y T AT - - GYPS T ST - - GYP Y T

mVα4.1 mVα4.2 mVα4.3, 4 mVα4.5, 6 mVα4.7 mVα4.9 mVα4.10 mVα4.11 mVα4.12

Y S AS - - GYPA E TK - - QYP Y T S ATS I AYPN Y Y T S TT - - GYP S ATS I GYPN Y Y S AS - - GYPA S ATS I AYPN Y WYP S TT - - WY W Y T S TT - - GYP Y T

50 hVα 2.1. 1 hVα 2.1. 2, 3 hVα 2.2 hVα 2.3 hVα 20.1 hVα 25.1 hVα 30.1

F I YSNGD Y S I YSNGD Y Y T YSSGN Y SVY Y S SGN QGYK Y T KV A L YKAGE Y T L YSAGE Y

mVα3.1, 5, 6, 7, 9 mVα3.2 mVα3.4, 8 mVα12.1

KYYSGDP Y KYYPGDP Y K Y Y SGNP QGYKDYV Y

Figure 5 CDRα sequences that contain other amino acids that frequently bind MHC at the same position. Listed are the human and mouse V region CDR1α sequences that include a Y at position 29 (a), or the human and mouse V region CDR2α sequences that contain a Y at position 50 (b). The amino acid of interest is highlighted in salmon pink. Vαs that have been reported in structures of TCRs bound to MHC are highlighted in yellow or blue, if the structure involved binding to MHCI or MHCII respectively. The sequences were selected and numbered as in Figure 3a and structures were selected from those in Figure 2.

(around A158) and MHCII (around βA73) (Figure 2b). Perhaps these amino acids are evolutionarily selected to do this.

Amino Acids Often Used by Vβs to Bind MHC As pointed out above, the published TCR/MHC structures are dominated by TCRs that use mVβ8 family members or the related human Vβ elements (hVβ3, hVβ12, hVβ13, and hVβ17). With this large pool of data for a related set of Vβs, a number of studies have already revealed particular amino acids that interact with MHC in a similar way from structure to structure (42, 67, 85, 86). These include VβN/Y29 in CDR1 and VβY/F46, VβY48, and VβD/E54 in CDR2 of Vβ (Figure 2c,d ). In these Vβ families and a few others there is often a Y or N in position 29 of Vβ CDR1 (Figure 6). In the published structures these amino acids often make 186

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contact with the α1 MHC helix, especially in the structures with MHCII in which there is often an H-bond to Q61. Thus, VβN/Y29 may be an amino acid selected by evolution to bind MHCα1. Also in VβCDR1, many different amino acids at position 28 often contact the MHC; however, there is no obvious conserved pattern of recognition for amino acids at this position. In fact, in some structures the rotation of the TCR has brought this amino acid over the peptide to contact the α2 helix of MHCI or the β1 helix of MHCII (Figure 2c); therefore, position 28 does not seem to be involved in an evolutionarily conserved interaction. The most striking example of a conserved point of interaction is position 48 of VβCDR2, which is a Y in the mVβ8 family. Originally, Maynard and coworkers (42) reported that this Y made very similar contacts with MHCII in the interactions of two different TCRs with two different MHC proteins (see 1U3H and 1D9K in Reference 42) and suggested that these residues might form an anchor point for TCRs that use mVβ8.2 to interact with MHCII. A recent paper from the Garcia group expanded on this idea (85). We subsequently found that in the B3K506 and YAe-62 TCRs (which use the closely related mVβs, 8.1 and 8.2) VβY48 binds to yet another MHCII molecule quite similarly. We and the Garcia group also pointed out a similar interaction in a human TCR that uses the related hVβ3 to bind to human HLADR1 and that when mVβ8-containing TCRs interact with MHCI, VβY48 also interacts with the MHCI α1 helix in a similar location (32, 84a, 85). The interaction of position 48 of VβCDR2 with MHC is similar to that discussed above for Vα, in that a conserved small amino acid on the top of the α1 helix at position 69 of MHCI and position 61 of MHCII creates an area for VβY48 to approach the helix. Mainly via Van der Waals interactions, VβY48 can pivot and slide somewhat on the helix without losing contact. In the Vβs of a number of structures, other amino acids (V, A, R, and Q) at Vβ48 contact

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a

b Vβ βs with an N/Y at aa29

c Vβs with a Y at aa48

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29 hVβ3.1 hVβ8.2 hVβ12.1 hVβ12.2 hVβ12.3 hVβ13.2 hVβ13.4, 7 hVβ13.6.1.6.4 hVβ14.1 hVβ16.1 hVβ20.1 hVβ22.1 hVβ25.1

D - MDHEN I - SGHDY T - ENHRY T - WNHNN T - WSHSY D - MNHEY D - MNHGY D - MNHNY N - MNHEY I - SGHDN EGT SNPN I - SNH L Y I - KGHSY

mVβ5.2 mVβ8.1 mVβ8.2a mVβ8.3 mVβ16 mVβ19a

I - SGHSN T - NNHDY T - NNHNN T - NSHNY V - SNH L Y I - NGHSY

Vβs with a D/E at aa54

48 hVβ1.1 hVβ1.2 hVβ3.1 hVβ5.2 hVβ5.3 hVβ5.6 hVβ11.1 hVβ12.1 hVβ12.2.1 hVβ12.2.2 hVβ12.2.3 hVβ22.1 hVβ23.1 hVβ24.1

QYY - NGEERA HYY - NGEERA F SY - DVKMKE QYY - EEEERQ QYY - EK E E RG QYY - R E E ENG YSY - GVNS T E YSY - GVKDTD YSY - GVQDTN YSY - GVKDTN YSY - GVHDTN S F Y - NN E I SE S F Y - EKMQSD HYYNKDF NN

mVβ5.1 mVβ5.2 mVβ7 mVβ8.1 mVβ8.2a mVβ8.3 mVβ9 mVβ16

QHY - - DKMER QHY - - EKVER I SY - DVDSNS YSY - VADS T E YSY - GAGS T E YSY - GAGN LQ F YYDK I L NRE NF Y - NGKVME

54 hVβ3.1 hVβ6.1 hVβ6.2 hVβ6.3 hVβ6.4 hVβ6.5 hVβ6.6 hVβ6.8 hVβ7.2 hVβ7.3 hVβ8.1, 2 hVβ8.3 hVβ11.1 hVβ12.1 hVβ12.3 hVβ13.1 hVβ13.3 hVβ13.4 hVβ13.6 hVβ13.7 hVβ14.1 hVβ16.1 hVβ18.1 hVβ21.1 hVβ21.2 hVβ21.3 hVβ22.1 hVβ23.1 hVβ25.1

F SY - DVKMKE Y FQ - GTGAAD Y FQ - NEAQL D Y FN - YEAQQD Y FQ - NEAQL E Y FQ - GNSAPD Y FN - YEAQPD YSQ - SDAQRD VYS - L EERVE VYN - F KEQT E Y FN - NNVP I D Y FR - NRAP L D YSY - GVNS T E YSY - GVKDTD YSA - AAD I TD YSV - GAG I TD YSA - SEGT TD YSV - AAG I TD YSV - GAG I TD YSA - AAGT TD YSM - NVEV TD HF V - KESKQD Y LQ - KEN I I D QFQ - DESVVD RYE - NEEAVD QFQ - NNGVVD S F Y - NNE I SE S F Y - EKMQSD S FQ - NENV FD

mVβ2a mVβ4 mVβ8.1 mVβ8.2a mVβ9 mVβ11 mVβ12 mVβ13 mVβ16 mVβ17a mVβ19a mVβ20

T L R - SPRDKE SYS - YQK LMD YSY - VADS T E YSY - GAGS T E F YYD K I L NRE Y FR - NQAP I D Y FR - SKS LME Y FR - DEAV I D NF Y - NGKVME NFR - NEE I ME Y FQ - NED I I D Y F Q - NQQP L D

Figure 6 TCR CDRβ sequences that contain amino acids that frequently bind to the same position of MHC. Listed are (a) the human and mouse V region CDR1β sequences that include an N/Y at position 29, (b) the human and mouse V region CDR2β sequences that contain a Y at position 48, and (c) the human and mouse V region CDR2β sequences that contain a D/E at position 54. Sequences are highlighted according to the criteria in Figure 5. The sequences were selected from 59 human Vβs and 23 mouse Vβs and were numbered as in Figure 3a.

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the MHC α1 helix in the same general area. Therefore, the amino acid at this position is a key determinant in anchoring the TCR to the MHC.

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a hVβ3.1 hVβ6.1 hVβ6.2 hVβ6.3 hVβ6.4 hVβ6.5 hVβ6.6 hVβ6.8 hVβ8.1, 2 hVβ8.3 hVβ11.1 hVβ12.1 hVβ12.2.1 hVβ12.2.2 hVβ12.2.3 hVβ12.3 hVβ13.1, 6 hVβ13.2 hVβ13.3 hVβ13.4 hVβ13.5 hVβ13.7 hVβ14.1 hVβ15.1 hVβ17.1 hVβ18.1 hVβ20.1.1 hVβ20.1.3 mVβ6a mVβ8.1 mVβ8.2 mVβ8.3 mVβ9 mVβ11 mVβ12 mVβ13 mVβ14 mVβ19a mVβ20

Another position in Vβ CDR2 worth consideration is Vβ46. As shown in Figure 7a, Vβ46 is a Y or F in a wide variety of Vβs, including 16 of the 22 structures analyzed

46 F S Y - D V KMK E Y FQ - GTGA A D Y FQ - NE AQL D Y F N - Y E A Q QD Y FQ - NE AQL E Y F Q - GN S A P D Y F N - Y E AQP D Y SQ - SDAQRD Y F N - NNV P I D Y F R - NRA P L D Y S Y - GVNS T E Y S Y - GV KD T D Y S Y - GVQD T N Y S Y - GV KD T N Y S Y - GVHD T N YSA - AAD I T D Y S V - GAG I T D Y S V - GEGT T A Y S A - S EGT T D Y S V - A AG I T D Y SN - T AGT T G Y S A - A AGT T D Y SM - N V E V T D YS F - DVKDI N Y SQ - I VND F Q Y LQ - KEN I I D Y S V - G I GQ I S Y S I - G I DQ I S Y S I - T END L Q YSY - VADS T E Y S Y - GAGS T E Y S Y - GAGN L Q F Y YDK I L N RE Y F R - NQA P I D Y F R - S K S L ME Y FR - DEAV I D Y S I - - - T V GQ Y FQ - NED I I D Y F Q - N QQ P L D

b

hVβ β12.1 (2NX5)

Q72

Q65

hVβ6.2 (1MI5)

Q72

hVβ13.1 (1BD2)

Q72

T69

HLA-B35

f

c

R65

A69

hVβ17.1 (1OGA)

hVβ13.1

Q72

(1AO7)

Q72

R65

A69

Q65 G69

R65

hVβ13.3 (2AK4)

Kb

l

mVβ8.2 (1D9K)

Q72 A69

T69

HLA-A2

n

mVβ8.2 (YAe62)

Q61

Q65

R65

HLA-B35

o

mVβ8.1 (B3K506)

Q61

Q57 A64

A64

IAb

A69

p

R65

HLA-A2 hVβ8.2 (2OI9)

Q65 Q72

m

G69

Kb mVβ8.2 (1U3H)

Q61

A64

Q57 A64

IAb

Q72

Q57

Q61 Q72

hVβ8.1 (1LP9)

i

hVβ8.2 (2CKB)

HLA-A2

k

e

HLA-A2

h

Q72 A69

HLA-B8

j

hVβ13.1 (2BNQ)

HLA-A2

g

Q65 T69

d

Q57

A64

IAk hVβ13.6 (2IAM)

A61

q

Q57

Q57 A64

HLA-DR1

IAu hVβ3.1 (1FTY)

A61

HLA-DR1

Figure 7 TCRβ Y/F46 in CDR2β is often used in TCRs and often engages MHC at the same site. (a) The CDR2 sequences of the human (h) and mouse (m) Vβ regions that contain a Y/F at position 46 (highlighted in salmon pink). V regions were omitted and numbered as in Figure 3a. (b–k) The arrangements of MHC amino acids around Y46β (orange with red hydroxyl group) in ten of the solved structures of TCRs bound to MHCI, with the α1 helix of MHCI in cyan. Also indicated are the Vβ region, MHC allele, and pdb number. The structures are arranged from b to k roughly according to the predicted strength of the interaction or alignment between S51α and MHCI; there is no predicted contact for Y46β in g–k. (l–q) Structures are shown as in b–k, but for TCRs bound to MHCII, with the α helix of MHCII in cyan. Structures were selected from the references in Figure 2. 188

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in Figure 2d. In 5 of the MHCI structures, VβY/F46 contacts the MHC α1 helix, usually via α65 (Figure 7b–f ). In the other 5 MHCI structures VβY/F46 is too far away to bind MHC, but it lies in roughly the same position (Figure 7g–k ). In all 6 VβY/F46-containing TCR/MHCII structures, this amino acid contacts Q57 of the α1 helix (Figure 7l–q). Finally, Vβ CDR2 very frequently has an acidic D or E at position 54 (Figure 6). Four of the 14 TCR structures with MHCI have VβD/E54 in contact with R65 of the α1 MHC helix. Because so many different mouse and human Vβs have D/E54, this residue is a candidate for a conserved and sometimesused interaction site. Six of the eight TCR structures with MHCII contain VβD/E54; in all cases D/E54 contacts Q57 of the MHCII α1 helix (Figure 2d ). As pointed out previously, in the ﬁve cases with an E at Vβ54, there is a salt bridge to αK39 of MHCII. αK39 is not on the α helix of MHCII, but rather lies on one of the β strands of MHCII as it protrudes beyond the peptide-binding site. This solvent-exposed amino acid is highly conserved in MHCII, although it is not involved in the structural integrity of MHCII or in peptide binding. Thus, D/E54 may represent a TCR CDR2 amino acid that has been evolutionarily selected to react with MHCI and MHCII (in this case in different ways).

The Biases for MHC Reaction Built into TCR Vαs and Vβs May Control the Orientation of TCRs on MHC Our purpose in this analysis was to determine whether the existing TCR/MHC structures could be used to make a case for germ line– encoded features of TCR Vα and Vβ elements that account for the obsession of TCRs with MHC ligands. Any analysis should account for both the conserved general diagonal orientation of the TCR on the MHC ligand as well as the considerable variation in angle and pitch of the TCR on the MHC sur-

face. We were guided in this analysis by the idea that the need to avoid negative selection in the thymus might select for TCR CDR3s that attenuated these germ line interactions and that in mice with limited negative selection highly MHC-cross-reactive T cells were common. Therefore, in examining the structures we looked for sites of interaction in which particular TCR amino acids were often but not always found and for interactions that might be ﬂexible and not necessarily lost by small shifts in orientation position. We conclude that such interactions can be identiﬁed. Our overall conclusions are summarized schematically in Figure 8. We propose that the anchor points on MHC molecules are not particular amino acid side chains, but rather two exposed areas on the tops of the MHC α-helices, centered about α169 and α2158 for MHCI and α1 64 and β173 for MHCII. These amino acids are highly conserved (mostly A and G) and contribute to dish-like areas that expose the backbones of the helices. The ﬂanking amino acids on the helices determine the size of this area. In the majority of the TCR/MHC structures, these exposed areas are major sites of interaction with speciﬁc amino acids in TCR CDR1 and CDR2 regions. Because these areas occur diagonally opposite one another on the helices, we propose that these exposed areas determine the general diagonal orientation of the TCR on the MHC. The TCR amino acids involved in these interactions are often, as previously noted (85), tyrosines that make many Van der Waals contacts with the backbones of helices and the amino acid side chains that ﬂank these areas. These types of interactions do not require a precise geometry and thus allow considerable ﬂexibility in the orientation and exact position of the TCR amino acid in question along the MHC helices, from one TCR/MHC combination to another. This may account for the ability of TCRs to bind MHCs in different structures with similar but not identical docking angles.

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Figure 8 The amino acids in Vα and Vβ that frequently contact the same areas of MHC can determine the diagonal aspect of the binding of the TCR to MHC. Ribbon representations of HLA-A2/Tax peptide (pdb 1BD2) and IAb /3K (pdb 1LNU) are shown as examples of MHCI and MHCII molecules, respectively, showing the α1/α domains (cyan), the MHCI α2 domain and the MHCII β1 domains (magenta), and the peptide ( yellow). The positions of two amino acids on the helices of each MHC molecule are labeled: α69 and α158 on MHC1 and α64 and β73 on MHCII. An area around each of these amino acids is circled. TCR amino acids most often found in contact with the MHC within these areas are listed next to each circle.

Interestingly, tyrosines are also often involved in these kinds of interactions between antibodies and protein antigens (87).

Why Are Not All the Evolutionarily Selected Rules Apparent in All TCR/MHC Complexes? If there are rules that govern the interactions between TCR V regions and MHC, then one might expect that these rules would always be manifest, particularly when the V region is involved in recognition of the same MHC allele, regardless of the peptide. However, this is not always observed. For example, investigators have reported the structures of three human TCRs containing Vβ13.1, bound to the class I protein, HLA-A2 (28, 29, 35). One TCR (1G4 190

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in 2BNQ) has many contacts between Vβ13.1 and HLA-A2, another (B7 in 1BD2) has a few, and the last (A6 in 1AO7) has none (Figure 2). These differences are due to changes in the CDR3 regions of the TCR. A6 has a very long CDR3β that lifts the TCR away from the MHC, so that contacts are not possible. We hypothesize that this ability of CDR3s to attenuate the evolutionarily selected ability of V regions to interact with MHC is needed for TCRs and the cells bearing them to escape negative selection in the thymus. The intrinsic reactivity of TCR V regions with MHC may then be even further obscured when the TCR binds MHC bound to foreign peptides, since, although these foreign peptides may provide additional positive interactions with TCRs, they may also interfere with the

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built-in TCR/MHC interactions. Such may be illustrated by the 2AK4 structure, in which TCR contacts with MHC were limited by a very pronounced bulge in the surface of MHC, caused by the extraordinary length of the engaged peptide (Figure 2). Thus, the CDR3 regions of the TCR and/or the peptide amino acids projecting out of the MHC groove determine whether or not the TCR will be lifted away from MHC or pitched such that it can contact only a portion of the MHC amino acids.

Do the Evolutionarily Selected Interactions Operate for Both MHCI and MHCII Recognition? Our analysis of highly cross-reactive T cells, such as YAe62, suggested that the same conserved MHC-reactive features of a particular CDR1 and CDR2 might apply to both MHCI and MHCII recognition. In general, we found evidence for this in our analysis, but there seem to be some differences. The rules seem somewhat more apparent with MHCII than with MHCI. For example, in TCRs with mVβ8.2, the limits of the area of contact between VβCDR2 Y48 and the α1 MHC helix appear to be more restricted on MHCII than on MHCI. Also, the MHC αK39 that forms a unique salt bridge with VβCDR2 E54 does not exist in MHCI. One factor might be that a number of the TCR/MHCI structures involve MHC alloantigens, and therefore the TCRs did not come from a repertoire shaped by negative selection by that particular MHC during development. Thus, as more TCR/MHCI structures that involve conventional peptide antigens appear, some of these differences may fade.

A Caveat TCR amino acid engagement of a particular site on MHC does not necessarily mean that the reaction is predetermined by the sequences of TCR and MHC. Instead, the reaction may be forced by other elements, such

as the docking sites of CD4 and CD8 or other phenomena (67). Another possibility is that there are limited numbers of amino acids in the CDR1 and CDR2 regions of TCRs and on the α helices of MHC, so some common reactions are bound to occur. We believe that these possibilities are unlikely for two reasons: First, the interactions are determined by the nature, not the position, of the TCR amino acid (Figure 2). Second, as proposed in Figure 8, the prominent availability of particular TCR amino acids does not in itself predict a particular area of MHC contact. Moreover, differences exist between MHCI and MHCII engagement (see above), which suggests that the system is built more subtly than suggested by the forced position argument. Ultimately, the hypothesis we propose simply focuses attention on particular amino acids whose function must be conﬁrmed in proper experiments, for example, by changing the relevant TCR amino acids in the mouse germ line and observing the effects of the changes on TCR selection and function.

DO THE BIASES OF TCRs FOR RECOGNITION OF MHC APPLY TO NONCLASSICAL MHC MOLECULES? In addition to the classical polymorphic MHC molecules, mammalian species express other relatively nonpolymorphic MHCIb and MHC-like molecules, such as HLA-E, H2M3, MR1, and members of the CD1 family (39, 88, 89). These molecules are also recognized by αβTCR-bearing T cells and are important in fending off infectious diseases. For example, HLA-E-restricted T cells have been identiﬁed in responses to bacteria and viruses, including Mycobacterium tuberculosis (Mtb) and cytomegalovirus (CMV) (90, 91). In mice, H2-M3-restricted T cells have a characteristic preactivated phenotype and mediate early T cell responses against Listeria monocytogenes (92). Similarly, T cells that recognize lipid antigens in the context of the CD1 family have been implicated in

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responses to Sphingomonas, Borrelia burgdorferi, and Mtb (93–98). One feature of this family of MHC-like proteins is that they often present nonpeptide antigens, such as glycolipids. The question then arises how TCRs that may have evolved to react with classical MHC/peptide cope with these nonconventional MHCs and their bound ligands. The nonpolymorphic MHCIb molecule, HLA-E, presents peptides from the leader sequences of conventional MHCIa molecules (99). These MHC/self-peptide complexes are recognized by inhibitory natural killer (NK) receptors as surrogate markers for MHCI ﬁdelity when MHCI expression is altered by speciﬁc pathogens (100, 101). Inhibitory NK receptors are quite tolerant of amino acid changes in HLA-E-presented peptides (39), whereas αβTCRs have conventional speciﬁcities both for HLA-E and its engaged peptides. This is exempliﬁed by the recent solution of the structure of a TCR, KK50.4, bound to HLA-E plus a CMV-encoded mimic of an MHCI leader peptide (39). The overall recognition of HLA-E by the KK50.4 TCR has the same topology as that of T cells reacting with conventional MHC, a similar diagonal binding mode, and the same TCR amino acids are used to dock with HLA-E (Figure 2). Thus, the proposed evolutionary biases of TCRs for MHC apply to reactions with both classical MHC and HLA-E. H2-M3 was originally identiﬁed as a minor histocompatibility molecule that presents a maternally linked factor (102). Like HLAE, H2-M3 is relatively nonpolymorphic but is unique to murine species. The peptidebinding groove of H2-M3 is unlike that of conventional MHCI molecules; it accommodates a formylmethionine moiety at the N terminus of the peptide that facilitates the presentation of bacterial- and mitochondrialproduced proteins (103, 104). The overall dimensions of the groove between the α-helices of H2-M3 are similar to those of conventional MHCI molecules, but the amino acids that line this pocket are primarily nonpolar, which facilitates the presentation of hy-

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drophobic peptides. The molecular basis for TCR recognition of H2-M3 remains undetermined, but the overall similarities to MHCIa suggest it may be receptive to conserved TCR interactions. MHC-related protein 1 (MR1) is another β2 m-associated, MHCIb molecule that is evolutionarily conserved among mammals (105). MR1 is associated with stimulation of, and is required for the development of, mucosal-associated invariant T (MAIT) cells. This population of T cells expresses a TCR with an invariant Vα19-Jα33 TCRα (Vα19i) in mice; in humans MAIT cells express the highly similar combination of Vα7.2 and Jα19 (Vα7.2i) (106). The natural antigens presented by MR1 in vivo are largely unknown, although researchers have suggested a role for gut ﬂora in the activation of these MAIT cells. Surprisingly, although the amino acid composition of the MR1 groove is not especially suited for glycolipid presentation [unlike that of CD1 molecules (see below)], αmannosylceramide stimulates Vα19i T cells in a MR1-dependent manner (107). The structure of MR1 is currently unknown, but, on the basis of the overall similarity to MHCIa and MHCIb, a computational analysis suggested an MHC-like fold and allowed a mutational analysis of the α-helices and the putative antigen-binding groove. The response of several T cell hybridomas to these mutants suggested both an antigen presentation function as well as a diagonal TCR docking mode similar to that of conventional T cells (108).

TCR Recognition of CD1d Molecules The human CD1 family and mouse CD1d are nonpolymorphic, β2 m-associated, MHCIlike molecules expressed predominately on hematopoetic cells. Compared with classical MHCI and MHCII molecules, the antigenbinding groove of CD1 family members is narrower, has a more pronounced bulge in its α2 helix, and is composed primarily of nonpolar amino acids to facilitate the presentation of

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diverse hydrophobic ligands, including lipids, glycolipids, and lipopeptides. Whereas the lipid tails serve to anchor these antigens in the groove, the polar head groups extend out of the groove for recognition by T cells (reviewed in Reference 58). Group I CD1 isoforms (CD1a, b, and c) present many different antigens, including lipids and lipopeptides that vary in size and shape (58, 109–113). Most human T cells reactive to CD1a, CD1b, and CD1c express diverse Vα and Vβ gene segments (110, 111, 113), which suggests that group I CD1reactive T cells may have a broad range of unique antigen speciﬁcities. Indeed, CD1aand CD1b-reactive T cell lines are highly speciﬁc for particular peptide moieties on didehydroxymycobactin antigens (for CD1a) and for carbohydrate moieties of mycolyl lipid antigens (for CD1b) (114, 115). Although the precise molecular basis for αβTCR recognition of CD1a, CD1b, or CD1c has not yet been resolved by crystallography, results from mutational analysis of the TCR-interacting face of CD1b suggest a diagonal orientation of TCR contacts, similar to the binding mode observed in conventional T cells (116). The interesting question remains how TCRs with apparently normal geometry can contact the group I CD1 proteins and classical MHC proteins in the same way, given the quite different geometry of the two types of MHC molecules. The situation is entirely different for the group II isoform of CD1d, the only CD1 isoform conserved between mouse and human (58). In contrast to the highly diverse TCR repertoire expressed by T cells reactive to other MHC molecules and group I CD1s, most CD1d-reactive cells in humans and mice are NKT cells (117, 118). NKT cells express an invariant TCRα composed of Vα24-Jα18 (Vα24i TCRα) in humans and the nearly identical Vα14-Jα18 (Vα14i TCRα) in mice (119, 120). The peripheral NKT population uses a diverse but limited number of TCRβ gene segments; the majority of the population expresses Vβ11 in humans and Vβs 2, 7, and

8.2 in mice (117, 118). The invariant TCRα chain and the complete lack of NKT cells in Jα18-deﬁcient mice strongly suggest a critical role for the TCRα in the recognition of glycolipid/CD1d complexes (121). Conversely, the role of the more diverse TCRβ is less clear. NKT cells react with a number of glycolipid antigens (118). Perhaps different TCRβs allow reaction with different glycolipids. We recently used mutagenesis analysis to assess the role of the mouse Vα14i TCRα and TCRβ chains in the recognition of different glycolipid/CD1d complexes (122). We showed that mouse Vα14i TCRα recognition of multiple α-linked glycolipids is conferred by a functional hot spot composed of germ line–encoded amino acids within CDR3α, CDR1α, and CDR2β. This functional hot spot does not differ between structurally distinct antigens, which suggests that the Vα14i TCRα functions as a pattern-recognition receptor. Results from the mutagenesis studies highlighted the critical role of germ line–encoded residues in the Vα14i TCRα,

CD1 α1

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Glycolipid α3 α2 β1 β3 CD1 α2 Figure 9 TCRs bind CD1d/glycolipid in an orientation that is completely different from that used to bind classical MHC/peptide. Shown is a plan of the contacts between the CDR1–3 loops of the α and β chains of the hVα24i TCR and a space-ﬁlling surface of CD1d and α-galactosylceramide (data taken from Reference 68, pdb 2PO6). Shown are the α1 region of CD1d (cyan), the α2 region of CD1d (magenta), the glycolipid ( yellow), as well as the CDR loops, color coded and labeled in their corresponding colors.

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CDR2β residue Mouse Vβ8.1

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Figure 10 Some of the CDR2β amino acids used to contact classical MHCI/MHCII and CD1d are identical. The amino acid sequences of mVβ8.1 and mVβ8.2 are shown in bold. Amino acids that have been shown to bind MHC in solved structures are indicated by blue squares, along with the names of the TCRs and pdb ﬁles used. White squares indicate amino acids that do not contact MHC in the indicated structure. Also shown are the results of mutational analyses, in which the amino acid in question was mutated as shown in each square. The inﬂuence of each mutation is indicated: red-ﬁlled squares indicate >1 log change in reactivity, whereas white squares indicate a <0.5 change in log reactivity (125, 126). Contact points identiﬁed by structural analysis (68) between a hVα24i TCR and hCD1d/αGC and identiﬁed by mutational analyses, using staining with CD1d/αGC tetramers, between a mVα14i TCR and mCD1d/αGC or hCD1d/αGC (122). Red-ﬁlled squares indicate >50% loss in tetramer mean ﬂuorescent intensity (MFI), pink indicates a change between 10% and 50% in tetramer MFI, and white indicates a <10% change in tetramer MFI.

and the requirement for these residues provided a basis for the extremely biased TCR repertoire of NKT cells. Although the mutagenesis studies alone could not provide the molecular basis of the interaction between the TCR and CD1d, the addition of structural 194

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data facilitated a precise deﬁnition of glycolipid recognition by NKT cells. Because CD1d has an MHC-like fold and TCRβ chains from conventional T cells could provide glycolipid/CD1d speciﬁcity, early models of the TCR-CD1d

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interaction postulated that the TCR would dock on CD1d as it does on MHCI/MHCII (55, 56). However, this hypothesis was disproved by the recently solved crystal structure of the Vα24i TCR bound to hCD1d/αGC (68). In this structure, the Vα24i TCR is oriented parallel to, rather than diagonally over, CD1d (Figure 9). The interaction of the Vα24i TCRα chain with CD1d is dominated by CDR3α (encoded by Jα18) residues bound to many conserved residues of CD1d, whereas glycolipid speciﬁcity is contributed primarily by CDR1α (encoded by Vα24/14). A question arises from the evolutionary arguments discussed above: What controls the ability of the Vα24i/14i TCRα to bind CD1d preferentially? The phenomenon may be explained by several observations: First, hJα18 residues in CDR3α interact with a large surface on CD1d and are needed for the speciﬁcity of the NKT TCRs because, although some Vα24 human CD1d/αGC TCRs have been isolated, these TCRs use Jα18 (55, 123). Second, residues in Vα24/14 CDR1α probably also play an important role. Overall, the CDR1α are nearly identical between mouse Vα14 and human Vα24 (63, 64), which suggests that evolutionary pressure has conserved these sequences, including the Vs at 26α, unique in human Vαs, and Ps at 28α, a combination that is unique in mouse Vα14 and human Vα24. These amino acids are crucial for the interaction between NKT TCRs and

CD1d/glycolipid (122) and may be one of the features that control the unique speciﬁcity of the Vα24i/14iTCRα. NKT TCR β chain residues are focused on CD1d rather than the antigen (68). Although the residues that contact CD1d (Y46β, Y48β, and E54β) also bind classical MHC (Figures 2 and 7), they do so at very different sites (compare Figures 1 and 9). The case is most striking for Vβ8.2 Y48β, which has been shown by both structural and mutational experiments to be absolutely required for Vβ8.2+ NKT TCRs to bind CD1d/glycolipid (Figure 10) (68, 122) and which is often crucial to the interaction of conventional TCRs with classical MHC (27, 30, 32, 34, 36, 42, 46, 84a, 124) (Figure 2). Vβ8.2 Y48β binds classical MHCs at a relatively conserved site (27, 30, 32, 34, 36, 42, 46, 85) (Figure 2). Nevertheless, Vβ8.2 Y48β binds CD1d at a totally different site (Figure 9) (68). Thus, Vα24i/14i NKT TCRs behave quite differently from most TCRs that recognize other MHC/ligands. Vα24i/14i TCRs use invariant TCRα chains and a restricted number of TCRβ chains. Vα24i/14i TCRs solve the problem of the different geometry of CD1d versus classical MHC by binding the two types of protein in quite different ways. Nevertheless, it is remarkable that some of the same TCR Vβ residues are crucial for binding both ligands—an example of evolutionary exploitation by the TCRs on NKT cells.

SUMMARY POINTS 1. Certain amino acids in TCR CDR1 and CDR2 regions often contact MHC. These contact points occur in similar positions on MHC in the complexes of different TCRs bound to different MHCs. 2. The target positions on MHC are often cups on the MHC α-helices that contain small amino acids. The TCR CDR amino acids can bind at variable positions within the cups and contact the MHC backbone and the side chains of adjacent amino acids.

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3. This arrangement allows ﬂexibility in the pitch of the CDR1/2 amino acid as it contacts MHC and allows the TCR to accommodate CDR3 regions and peptide ligands of different sequences and lengths and yet still bind the MHC/peptide ligand in approximately the same orientation. 4. TCRs do not usually use all their built-in abilities to react with MHC because negative selection in the thymus removes TCRs that react too well with MHC from mature repertoires. 5. The CDR1/2 amino acids often involved in binding MHC are not the same for all families of variable (V) regions. Annu. Rev. Immunol. 2008.26:171-203. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

6. The frequently used CDR1/2 amino acids may not always be the same in reactions with MHCI and MHCII. 7. The positions of the frequently used CDR1/2 amino acids and their targets on MHC suggest that these contacts may impose the usual diagonal mode of TCR binding on MHC. 8. The Vα24i/14i TCRs that bind CD1d and conventional TCRs that bind classical MHC use some of the same CDR2β residues to contact their ligands, even though the mode of binding is quite different. Vα24i/14i TCRs have adopted evolutionarily selected TCR residues for their own purposes.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS The authors thank Frances Crawford, Janice White, and Rachel Frugge for their technical help in the work that led to this analysis and Drs. Whitney MacDonald and K.C. Garcia for many helpful discussions. This work was supported in part by USPHS grants AI-17134, AI-19785, AI-22295, AI-057485, and Cancer Center CA-046934.

LITERATURE CITED 1. Claman HN, Chaperon EA, Triplett RF. 1966. Thymus-marrow cell combinations. Synergism in antibody production. Proc. Soc. Exp. Biol. Med. 122:1167–71 2. Mitchell GF, Miller JF. 1968. Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J. Exp. Med. 128:821–37 3. Mitchison NA. 1971. The carrier effect in the secondary response to hapten-protein conjugates. V. Use of antilymphocyte serum to deplete animals of helper cells. Eur. J. Immunol. 1:68–75 4. Rajewsky K. 1971. The carrier effect and cellular cooperation in the induction of antibodies. Proc. R. Soc. London Ser. B 176:385–92 196

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:171-203. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Contents

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T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs ∗

∗

Benjamin D. Medoff,1,2, Seddon Y. Thomas,1, and Andrew D. Luster1 1

Center for Immunology and Inﬂammatory Diseases, Division of Rheumatology, Allergy and Immunology, 2 Pulmonary and Critical Care Unit, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129; email: [email protected]

Annu. Rev. Immunol. 2008. 26:205–32

Key Words

First published online as a Review in Advance on November 19, 2007

asthma, T lymphocytes, chemotaxis, chemokines

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090312 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0205$20.00 ∗

These authors contributed equally to this work.

Abstract T cells are critical mediators of the allergic airway inﬂammation seen in asthma. Pathogenic allergen-speciﬁc T cells are generated in regional lymph nodes and are then recruited into the airway by chemoattractants produced by the asthmatic lung. These recruited effector T cells and their products then mediate the cardinal features of asthma: airway eosinophilia, mucus hypersecretion, and airway hyperreactivity. There has been considerable progress in delineating the molecular mechanisms that control T cell trafﬁcking into peripheral tissue, including the asthmatic lung. In this review, we summarize these advances and formulate them into a working model that proposes that T cell trafﬁcking into and out of the allergic lung is controlled by several discrete regulatory pathways that involve the collaboration of innate and acquired immune cells.

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INTRODUCTION

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Airway hyperresponsiveness (AHR): increase in narrowing of airways from airway smooth muscle hypercontraction in response to stimuli such as histamine or allergen exposure Mucus hypersecretion: tendency of the mucus-secreting cells in the airway lining of asthmatics to hypertrophy and increase mucus secretion DC: dendritic cell

Asthma is a complex syndrome broadly deﬁned by inﬂammation of the airways associated with airway hyperresponsiveness (AHR) and mucus hypersecretion (1). Asthma has a prevalence estimated at 5% of the population, making it one of the most common chronic diseases worldwide (2). Despite effective therapies, the incidence of this disease and the frequency of its signiﬁcant complications are increasing. New therapeutic approaches based on our understanding of the pathophysiology of asthma could have profound repercussions for the care of asthmatics and the health of the public in general. T lymphocytes are important mediators of adaptive immune responses and are vital for host defense against infection. The lung has a large network of antigen-presenting cells and lymphatics that aid in antigen presentation to T cells in associated lymphoid tissue located within the lung parenchyma and mediastinum. In addition, the adult lung contains T cells (the majority of which are memory cells) residing in the alveoli, airway (bronchial) lumen, intraepithelial layer, submucosa, and interstitium (3). Although important for immunity, aberrant accumulation of T cells in the lung is seen in numerous noninfectious pulmonary inﬂammatory diseases such as asthma, where T lymphocytes in the lung are believed to orchestrate an abnormal inﬂammatory process (4, 5). In asthma, the airways develop prominent inﬂammation with accumulation of activated effector T cells around the airways and in the airway lumen. It is thought that these cells are recruited into the lung and serve as the critical controllers of airway inﬂammation. In this review, we explore the mechanisms of T cell homing into the lung during asthma and present a paradigm for the mechanisms that control the movement of these cells into and out of the airways.

ASTHMA PATHOGENESIS The hallmark of asthma is chronic airway inﬂammation associated with reversible air206

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way dysfunction. This causes the classic symptoms and signs of asthma: shortness of breath, cough, mucus production, and wheezing. Although there is a growing recognition that the type of inﬂammation in the airways can be heterogeneous (6), in most cases the airway inﬂammation characteristic of asthma results from an allergic-type reaction to an inhaled antigen from the environment (socalled allergic asthma). In allergic asthma, the airways develop predominantly eosinophilic inﬂammation with prominent edema and mucus production in response to inhalation of antigen. In addition, smooth muscle cells of the airways become hyperreactive, leading to reversible bronchoconstriction in response to various stimuli. In nonallergic asthma, neutrophilic and pauci-immune forms have been described, but it is unclear if these represent distinct clinical phenotypes, as they often respond to conventional asthma therapy (6). In addition, the role of T cells in nonallergic (or noneosinophilic) asthma is currently unknown. For these reasons, in this review, we focus on the homing of T cells only in the allergic form of asthma.

Sensitization One of the earliest steps in the establishment of allergic sensitization to an antigen is the generation of an antigen-speciﬁc T cell response (Figure 1a). Antigens from the environment are constantly introduced into the airways with every breath. Multitudes of airway DCs (dendritic cells) are located under the airway epithelium, where they form an antigen-sampling network in the airway mucosa (7–9). It is thought that adjuvant signals from airway epithelium such as TSLP (thymic stromal lymphopoietin) and GM-CSF (granulocyte-macrophage colony-stimulating factor), generated in response to inhaled stimuli, inﬂuence the activation/maturation state of DCs and help determine whether a particular allergen will trigger a Th2-type inﬂammatory response or will lead to tolerance (5, 8, 10–12). Once DCs mature

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Figure 1 Asthma pathogenesis. (a) Sensitization to an allergen results from uptake by airway dendritic cells (DCs), maturation of the DCs, migration to lymphoid tissue, and antigen presentation to T cells. Maturation of the DCs requires secondary stimuli generated from epithelial cells following antigen exposure. These primed T cells then reenter the lung, where they provide surveillance for the allergen. (b) Exacerbation of allergic airway inﬂammation occurs when there is reexposure to allergen and uptake by airway DCs with presentation of antigen to airway-associated T cells as well as T cells in lymphoid tissue. T cells in the lymph node then proliferate and home to the lung, where they amplify the airway inﬂammation. (c) The full allergic airway phenotype results from cytokine production from T cells as well as from inﬂammatory mediators released from recruited eosinophils and other cells in the lung. This results in mucus hypersecretion, smooth muscle cell hyperreactivity, and airway remodeling with chronic inﬂammation. (PMN, polymorphonuclear cell; Eos, eosinophil; M, macrophage; Treg, T regulatory cell; NKT, natural killer cell.)

and migrate to the draining lymphoid tissue, they can activate antigen-speciﬁc T cells, thus sensitizing the individual to the inhaled antigen. Allergen-speciﬁc memory T cells then migrate into the airways where they can reside long term, even during asymptomatic periods (13–16).

Exacerbation Once an individual is sensitized to an antigen, reexposure in the lung rapidly leads to an exacerbation of allergic airway inﬂammation (Figure 1b). Allergic responses in the airways of sensitized individuals begin with deposition of allergen in the airway mucosa. Once in the www.annualreviews.org • T Cell Trafficking in Asthma

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Early asthmatic response (EAR): initial wave of inﬂammation, airway edema, and airway narrowing occurs within minutes following allergen exposure. Results predominantly from mast cell degranulation Late asthmatic response (LAR): second wave of inﬂammation and airway narrowing occurs within hours to days. Results largely from T cell–mediated inﬂammation Chemokine receptor: seventransmembrane G protein–coupled receptor (GPCR) that induces chemotaxis and adhesion in cells after encounter with a cognate chemokine ligand CRTH2: chemoattractant receptorhomologous molecule expressed on T helper type 2 LTB4 : leukotriene B4 PGD2 : prostaglandin D2

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airway, the allergen can react with various innate immune cells and can bind to preformed IgE and IgG antibodies in the airways, leading to immediate mediator release from mast cells and causing the so-called early asthmatic response (EAR). These mediators lead to airway edema and bronchoconstriction and initiate the inﬂammatory inﬁltrate. Inhaled allergen is also taken up by the airway DCs, which are then stimulated to mature and migrate to local lymphoid tissue (8). Once in the lymph node, the DCs present processed antigen to both memory and naive T lymphocytes, leading to T cell activation and differentiation. These activated effector T lymphocytes then migrate into the airways, where they secrete cytokines and other mediators, which direct the late asthmatic response (LAR) in the airway.

Asthma Inflammation In response to cytokines secreted by effector T cells, structural cells as well as other leukocytes in the lung are stimulated to release further inﬂammatory mediators (Figure 1c). This release then stimulates the phenotypic endpoints in asthma: recruitment of eosinophils, mucus hypersecretion by goblet cells in the airway epithelium, smooth muscle cell hyperreactivity, and airway remodeling with chronic inﬂammation. Eosinophils release further mediators and cytokines into the airways and are critical for the full manifestation of the asthma phenotype (17–20).

RECRUITMENT OF T CELLS INTO THE ASTHMATIC LUNG After exposure to allergen, the number of T cells in the airways increases dramatically, amplifying allergic inﬂammation (21, 22). Experiments in animal models have demonstrated that, following allergen challenge, antigenspeciﬁc T cells in the airways do not proliferate but are recruited into the lung from regional lymph nodes, leading to the increase in T cells in the airways (23). Allergen-speciﬁc naive and memory T cells in the lymph nodes are stimulated to proliferate by antigenMedoff

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loaded and -activated DCs that have recently migrated from the airways. Thus, a therapeutic strategy that interrupts the recruitment of pathogenic effector T cells generated in lymphoid tissue from homing to the lung or that enhances the recruitment of regulatory T cells (Tregs) into the lung should effectively interrupt airway inﬂammation in asthma (24). To accomplish this goal, the molecular mechanisms that regulate T cell homing into and out of the asthmatic lung need to be fully elucidated.

T CELL TRAFFICKING IN THE ALLERGIC ASTHMATIC RESPONSE Chemokines, Lipid Chemoattractants, and Cognate Receptors The recruitment of leukocytes into tissue during the asthmatic response is controlled by chemokines and lipid chemoattractants (14, 25). Chemokines (chemotactic cytokines) are 8- to 10-kDa proteins, which are secreted from cells to form a gradient. Chemokines signal through speciﬁc cognate chemokine receptors [seven-transmembrane G protein– coupled receptors (GPCRs)] to induce functional responses, such as adhesion and chemotaxis. Engagement of chemokine receptors with cognate chemokine ligand leads to the movement of cells toward the chemokine gradient. In addition to chemokine receptors, subsets of T cells also express speciﬁc seventransmembrane GPCR lipid chemoattractant receptors, including BLT1 and CRTH2, which play important roles in recruitment during the early asthmatic response. Leukotriene B4 (LTB4 ) and prostaglandin D2 (PGD2 ) signal through BLT1 and CRTH2, respectively, to induce chemotactic recruitment of T cells and other leukocytes.

Chemokine-Mediated T Cell Trafficking into Tissue T cell trafﬁcking is a tightly regulated and complex process that involves expression of

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Homeostatic and tissue-speciﬁc T cell trafﬁcking is required for regulated immune surveillance. Speciﬁc chemokine receptors have been associated with T cell homing to tissue-speciﬁc sites, including CCR7 and CXCR5 for the lymph node, CCR9 for the small intestine, and CCR4, CCR8, and CCR10 for the skin (27, 28) (Figure 2). It was initially proposed that CD4+ T cells could be divided into naive (CD45RA+

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numerous different adhesion molecules and chemokines (26). These signals allow T cells to arrest on endothelial cells and migrate into the tissue. Once in the tissue, further signals generated from chemokine gradients can then guide T cells into speciﬁc microcompartments, such as the alveoli or bronchial lumen (3). Thus, the signals that determine the migration and homing of T cells into the lung are crucial for effective immune function. However, inappropriate homing of T cells into the lung may also be a critical component of asthma.

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Figure 2 Homeostatic- and organ-speciﬁc T cell homing. Speciﬁc chemokine receptors have been associated with organ-speciﬁc trafﬁcking and recirculation. CCR7 has been associated with T cell homing and recirculation into lymphoid tissue. Initially, it was proposed that “central” memory CCR7+ T cells must lose CCR7 expression and gain tissue-speciﬁc chemokine receptors in order to home to tissue-speciﬁc locations. However, CCR7+ memory T cells have been found in the tissue, and it is now believed that CCR7 deﬁnes T cells with lymph node–homing potential. In addition, CCR10 is enriched on CLA+ skin homing T cells (S), whereas CCR9 is enriched on α4 β7 hi gut (G) homing T cells. These receptors are believed to deﬁne T cells with either skin- or gut-homing potential. No such chemoattractant receptor has yet been identiﬁed for lung-speciﬁc T cell homing (L). www.annualreviews.org • T Cell Trafficking in Asthma

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CCR7+ ), central memory (CD45RA− CCR7+ ), and effector memory (CD45RA− CCR7− ) populations based on CD45RA and CCR7 expression patterns (29). According to this model, naive and central memory T cells express CCR7 and can home to lymph nodes, whereas effector memory T cells must lose expression of CCR7 and gain tissuespeciﬁc homing receptors. This model has subsequently been reﬁned by observations that CCR7+ T cells are found in tissue sites, including the lung (22, 30–32). We and others have found that CCR7 expression on T cells in the tissue marks T cells capable of recirculation to the lymph nodes (33, 34). CCR7 expression is likely regulated dynamically in vivo and may be responsible for the return of peripheral tissue T cells to the lymph node in the process of antigen surveillance. Other examples of tissue-speciﬁc homing receptors include CCR4, CCR8, and CCR10 for the skin and CCR9 for the small intestine. CCR4, CCR8, and CCR10 expression is associated with CLA+ skin-homing T cells, and the cognate ligand for CCR10, CCL27, is highly expressed in the skin at baseline (35). The CCR4 ligand, CCL17, and the CCR8 ligand, CCL1, are also found in the skin, but their pattern of expression is less restricted than that of CCL27 (28, 36–38). Similarly, CCR9 is associated with α4 β7 hi small intestine–homing T cells, and its cognate ligand CCL25 is found in epithelial crypts of the small intestine (39, 40). Currently, no chemoattractant receptor has been speciﬁcally associated with homing to the lung or airways. This suggests that homing to the lung or airway at baseline may require a combination of chemoattractant receptors or that there is an as yet unidentiﬁed chemoattractant receptor involved in this process.

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Inflammation-Driven Migratory Patterns of Memory T Cells In addition to this model of homeostaticand tissue-speciﬁc trafﬁcking, T lymphocytes 210

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must be able to respond to sites of inﬂammation (Figure 3). This inﬂammatory response can lead to control of viral or bacterial infection but can also be dysregulated and lead to an overexuberant response to an allergen in allergic asthma. In Th2-type inﬂammation, production of STAT6-inducible chemokines from resident cells at sites of inﬂammation recruits IL-4-, IL-5-, or IL-13-secreting Th2 cells through the cognate Th2-associated receptors CCR4 and CCR8 (41). This serves to locally amplify Th2-type inﬂammation. In contrast, in Th1 inﬂammation, production of STAT1-inducible chemokines from resident cells at sites of inﬂammation recruits IFN-γ-secreting Th1 and CD8 effector T cells through the cognate chemokine receptors, CCR5, CXCR3, and CXCR6 (42). This serves to locally amplify Th1-type inﬂammation. Although these receptors have been observed on in vitro polarized T cells, the ex vivo chemoattractant receptor expression pattern is more complex. For example, in human peripheral blood Th1, but not Th2, cells are CXCR3+ CCR4− , whereas Th2, but only rarely Th1, cells are CXCR3− CCR4+ (32). However, the population of CXCR3+ CCR4+ T cells is a mixture of Th1, Th2, and Th0 cells. This pattern illustrates the important interrelationship of chemokine receptor expression proﬁles on T cells in vivo. Th17 inﬂammation is only beginning to be understood. Because Th17 inﬂammation is STAT3 inducible, it will be of interest to identify STAT3-inducible chemokines that could be involved in Th17 cell recruitment (43). A recent study has shown that human Th17 cells are enriched in the CCR6+ CCR4+ subset of CD4+ T cells, whereas Th1 cells are enriched in the CCR6+ CXCR3+ and CCR6− CXCR3+ subsets and Th2 cells in the CCR6− CCR4+ subset (44). CCR6 is highly enriched on memory T cells and immature, but not mature, DCs (45, 46). The ligand for CCR6, CCL20, is highly expressed on inﬂamed epithelium, which could bring together memory T and immature DCs in the tissue

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Figure 3 Inﬂammation-driven migratory patterns of effector T cells. CD4+ T cell–driven inﬂammation is characterized by the predominant type of cytokines secreted by inﬁltrating T cells. In Th1-type inﬂammation, IFN-γ predominates; in Th2-type inﬂammation, IL-4, IL-5, and IL-13 predominate; and in Th17-type inﬂammation, IL-17 predominates. These cytokines in turn induce speciﬁc subsets of IFN-γ-inducible chemokines; IL-4- and IL-13-inducible chemokines; or IL-17-inducible chemokines at the site of inﬂammation. These chemokines then induce the recruitment of T cells that express the speciﬁc cognate receptors for these inﬂammatory chemokines.

(47, 48). Another recent study also identiﬁed Th17 cells as enriched in the CCR2+ CCR5− subset of CD4+ memory T cells (49). Further studies are required to more fully differentiate chemoattractant receptors on Th17 cells from other subsets of T cells. In contrast to Th1, Th2, and Th17 cells, Tregs are not responsible for promoting cytokine-induced inﬂammation but rather for regulating inﬂammation generated by other T cells subsets. Tregs can trafﬁc as part of immune surveillance homeostatically, but also respond to inﬂammation-induced chemokine gradients to inhibit overly exuberant T cell responses. It is necessary to understand Treg trafﬁcking patterns in the context of the asthmatic response because these CD4+ T cells

could play an important role in controlling allergic asthma. In mouse models of disease, the chemokine receptors CCR4 and CCR7 play critical roles in Treg trafﬁcking and function in the draining lymph node (50–52), whereas CCR5 is important for Treg trafﬁcking and function at sites of inﬂammation (53, 54). In addition, CCR8 has been observed on human and mouse FoxP3+ Tregs (52, 55, 56). Tregs likely function in peripheral tissue as well as in draining lymphoid tissue and utilize different sets of chemokine receptors to accomplish these functions. A summary of the key factors for Th1, Th2, Th17, and Treg cell development and the chemokine receptors expressed on these different T cell subsets is shown in Figure 4.

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Figure 4 Chemokine receptors and CD4+ T cell subsets. Chemokine receptors associated with the differentiation of CD4+ T cells under the inﬂuence of the indicated cytokines and transcription factors. The pattern of chemokine receptors expressed by a given CD4+ T cell subset indicated in the ﬁgure does not deﬁne that subset nor is it necessarily speciﬁc for that subset, but it does dictate the speciﬁc homing potential of that speciﬁc T cell subset. Although certain chemokine receptors have been associated with a speciﬁc CD4+ T cell subset, chemokine receptor expression in vivo is complex and overlapping. This expression allows for recruitment of a variety of T cells under inﬂammatory conditions for immune control.

CD4+ T CELL SUBSETS IN THE ALLERGIC ASTHMATIC RESPONSE Segmental allergen challenge: instillation of known allergen into a segment of the lung to induce an in vivo allergic response in sensitized individuals Bronchoalveolar lavage (BAL): wash of alveolar and bronchial airspaces using a bronchoscope to instill ﬂuid and retrieve cells and protein secretions

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Allergic airway inﬂammation is thought to be mediated by Th2 cells and their cytokines. However, it is also becoming increasingly clear that other subsets of CD4+ T cells, such as Th1 (57–59), Th17 (60), Treg (61– 63), and CD1d-restricted NKT cells (64, 65), may also play a role in the modulating allergic pulmonary inﬂammation. Other non-CD4+ T cell subsets, such as CD8+ T cells and γδ T cells, are also present in the airway, although at numbers 2-fold and 20-fold less than CD4+ T cells, respectively (66). However, the role of CD8+ T (67, 68) and γδ T (69, 70) cells in the asthmatic response is less clear. Therefore, we focus on CD4+ T cells in this review. Medoff

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Th2 Cells In support of this central role of Th2 cells, levels of Th2-type cytokines, including IL4, IL-5, and IL-13, were elevated at baseline in the airways of human asthma patients (71, 72). Following segmental allergen challenge, increases in Th2 cytokines have been measured in allergic asthmatic subjects (21, 73–76). In addition, murine models of allergic pulmonary inﬂammation and AHR have elegantly demonstrated that Th2 cells and the cytokines they secrete were essential for the development of eosinophilia and AHR (77–80). Furthermore, adoptive transfer of effector Th2 cells into naive mice followed by exposure to inhaled antigen induced the pathophysiologic features of asthma, including eosinophilic inﬂammation, mucus hypersecretion, and AHR (81–83), demonstrating that these cells were fully capable of producing the asthma phenotype. Taken together, these data reveal that Th2 cells and the cytokines they secrete are central to the pathogenesis of asthma. Although Th2 cells play a critical role in asthma pathogenesis, they are not the predominant T cells in human BAL (bronchoalveolar lavage) from asthmatic subjects, as measured by intracellular cytokine staining (22, 84–86). Following segmental allergen challenge, the total number of IL4-producing T cells was increased, but the percentage of IL-4 producing Th2 cells was unchanged from prechallenge levels (22, 66, 84). Thus, other T cell subsets must be recruited to the asthmatic BAL along with Th2 cells.

Th1 In addition to Th2 cells, Th1 lymphocytes are also recruited into the lung, where they are capable of either increasing or decreasing the severity of the asthmatic response (57–59). In an adoptive transfer murine model of asthma, it was demonstrated that antigen-speciﬁc Th1 cells could suppress Th2-mediated airway

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eosinophilia but did not prevent Th2 cell recruitment or eosinophilia in the lung (57). However, other adoptive transfer mouse models have shown that Th1 cells actually enhance Th2 trafﬁcking and airway eosinophilia (58, 59). Additionally, in human subjects, inhaled IFN-γ led to an increase in lymphocyte recruitment to the airway (87). Following human segmental allergen challenge, the total number of IFN-γ-producing T cells as measured by intracellular cytokine staining increased, but the percentage of IFN-γ Th1 cells decreased somewhat in comparison to prechallenge levels (22, 66, 84). This indicates that Th1 cells trafﬁc to the asthmatic lung, but the decrease in percentage of Th1 cells indicates that these cells may not be preferentially recruited in comparison to other T cell subsets. Th1 cells appear to play an important role in Th2-mediated allergic response and could be recruited through expression of CXCR3 and CCR6, as well as other chemoattractant receptors.

Th17 The role of Th17 cells in the allergic response is only beginning to be investigated. Recently, a murine model of asthma demonstrated that IL-17 is required for the induction of allergic asthma but negatively regulates established asthma (60). IL-17 production was increased following allergen challenge in this model and was further increased in the absence of regulation by the IL-4Rα. Similar to the mouse model, human IL-17 was produced by peripheral blood T cells from allergic asthmatic subjects in response to allergic stimulus and was increased in the human asthmatic airway as compared to healthy controls (88–90). These data, along with observed patterns of chemoattractant receptor expression, suggest that Th17 cells are recruited to the asthmatic airway following allergen challenge through CCR4 and/or CCR6 and may be involved in both the initiation and regulation of the asthmatic response.

Treg Recruitment of Tregs may play a role in the suppression of allergic inﬂammation. Depletion of CD4+ CD25hi Tregs before sensitization with allergen led to an increase in Th2 cytokines, IgE concentrations, eosinophilia, and AHR in C3H mice normally resistant to the allergic asthmatic response (61). Adoptive transfer of antigen-speciﬁc Tregs into allergen-sensitized mice before allergen challenge inhibited AHR, eosinophil recruitment, and Th2 cytokine production through an IL10-dependent process (63). These studies support the notion that Tregs play a key role in regulating the allergic asthmatic process. The balance of Tregs likely regulates the allergic asthmatic response in humans, although this is still being investigated. A recent study showed that the frequency of allergenspeciﬁc IL-10-producing peripheral blood T cells was increased in healthy controls, whereas the frequency of allergen-speciﬁc IL4-producing peripheral blood T cells was increased in atopic asthmatics (91). Because Tregs utilize IL-10 in regulation of T cell inﬂammatory processes, this study suggests that allergic asthmatics may lack high enough levels of IL-10 and thus Treg activity. In addition, a recent study has demonstrated that pediatric asthmatic subjects have a lower percentage of CD4+ CD25hi BAL T cells than do children with chronic cough, healthy controls, or even asthmatic subjects treated with corticosteroids (92). Tregs may play a signiﬁcant role in allergic asthma responsiveness following encounter with antigen. The chemokine receptors expressed on Tregs, including CCR4, CCR5, CCR7, and CCR8, allow these cells to be recruited to either the allergic lung or lymph node to exert their regulatory activity following allergen challenge. Tregs appear to play an important role in regulating the allergic asthmatic response, but further study is required to understand the mechanisms by which they exert their activity and their requirements for tissue localization in this process.

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CD1d-Restricted NKT Cells

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A recent report has suggested that CD1drestricted natural killer T (NKT) cells comprise the majority (>60%) of T cells in the asthmatic BAL as compared to healthy controls or subjects with sarcoidosis (64). However, although CD1d-restricted NKT cells were signiﬁcantly enriched in the BAL as compared to blood, we have found that they made up only <2.5% of the lymphocyte gate (93). We believe that the discrepancy between the study by Akbari et al. (64) and our data (93) may be due to nonspeciﬁc staining from alveolar macrophages and cellular debris in the BAL. Further, following segmental allergen challenge, the percentage of BAL CD1d-restricted NKT cells was unchanged from prechallenge levels (22). This suggests that while CD1d-restricted NKT cells are enriched tenfold in the BAL as compared to peripheral blood, CD1d-restricted NKT cells are not the predominant T cell in the asthmatic BAL before or after segmental allergen challenge. Because CD1drestricted NKT cells express high levels of CXCR3, CCR5, and CCR6 as well as intermediate levels of CCR4 (94), these receptors would be predicted to recruit CD1drestricted NKT cells to the asthmatic lung but not with >1000-fold enrichment compared to peripheral blood NKT concentrations, as suggested by Akbari et al. (64). More recent data are consistent with the conclusion that the frequency of CD1drestricted NKT cells in the BAL may have been substantially overestimated, possibly for technical reasons (95). A number of other groups have also observed low numbers of BAL NKT cells in persons with mild, moderate, and severe asthma, subjects with chronic obstructive pulmonary disease (COPD), and healthy controls (95–99), in contrast to the ﬁndings of Akbari et al. (64). In mouse models of asthma, CD1drestricted NKT cells have also been shown to be critical for generating the allergic airway response through production of IL-4 and

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IL-13 (65, 100). However, the requirement for CD1d-restricted NKT cells (and CD8 T cells) in the development of T cell–dependent airway inﬂammation has been called into question by a recent study that shows that CD4+ T cells, but not CD1d-restricted NKT or CD8 T cells, are sufﬁcient to induce allergic airway inﬂammation (68). Thus, although CD1d-restricted NKT cells likely contribute to the asthmatic response through secretion of Th2 cytokines, we believe that conventional T cells make up the predominant T cell population in the asthmatic BAL and play the predominant role in promoting the allergic asthmatic response.

CHEMOKINES AND CHEMOATTRACTANT RECEPTORS IN THE ALLERGIC ASTHMATIC RESPONSE Although the recruitment of eosinophils into the airways is an important component in the pathogenesis of asthma (see the sidebar, Eosinophil Recruitment into the Lung During Allergic Inﬂammation), the trafﬁcking of activated T lymphocytes into the airways clearly orchestrates the asthmatic inﬂammatory response (101). Consistent with this, after exposure to allergen, there is a dramatic increase in the number of T cells in the airways of humans and in murine models of asthma (1, 21, 22, 41, 71, 102, 103). In a murine model of asthma, we determined that the trafﬁcking of Th2 cells into the airway both before and following allergen challenge is controlled by G protein–coupled chemoattractant receptors (104). Although CCR4 and CCR8 are expressed on Th2 cells, deletion of these receptors in mouse models of asthma has yielded conﬂicting results (105–110). The preservation of Th2 cell trafﬁcking in the absence of individual chemoattractants or their receptors has led us to hypothesize that multiple pathways, acting through multiple chemoattractants and their receptors, direct Th2 cell trafﬁcking into the airways.

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Comparisons between normal controls and human asthmatics, as well as human segmental allergen challenge studies, have yielded information about T cell trafﬁcking and chemoattractant receptor expression. At baseline, healthy controls and asthmatic subjects express similar chemokine receptors on BAL T cells (31, 84, 86, 111, 112). BAL T cells were enriched in expression of speciﬁc receptors as compared to peripheral blood T cells and expressed high levels of CCR5, CXCR3, and CXCR4; intermediate levels of CCR4, CCR6, CCR7, and CXCR6; and low levels of BLT1, CCR1, CCR2, CCR3, and CXCR1 along with other chemoattractant receptors. The increased expression of speciﬁc chemoattractant receptors on BAL T cells as compared to peripheral blood T cells may be related to the larger memory T cell population in the BAL and to the requirement for speciﬁc chemokine receptors in trafﬁcking to tissue sites. Following allergic stimulus, a number of chemokines are produced and lead to recruitment of T cells along with other leukocytes to the allergic lung. Human CCR2, CCR3, CCR4, CCR5, CCR6, CXCR1/2, CXCR3, CRTH2, but not CCR8 or CXCR6 ligands were increased following segmental allergen challenge (22, 102, 113–118). Similarly, in the mouse models of asthma, ligands of the chemoattractant receptors, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR1, CXCR3, and BLT1 were induced in the lung at early time points (119–122). These data suggest that a number of chemokine receptors play a role in recruitment of T cells and other allergic mediators. We and others have examined the proﬁles of chemoattractant receptor expression on T cells before and after segmental allergen challenge to determine whether speciﬁc chemoattractant receptors were preferentially utilized in T cell recruitment (22, 31). We hypothesized that the Th2-associated receptors CCR3, CCR4, and CCR8 would be increased following allergen challenge, whereas the Th1-associated receptors CCR5,

EOSINOPHIL RECRUITMENT INTO THE LUNG DURING ALLERGIC INFLAMMATION Although T cells likely orchestrate allergic asthmatic inﬂammation, it is the eosinophil that remains the primary leukocyte recruited into the airway during allergic inﬂammation. Recent data indicate that eosinophils are necessary for the full manifestations of the asthma phenotype (17, 18), and thus the mechanisms that control their recruitment into the airway are important for asthma pathogenesis. Eosinophils express the chemokine receptor CCR3 and are likely recruited via production of the STAT6-dependent chemokines CCL11 and CCL24 (in both mice and humans) and CCL26 (in humans only), all of which are upregulated in the airways of asthmatics (117, 173–175). Data in animal models indicate that both CCL11 and CCL24 contribute to eosinophil recruitment, with CCL24 likely being the dominant eosinophilactive chemokine (176). STAT6 expression in a lung cell is necessary for eosinophil recruitment (41), and recent data (159) suggest that alternatively activated macrophages may mediate eosinophil recruitment. We also have data suggesting that myeloid DCs can express CCL24 and may also help mediate eosinophil recruitment (unpublished observation). These data demonstrate that eosinophil recruitment into the airway in allergic inﬂammation is largely controlled by myeloid cells in the lung via a STAT6-dependent mechanism.

CXCR3, and CXCR6 would be decreased following challenge. Although total numbers of T cells were increased following allergen challenge, we did not observe an increase in the percentage of CD4+ or CD8+ T cells expressing any chemoattractant receptor (22). Instead, we noted a statistically signiﬁcant decrease in CCR6 and CXCR3 expression on CD4+ and CD8+ BAL T cells 24 h following allergen challenge. We believe that CCR6 and CXCR3 were downregulated following encounter with cognate ligand, indicating that these two receptors might play an underappreciated role in T cell recruitment to the asthmatic airway. However, we would also argue that these data have not ruled out the signiﬁcance of other chemoattractant receptors in the recruitment of T cells to the allergic lung. www.annualreviews.org • T Cell Trafficking in Asthma

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Overall, these data suggest that T cells are recruited to the asthmatic BAL through a number of different chemoattractant receptors. Because the chemokines expressed following the asthmatic response can recruit a variety of T cells, it is important to understand the role of different T cell subsets in the asthmatic response.

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THE PHASES OF T CELL TRAFFICKING IN ALLERGIC ASTHMA: A NEW PARADIGM In the past few years, we and others have made considerable progress in delineating the mechanisms that control T cell trafﬁcking in asthma. These data have led us to the central hypothesis of this review: T cell trafﬁcking into the allergic lung is controlled by several discrete regulatory pathways that involve the collaboration of innate and acquired immune cells. We propose a four-step model that includes an initiation phase, a propagation phase, an ampliﬁcation phase, and a resolution phase (Figure 5). The ﬁrst two phases of T cell recruitment into the airways are mediated by chemoattractants produced by innate immune cells stimulated by the direct interactions of inhaled allergens. Binding and cross-linking of mast cell–associated IgE by allergens lead to mast cell activation, resulting in the release of a ﬁrst set of chemoattractants that initiate T cell recruitment (phase I). We propose that early T cell recruitment is then propagated by the recognition of allergen by PAMP (pathogen-associated molecular pattern) receptors on innate immune cells in the lung, which leads to the release of chemokines (phase II). The third phase of T cell recruitment is mediated by chemoattractants produced in response to the interactions of resident airway cells with the Th2 cells arriving in the initiation and propagation phases of recruitment. These early recruited Th2 cells elaborate Th2-type cytokines that stimulate resident innate immune cells of the airways to release this third set of STAT6inducible chemoattractants that dramatically 216

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amplify Th2 cell recruitment (phase III), and also mediate eosinophil recruitment. Finally, a subset of T cells leaves the airways and enters the lymphatics during a resolution phase (phase IV) that we believe may be involved in both attenuating allergic pulmonary inﬂammation and in establishing immunologic memory. In this review, we present data that support this paradigm for the control of T cell migration into and out of the lung.

PHASE I: INITIATION—MAST CELL–DEPENDENT TH2 CELL RECRUITMENT Within minutes of inhaling allergen, IgE on mast cells binds the foreign substance, leading to cross-linking of mast cell IgE Fc receptors (Figure 5a). This cross-linking stimulates the mast cells to release preformed inﬂammatory mediators, such as histamine, and also stimulates the cells to produce and release other mediators, including leukotrienes, TNF-α, chemokines, and Th2-type cytokines. These mediators start the development of mucosal edema, mucus secretion, and airway reactivity. In addition, some of these mediators initiate the recruitment of T cells into the airways (123, 124). We have recently shown that one of these mediators, LTB4 , is a potent chemoattractant for Th2 and Th1 cells (125– 127). Furthermore, we have demonstrated that deletion of the LTB4 receptor, BLT1, leads to an early defect in T cell accumulation in the airways in a model of allergic pulmonary inﬂammation that involves active immunization to a foreign antigen (chicken egg albumin or OVA) with IgE formation. However, this defect was not seen when OVA-speciﬁc Th2 cells were adoptively transferred into unimmunized mice, which do not form OVAspeciﬁc IgE (127). Because one of the important differences between these two models is the formation of IgE and mast cell stimulation with OVA challenge, these results led us to hypothesize that in response to allergen challenge, IgE-activated mast cells release LTB4 into the airway, which mediates the

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Figure 5 Proposed multiphase model of T cell trafﬁcking into the asthmatic airway. (a) Phase I: initial T cell recruitment into the airways. Antigen-IgE complexes stimulate airway mast cells to release various mediators, such as leukotriene B4 (LTB4 ) and CCL1, which leads to Th2 cell recruitment through BLT1 and CCR8. (b) Phase II: propagation of T cell recruitment into the airways. Antigen directly interacts with innate immune cells, such as macrophages via TLRs, to stimulate the release of chemokines, such as CXCL10 and CCL20. These chemokines then recruit more T cells into the airways via their receptors CXCR3 and CCR6. (c) Phase III: proposed mechanism for ampliﬁcation of T cell recruitment into the airways. IL-4 and IL-13 produced by Th2 cells already recruited into the airways stimulate innate immune cells, such as myeloid DCs, to release the STAT6-dependent chemokines CCL17 and CCL22. These chemokines amplify Th2 cell recruitment into the airways through their receptor CCR4. (d ) Phase IV: proposed mechanism for resolution of T cell accumulation in the airways. Th2 cells in the airways that express CCR7 are recruited into the lymphatics through the action of CCL21. This then leads to efﬂux of the Th2 cells out of the lung and into the lymph node.

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initial recruitment of Th2 cells into the lung (126). Mast cells also produce and release the chemokines CCL1, CCL17, CCL22, and the chemical histamine, all of which are chemotactic for Th2 cells through interactions with their G protein–coupled receptors CCR8 (CCL1), CCR4 (CCL17, CCL22), and H1R (histamine) (123, 128–130). CCL1 release may be particularly important for early Th2 cell recruitment, as recently demonstrated in a murine model of mast cell-dependent allergic airway inﬂammation (130). Mast cell stimulation by allergen-IgE complexes thus leads to the release of multiple chemoattractants able to initiate the recruitment of T lymphocytes into the airways. The sequential release of LTB4 and chemokines from mast cells is consistent with the differing time courses of the generation of these chemoattractants: LTB4 is rapidly generated enzymatically from membrane lipids within minutes, whereas the release of chemokines may require transcription and translation and consequently take several hours. In addition to generating leukotrienes, activated mast cells also generate prostaglandins, including PGD2 , which could lead to Th2 cell recruitment through the CRTH2 receptor (131–133). In addition to preformed IgE antibodies, IgG antibodies to allergens can also stimulate mast cells and macrophages to release inﬂammatory mediators (134–136). These mediators, which include LTB4 , can further enhance the recruitment of T cells into the airways following allergen exposure. The initial recruitment of T cells in the airways allows the immune system to respond to a perceived threat within minutes as opposed to the hours it would take to mount an adaptive immune response. DC maturation and function may also depend on an initial interaction with memory T cells in the airways (8). Thus, the almost immediate recruitment of memory T cells into the airways following allergen challenge may also help prime the adaptive immune response by increasing potential DC–T cell interactions. Furthermore,

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mast cell degranulation may contribute to the efﬁciency and intensity of the LAR by enhancing T cell recruitment into the airways (124, 127). In summary, soon after the inhalation of allergen, antigen interaction with preformed IgE and IgG antibodies leads to early mediator release, including chemoattractants. These chemoattractants then rapidly induce the initial recruitment of T cells into the airways.

PHASE II: PROPAGATION—INNATE IMMUNE-DEPENDENT TH2 CELL RECRUITMENT In addition to binding to antigen-speciﬁc antibodies, components in allergens, or associated viral infections or particles in the air, can stimulate innate immune cells in the airways through interactions with PAMP receptors, such as the TLRs (Figure 5b). TLR stimulation in asthma exacerbations may occur through LPS (lipopolysaccharide) found in antigens. In addition, viral infections (a common cause of asthma exacerbations) can stimulate innate immune cells as well as epithelial cells via TLR stimulation (137– 140). We have previously shown that most Th2 cell recruitment into the lung following allergen challenge is dependent on tissuederived STAT6-induced chemokines, such as CCL22 and CCL17. Although STAT6 plays a signiﬁcant role in the regulation of antigen-speciﬁc Th2 cell trafﬁcking, there is evidence that STAT6-independent pathways contribute to T cell trafﬁcking into the allergic lung. Namely, after the adoptive transfer of antigen-speciﬁc Th2 cells and antigen challenge, a small number of antigen-speciﬁc Th2 cells do enter the lung of STAT6−/− mice (41, 104). We hypothesize that PAMP receptorinducible chemokines and their corresponding chemokine receptors represent a STAT6independent pathway of antigen-speciﬁc Th2 cell and Th1 cell trafﬁcking. This could have direct relevance to upper respiratory viral

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infections, such as the inﬂuenza virus (141) or respiratory syncytial virus (142), which can activate TLR7 and TLR3. In addition, aeroallergens, like animal dander and house dust mites, contain LPS. The importance of TLRs in asthma is indicated by studies demonstrating that the inﬂammatory response in a murine model of asthma is partially dependent on the adaptor molecule, MyD88, a downstream mediator of TLR signaling (12). In addition, LPS in allergens can stimulate antigen-presenting cells in the lung and may play role in Th2 cell polarization (143). Recently, Jung et al. (144) have shown that TLR4 stimulation in an animal model of allergic airway inﬂammation enhances both Th1 and Th2 cell recruitment into the lung. Consistent with this, there is evidence in the published literature that TLR activation leads to CXCL10 and CCL20 expression (145, 146) and that CXCL10 and CCL20 are increased in the context of allergen exposure (22, 102, 121, 122, 147, 148). CXCR3 and CCR6, the receptors recognizing CXCL10 and CCL20, are expressed on T cells that inﬁltrate the lung during allergic inﬂammation (22, 31, 111). Furthermore, CCR6-deﬁcient mice have decreased airway resistance, fewer lung eosinophils, and reduced lung IL-5 levels compared with their wild-type counterparts (121, 149), and the transfer of CCR6+/+ T cells from sensitized wild-type mice into CCR6−/− mice results in an increase in airway eosinophilia and IL5 levels (149). In addition, studies with the CXCL10 knockout and transgenic mice suggest that recruitment of CXCR3+ T cells exacerbates the allergic asthmatic response (122). These data are also consistent with some of our recent ﬁndings suggesting that CXCR3 and CCR6 are mediators of T cell recruitment into the airways of asthmatics following allergen challenge (22). Although Th2 and Th1 cells are likely the primary mediators of allergic airway inﬂammation, some data suggest that other T cell subsets may also be involved in asthma pathogenesis. These include Th17 cells, NKT cells,

CD8+ T cells, and γδ T cells. Th17 cells express CCR6, for which the ligand (CCL20) is made in this phase (44, 150, 151). In addition, NKT cells, CD8+ T cells, and γδ T cells may all be recruited by similar mechanisms as Th1 cells in this phase, with CXCL10 and CCL20 both potentially mediating the recruitment of these cells (22, 94, 152). Allergen deposition in the lung may also interact with other inﬂammatory pathways, such as the complement cascade, and enzymes in the airways, such as chitinases, which could then inﬂuence the production of chemokines (153, 154). For example, the activation of the C5a receptor following allergen challenge increases the secretion of the CCL17 and CCL22 by airway DCs in a murine model of asthma. These data suggest that allergen-induced pattern recognition receptor stimulation on innate immune cells in the lung may contribute to T cell recruitment. We hypothesize that allergen or viral components directly stimulate innate immune cells, activating these cells to present antigen and to produce chemokines and other inﬂammatory cytokines. These mediators then propagate the inﬂammatory response and enhance the recruitment of Th2 cells into the lung. In addition, these mediators may also initiate the recruitment of Th1 cells and other T cell subsets into the lung, which could further enhance the homing of Th2 cells into the lung (58, 59, 144).

PHASE III: AMPLIFICATION— STAT6-DEPENDENT TH2 CELL RECRUITMENT Following the early trafﬁcking of Th2 cells into the airways, there is a dramatic ampliﬁcation of Th2 cell recruitment (Figure 5c). Previous research by our group has shown that deletion of STAT6 in the lung eliminates more than 80% of Th2 cell trafﬁcking following allergen (41, 42). These studies also demonstrated that STAT6 expression in a resident lung cell is necessary for effective www.annualreviews.org • T Cell Trafficking in Asthma

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Th2 cell and eosinophil recruitment into the airways. Consistent with this ﬁnding, STAT6−/− mice had signiﬁcantly lower production of the Th2-active chemokines CCL17 and CCL22 and the eosinophil-active chemokines CCL11 and CCL24. Using a model of pulmonary Th2 inﬂammation induced by infection by Nippostrongylus brasiliensis, Voehringer et al. (19, 20) demonstrated that STAT6 expression in a bone marrow– derived myeloid cell is necessary for Th2 cell trafﬁcking into the lung. These data suggest that the critical cellular mediator of Th2 cell recruitment is a bone marrow–derived nonlymphocyte cell in the lung, possibly a myeloid-derived cell such as a pulmonary macrophage or DC. We hypothesize that Th2 cells recruited into the lung in the initial and propagation phases produce IL-4 and IL-13 (155), which stimulate myeloid-derived cells in the lung, such as pulmonary macrophages and/or DCs, to produce STAT6-dependent chemokines. These chemokines then serve to amplify the recruitment of Th2 cells and initiate eosinophil trafﬁcking into the lung. Previous work by others has demonstrated that depletion of CD11c+ cells in the lung after sensitization to OVA but prior to challenge prevents the development of allergic airway inﬂammation, suggesting that airway DCs and/or alveolar macrophages (both are CD11c+ ) control key aspects in the pathogenesis of the inﬂammatory response (156). When alveolar macrophages or DCs were added back to the lung in those experiments, the phenotype was restored only with DC transfer, suggesting that DCs were necessary for allergic inﬂammation. In addition, pulmonary DCs accumulate around the airways during allergic inﬂammation and produce CCL17 and CCL22 (9, 153, 156, 157, 158). We have recently found that selective depletion of a population of CD11b+ /CD11c+ myeloid DCs in the lung signiﬁcantly impairs Th2 cell trafﬁcking into the lung following Th2 cell transfer and antigen challenge (B.D. Medoff and A.D. Luster, unpublished observation). Our data demonstrate that myeloid

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DCs in the lung are both necessary and sufﬁcient for Th2 cell trafﬁcking in this model. Based on these data, we propose that Th2 cells recruited into the airways in the initiation and propagation phase produce cytokines that stimulate pulmonary myeloid DCs to make CCL17 and CCL22, which ampliﬁes Th2 cell recruitment into the airways. In addition, these cells, as well as alternatively activated pulmonary macrophages (159), produce CCL24, which stimulates eosinophil recruitment into the airways. Tregs likely are involved in the control of airway inﬂammation and have been shown to accumulate in the airway during the development of allergic inﬂammation in an animal model of asthma (62). Tregs express the chemokine receptors CCR4 and CCR8 (56) and thus may be recruited by the release of CCL1 during phase I, and CCL17 and CCL22 during phase III of the paradigm.

PHASE IV: RESOLUTION/ MEMORY—CCR7-DEPENDENT TH2 CELL EXIT In murine models of asthma, intense airway inﬂammation produced by sensitization and challenge with allergen decreases over time following the cessation of allergen exposure (160–162) (Figure 5d ). The resolution of airway inﬂammation following allergen challenge is clearly an important process for limiting the allergic response and may prevent complications of inﬂammation. For example, deletion of matrix metalloproteinase2 expression in a murine model of asthma reduced the efﬂux of inﬂammatory cells out of the lung, which slowed the resolution of airway inﬂammation and was associated with increased mortality from asphyxiation (163). Despite its potential importance in asthma, the mechanisms of Th2 cell clearance from the lung are poorly understood. Some cells may become apoptotic (160), and others are likely cleared with expectoration (163). Recently, we have demonstrated that a subset of Th2 effector cells express CCR7 and migrate

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into the lymphatics under the guidance of CCL21 expression (33). Although the exact contribution of this CCR7-directed migration for Th2 cell egress from the lung remains to be determined, this may be one of several mechanisms involved in the resolution of allergic airway inﬂammation. In addition, migration of Th2 cells from the lung into the lymphatics may help establish immunologic memory and, importantly, contribute to future exacerbations following allergen reexposure. We propose that CCR7-mediated T cell egress from the asthmatic lung contributes to both the resolution of a primary allergic response as well as to the establishment of T cell memory and generation of local lung recall immune responses.

EXPANSION OF THE PARADIGM FOR OTHER LUNG CELL TYPES The contributions of structural lung cells, such as epithelial cells, endothelial cells, ﬁbroblasts, and smooth muscle cells, in asthma pathogenesis remain less well deﬁned than other innate immune cells in the lung. All these cell types produce chemokines when properly stimulated (164–173) and thus may

also contribute to T cell recruitment into the lung in asthma. However, our data and data from others indicate that mast cells and myeloid cells in the lung are likely the primary mediators of T cell recruitment (19, 20, 156)

CONCLUSIONS In this review, we have tried to identify the critical mediators of T cell migration into and out of the lung during the development and resolution of allergic airway inﬂammation. On the basis these data, we propose a new paradigm in T cell trafﬁcking in asthma, involving three phases of chemoattractant-directed T cell recruitment into the lung, followed by a resolution phase of chemoattractant-directed T cell exit (Figure 5). This paradigm involves extensive interactions between the innate immune cells in the lung and T cells. In addition, each phase is regulated by distinct mechanisms, providing an overlapping network of pathways involved in T cell recruitment. This new paradigm may help provide novel insights into the pathogenesis of asthma and identify potential therapeutic targets to treat this common disorder.

SUMMARY POINTS 1. Asthma is a chronic inﬂammatory disease of the airways that is often caused by an allergic-type reaction to antigen. 2. In most cases the inﬂammatory reaction is orchestrated by CD4+ T cells that are recruited into the airways. 3. Recruitment of T cells into the lung is orchestrated by secretion of chemokines and chemoattractants in response to allergen exposure. 4. IgE-stimulated mast cells mediate early T cell recruitment through the secretion of leukotriene B4 and the chemokines CCL1, CCL17, and CCL22. 5. Allergen may also directly stimulate early T cell recruitment into the airway through interaction with PAMP receptors on macrophages, leading to secretion of chemokines, such as CXCL10 and CCL20. 6. The great majority of T cell recruitment is mediated by exposure of mucosal DCs to the cytokines IL-4 and IL-13, which leads to STAT6-mediated secretion of CCL17 and CCL22. www.annualreviews.org • T Cell Trafficking in Asthma

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7. Tregs may also be recruited into the airways via the action of chemokines. 8. Some T cells in the airways express CCR7 and are recruited out of the lung back into lymphoid tissue, which may reduce airway inﬂammation but may also provide a mechanism for memory responses to allergens.

DISCLOSURE STATEMENT

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The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS This work was supported by the National Institutes of Health grants K08 HL072775 to B.D. Medoff and R01 AI40618 and T32 AI060548 to A.D. Luster and a Dana Foundation Award in Human Immunology to A.D. Luster.

LITERATURE CITED

7. Established that specialized DCs lie beneath the airway epithelium and provide a sampling network for the airway that serves as the basis for allergen sensitization. 8. Demonstrated that airway DCs interact with airway T cells following antigen uptake, which helps stimulate DC maturation.

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19 and 20. These two papers demonstrated that bone marrow–derived myeloid cells control Th2 cell recruitment into the lung during the establishment of a type 2 immune response.

22. Found that multiple chemokine receptors are expressed on T cells recruited into the airway of allergic asthmatic individuals following allergen challenge, including chemokine receptors associated with Th1 cells. 23. Demonstrated that adoptively transferred Th2 cell effectors migrated into the lymph node and lung parenchyma. Following allergen challenge, lymph node T cells proliferated and migrated into lung tissue; T cells in the lung did not proliferate but produced cytokines.

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41. Established that Th2 cell trafficking into the lung during allergic airway inflammation is both STAT6 dependent and independent.

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116. Holgate ST, Bodey KS, Janezic A, Frew AJ, Kaplan AP, Teran LM. 1997. Release of RANTES, MIP-1α, and MCP-1 into asthmatic airways following endobronchial allergen challenge. Am. J. Respir. Crit. Care Med. 156:1377–83 117. Lilly CM, Nakamura H, Belostotsky OI, Haley KJ, Garcia-Zepeda EA, et al. 2001. Eotaxin expression after segmental allergen challenge in subjects with atopic asthma. Am. J. Respir. Crit. Care Med. 163:1669–75 118. Sur S, Kita H, Gleich GJ, Chenier TC, Hunt LW. 1996. Eosinophil recruitment is associated with IL-5, but not with RANTES, twenty-four hours after allergen challenge. J. Allergy Clin. Immunol. 97:1272–78 119. Henderson WR Jr, Lewis DB, Albert RK, Zhang Y, Lamm WJ, et al. 1996. The importance of leukotrienes in airway inﬂammation in a mouse model of asthma. J. Exp. Med. 184:1483–94 120. Fulkerson PC, Zimmermann N, Hassman LM, Finkelman FD, Rothenberg ME. 2004. Pulmonary chemokine expression is coordinately regulated by STAT1, STAT6, and IFNγ. J. Immunol. 173:7565–74 121. Lukacs NW, Prosser DM, Wiekowski M, Lira SA, Cook DN. 2001. Requirement for the chemokine receptor CCR6 in allergic pulmonary inﬂammation. J. Exp. Med. 194:551–55 122. Medoff BD, Sauty A, Tager AM, Maclean JA, Smith RN, et al. 2002. IFN-γ-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inﬂammation in a mouse model of asthma. J. Immunol. 168:5278–86 123. Bryce PJ, Mathias CB, Harrison KL, Watanabe T, Geha RS, Oettgen HC. 2006. The H1 histamine receptor regulates allergic lung responses. J. Clin. Invest. 116:1624–32 124. Stephens R, Chaplin DD. 2002. IgE cross-linking or lipopolysaccharide treatment induces recruitment of Th2 cells to the lung in the absence of speciﬁc antigen. J. Immunol. 169:5468–76 125. Goodarzi K, Goodarzi M, Tager AM, Luster AD, von Andrian UH. 2003. Leukotriene B4 and BLT1 control cytotoxic effector T cell recruitment to inﬂamed tissues. Nat. Immunol. 4:965–73 126. Luster AD, Tager AM. 2004. T-cell trafﬁcking in asthma: lipid mediators grease the way. Nat. Rev. Immunol. 4:711–24 127. Tager AM, Bromley SK, Medoff BD, Islam SA, Bercury SD, et al. 2003. Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat. Immunol. 4:982–90 128. Gombert M, Dieu-Nosjean MC, Winterberg F, Bunemann E, Kubitza RC, et al. 2005. CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhanstype dendritic cells to sites of atopic skin inﬂammation. J. Immunol. 174:5082–91 129. Oliveira SH, Lukacs NW. 2001. Stem cell factor and IgE-stimulated murine mast cells produce chemokines (CCL2, CCL17, CCL22) and express chemokine receptors. Inﬂamm. Res. 50:168–74 130. Gonzalo JA, Qiu Y, Lora JM, Al-Garawi A, Villeval JL, et al. 2007. Coordinated involvement of mast cells and T cells in allergic mucosal inﬂammation: critical role of the CC chemokine ligand 1:CCR8 axis. J. Immunol. 179:1740–50 131. Hirai H, Tanaka K, Yoshie O, Ogawa K, Kenmotsu K, et al. 2001. Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J. Exp. Med. 193:255–61 132. Nagata K, Hirai H, Tanaka K, Ogawa K, Aso T, et al. 1999. CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cellderived factor(s). FEBS Lett. 459:195–99 www.annualreviews.org • T Cell Trafficking in Asthma

127. Established leukotriene B4 as a potent chemoattractant for effector T cells and as a mediator of early T cell recruitment following allergen challenge in sensitized mice.

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133. Nagata K, Tanaka K, Ogawa K, Kemmotsu K, Imai T, et al. 1999. Selective expression of a novel surface molecule by human Th2 cells in vivo. J. Immunol. 162:1278–86 134. DuBourdieu DJ, Morgan DW. 1990. Multiple pathways for signal transduction in the regulation of arachidonic acid metabolism in rat peritoneal macrophages. Biochim. Biophys. Acta 1054:326–32 135. Hsueh W, Gonzalez-Crussi F, Henderson S. 1987. LTB4 production and lysosomal enzyme release by rat alveolar macrophages: effects of phagocytosis, receptor binding, and ionophore stimulation. Exp. Lung Res. 13:385–99 136. Rankin JA, Schrader CE, Smith SM, Lewis RA. 1989. Recombinant interferon-γ primes alveolar macrophages cultured in vitro for the release of leukotriene B4 in response to IgG stimulation. J. Clin. Invest. 83:1691–700 137. Everard ML. 2006. The relationship between respiratory syncytial virus infections and the development of wheezing and asthma in children. Curr. Opin. Allergy Clin. Immunol. 6:56–61 138. Heymann PW, Platts-Mills TA, Johnston SL. 2005. Role of viral infections, atopy and antiviral immunity in the etiology of wheezing exacerbations among children and young adults. Pediatr. Infect. Dis. J. 24:S217–22, discussion S220–21 139. Platts-Mills TA, Erwin EA, Woodfolk JA, Heymann PW. 2006. Environmental factors inﬂuencing allergy and asthma. Chem. Immunol. Allergy 91:3–15 140. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, et al. 2006. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441:101–5 141. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. 2004. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303:1529–31 142. Rudd BD, Smit JJ, Flavell RA, Alexopoulou L, Schaller MA, et al. 2006. Deletion of TLR3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection. J. Immunol. 176:1937–42 143. Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K. 2002. Lipopolysaccharide-enhanced, Toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J. Exp. Med. 196:1645–51 144. Jung YW, Schoeb TR, Weaver CT, Chaplin DD. 2006. Antigen and lipopolysaccharide play synergistic roles in the effector phase of airway inﬂammation in mice. Am. J. Pathol. 168:1425–34 145. Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, et al. 2001. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167:5887–94 146. Reibman J, Hsu Y, Chen LC, Bleck B, Gordon T. 2003. Airway epithelial cells release MIP-3α/CCL20 in response to cytokines and ambient particulate matter. Am. J. Respir. Cell Mol. Biol. 28:648–54 147. Pichavant M, Charbonnier AS, Taront S, Brichet A, Wallaert B, et al. 2005. Asthmatic bronchial epithelium activated by the proteolytic allergen Der p 1 increases selective dendritic cell recruitment. J. Allergy Clin. Immunol. 115:771–78 148. Thomas MS, Kunkel SL, Lukacs NW. 2002. Differential role of IFN-γ-inducible protein 10 kDa in a cockroach antigen-induced model of allergic airway hyperreactivity: systemic vs local effects. J. Immunol. 169:7045–53 149. Lundy SK, Lira SA, Smit JJ, Cook DN, Berlin AA, Lukacs NW. 2005. Attenuation of allergen-induced responses in CCR6−/− mice is dependent upon altered pulmonary T lymphocyte activation. J. Immunol. 174:2054–60

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150. Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, et al. 2007. Phenotypic and functional features of human Th17 cells. J. Exp. Med. 204:1849–61 151. Kao CY, Huang F, Chen Y, Thai P, Wachi S, et al. 2005. Up-regulation of CC chemokine ligand 20 expression in human airway epithelium by IL-17 through a JAK-independent but MEK/NF-κB-dependent signaling pathway. J. Immunol. 175:6676–85 152. Glatzel A, Wesch D, Schiemann F, Brandt E, Janssen O, Kabelitz D. 2002. Patterns of chemokine receptor expression on peripheral blood γδ T lymphocytes: strong expression of CCR5 is a selective feature of Vδ2/Vγ9 γδ T cells. J. Immunol. 168:4920–29 153. Kohl J, Baelder R, Lewkowich IP, Pandey MK, Hawlisch H, et al. 2006. A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma. J. Clin. Invest. 116:783–96 154. Zhu Z, Zheng T, Homer RJ, Kim YK, Chen NY, et al. 2004. Acidic mammalian chitinase in asthmatic Th2 inﬂammation and IL-13 pathway activation. Science 304:1678–82 155. Venkayya R, Lam M, Willkom M, Grunig G, Corry DB, Erle DJ. 2002. The Th2 lymphocyte products IL-4 and IL-13 rapidly induce airway hyperresponsiveness through direct effects on resident airway cells. Am. J. Respir. Cell Mol. Biol. 26:202–8 156. van Rijt LS, Jung S, Kleinjan A, Vos N, Willart M, et al. 2005. In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma. J. Exp. Med. 201:981–91 157. Hammad H, Smits HH, Ratajczak C, Nithiananthan A, Wierenga EA, et al. 2003. Monocyte-derived dendritic cells exposed to Der p 1 allergen enhance the recruitment of Th2 cells: major involvement of the chemokines TARC/CCL17 and MDC/CCL22. Eur. Cytokine Netw. 14:219–28 158. Beaty SR, Rose CE, Sung SJ. 2007. Diverse and potent chemokine production by lung CD11bhigh dendritic cells in homeostasis and in allergic lung inﬂammation. J. Immunol. 178:1882–95 159. Voehringer D, van Rooijen N, Locksley RM. 2007. Eosinophils develop in distinct stages and are recruited to peripheral sites by alternatively activated macrophages. J. Leukoc. Biol. 81:1434–44 160. Duez C, Tomkinson A, Shultz LD, Bratton DL, Gelfand EW. 2001. Fas deﬁciency delays the resolution of airway hyperresponsiveness after allergen sensitization and challenge. J. Allergy Clin. Immunol. 108:547–56 161. Mojtabavi N, Dekan G, Stingl G, Epstein MM. 2002. Long-lived Th2 memory in experimental allergic asthma. J. Immunol. 169:4788–96 162. Tomkinson A, Cieslewicz G, Duez C, Larson KA, Lee JJ, Gelfand EW. 2001. Temporal association between airway hyperresponsiveness and airway eosinophilia in ovalbuminsensitized mice. Am. J. Respir. Crit. Care Med. 163:721–30 163. Corry DB, Rishi K, Kanellis J, Kiss A, Song LZ, et al. 2002. Decreased allergic lung inﬂammatory cell egression and increased susceptibility to asphyxiation in MMP2-deﬁciency. Nat. Immunol. 3:347–53 164. Gounni AS, Hamid Q, Rahman SM, Hoeck J, Yang J, Shan L. 2004. IL-9-mediated induction of eotaxin1/CCL11 in human airway smooth muscle cells. J. Immunol. 173:2771–79 165. Kumagai N, Fukuda K, Nishida T. 2000. Synergistic effect of TNF-α and IL-4 on the expression of thymus- and activation-regulated chemokine in human corneal ﬁbroblasts. Biochem. Biophys. Res. Commun. 279:1–5 166. Li L, Xia Y, Nguyen A, Lai YH, Feng L, et al. 1999. Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells. J. Immunol. 162:2477–87 www.annualreviews.org • T Cell Trafficking in Asthma

156. Suggests that CD11c+ DCs in the airway control Th2 cell recruitment into the lung during allergic inflammation.

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167. Lilly CM, Nakamura H, Kesselman H, Nagler-Anderson C, Asano K, et al. 1997. Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids. J. Clin. Invest. 99:1767–73 168. Matsukura S, Stellato C, Plitt JR, Bickel C, Miura K, et al. 1999. Activation of eotaxin gene transcription by NF-κB and STAT6 in human airway epithelial cells. J. Immunol. 163:6876–83 169. Miyamasu M, Misaki Y, Yamaguchi M, Yamamoto K, Morita Y, et al. 2000. Regulation of human eotaxin generation by Th1-/Th2-derived cytokines. Int. Arch. Allergy Immunol. 122(Suppl. 1):54–58 170. Rothenberg ME, Luster AD, Leder P. 1995. Murine eotaxin: an eosinophil chemoattractant inducible in endothelial cells and in interleukin 4-induced tumor suppression. Proc. Natl. Acad. Sci. USA 92:8960–64 171. Sekiya T, Miyamasu M, Imanishi M, Yamada H, Nakajima T, et al. 2000. Inducible expression of a Th2-type CC chemokine thymus- and activation-regulated chemokine by human bronchial epithelial cells. J. Immunol. 165:2205–13 172. Shore SA. 2004. Direct effects of Th2 cytokines on airway smooth muscle. Curr. Opin. Pharmacol. 4:235–40 173. Ying S, Meng Q, Zeibecoglou K, Robinson DS, Macfarlane A, et al. 1999. Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic (Intrinsic) asthmatics. J. Immunol. 163:6321–29 174. Lilly CM, Woodruff PG, Camargo CA Jr, Nakamura H, Drazen JM, et al. 1999. Elevated plasma eotaxin levels in patients with acute asthma. J. Allergy Clin. Immunol. 104:786–90 175. Yuan Q, Campanella GS, Colvin RA, Hamilos DL, Jones KJ, et al. 2006. Membranebound eotaxin-3 mediates eosinophil transepithelial migration in IL-4-stimulated epithelial cells. Eur. J. Immunol. 36:2700–14 176. Pope SM, Zimmermann N, Stringer KF, Karow ML, Rothenberg ME. 2005. The eotaxin chemokines and CCR3 are fundamental regulators of allergen-induced pulmonary eosinophilia. J. Immunol. 175:5341–50

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:205-232. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania 19104; email: [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:233–59

Key Words

First published online as a Review in Advance on November 19, 2007

immunological synapse, distal pole complex, signaling, lymphocyte

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090347 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0233$20.00

Abstract T cell cytoarchitecture differs dramatically depending on whether the cell is circulating within the bloodstream, migrating through tissues, or interacting with antigen-presenting cells. The transition between these states requires important signaling-dependent changes in actin cytoskeletal dynamics. Recently, analysis of actin-regulatory proteins associated with T cell activation has provided new insights into how T cells control actin dynamics in response to external stimuli and how actin facilitates downstream signaling events and effector functions. Among the actin-regulatory proteins that have been identiﬁed are nucleation-promoting factors such as WASp, WAVE2, and HS1; severing proteins such as coﬁlin; motor proteins such as myosin II; and linker proteins such as ezrin and moesin. We review the current literature on how signaling pathways leading from diverse cell surface receptors regulate the coordinated activity of these and other actin-regulatory proteins and how these proteins control T cell function.

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INTRODUCTION APC: antigen-presenting cell IL-2: interleukin-2

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TCR: T cell receptor

Regulated reorganization of the actin cytoskeleton is important for multiple aspects of T cell function, including signaling and differentiation along discrete developmental lineages, migration through tissues, and execution of effector functions. Early studies testing the role of actin in T cell activation were based on the use of actin-disrupting agents such as cytochalasin D (CytoD) (see sidebar: Actin Depolymerizing Agents). These studies showed that actin is required for adherence to target cells and cytotoxic activity (1), as well as for signaling events associated with Ca2+ ﬂux and downstream changes in gene expression (2). However, these studies revealed a complex role for actin. For example, in T cells stimulated with superantigen-pulsed antigenpresenting cells (APCs), CytoD treatment results in diminished Ca2+ ﬂux, but stimulation with anti-CD3 antibodies results in a prolonged Ca2+ response (2). Similarly, although one study showed that CytoD treatment impairs the activation of the interleukin-2 (IL-2) promoter (3), another found that at low doses of CytoD, Ca2+ signaling is prolonged and IL2 production increases (4). These ﬁndings and more recent data described below suggest that actin ﬁlaments play a dual role, enhancing T cell activation by promoting conjugate formation and the assembly of signaling complexes, but also downregulating activation, perhaps by facilitating molecular movements that culminate in the internalization of the T cell receptor (TCR).

ACTIN DEPOLYMERIZING AGENTS The two most commonly used pharmacological agents to perturb actin dynamics are cytochalasin D and latrunculin A. Neither inhibitor actually disassembles polymerized actin ﬁlaments. Instead, they induce net actin depolymerization by preventing new actin polymerization. Latrunculin A sequesters actin monomers, whereas cytochalasin D binds to the fast-growing (barbed) ends of actin ﬁlaments, preventing monomer addition.

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Recent studies focusing on individual actin-regulatory proteins have greatly advanced our understanding of how actin dynamics are regulated and have provided new insights into how actin functions to facilitate or terminate T cell activation. This review focuses on the roles of those proteins in T cell function and on the interplay between cytoskeletal changes and the signaling events associated with T cell activation.

T CELL MOVEMENTS AND SHAPE CHANGES The actin cytoskeleton controls T cell shape, which changes dramatically depending on whether the cell is circulating in the bloodstream, migrating through tissues, or interacting with APCs (Figure 1).

Microvillar Dynamics in Circulating T Cells Circulating T cells are covered in short microvilli containing parallel bundles of highly dynamic actin ﬁlaments (Figure 1, cell 1) (5). Low-afﬁnity adhesion molecules such as Lselectin and α4β7 integrin (VLA-4) are concentrated at the tips of the microvilli (6), whereas molecules involved in tight adhesion, e.g., leukocyte function–associated antigen 1 (LFA-1, αLβ2), are distributed randomly or excluded from microvilli (7, 8). This segregation is thought to facilitate tethering and rolling along vessel walls and to minimize nonspeciﬁc adhesion (Figure 1, cell 2) (9). Interaction of T cells with soluble and endothelium-displayed chemokines triggers rapid microvillar collapse as well as upregulation of integrin avidity through a process involving release of integrins from the cortical actin cytoskeleton (9, 10). This leads to the arrest of the lymphocyte on the endothelium and, ultimately, to transmigration across the endothelial wall (Figure 1, cells 3 and 4). The mechanisms that control microvillar formation and collapse in T cells are not fully understood. Several studies point to a key role

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Target cell

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Figure 1 Actin remodeling in response to environmental cues. 1. Circulating T cells are decorated with microvilli containing parallel actin bundles anchored in cortical actin ﬁlaments. 2. Segregation of adhesion molecules with respect to microvilli facilitates tethering and rolling along the vascular endothelium. 3. Chemokine stimulation induces microvillar collapse and a switch from tethering and rolling to tight adhesion mediated by high-afﬁnity integrin interactions. 4. The T cell must then squeeze between endothelial junctions to enter the underlying tissue. 5. As the T cell migrates through tissues, a leading edge rich in branched actin ﬁlaments pushes the cell forward, while the uropod trails behind. 6. Upon recognizing an APC, the T cell forms a tight contact rich in branched actin ﬁlaments. This contact leads to the formation of the immunological synapse (IS), where active actin polymerization is associated with formation and inward movement of TCR microclusters. Cortical actin, most likely comprised of linear ﬁlaments, remains at sites peripheral to the IS, forming a scaffold for protein sequestration at the distal pole complex (DPC).

for proteins of the ezrin, radixin, and moesin (ERM) family, which link plasma membrane proteins to cortical actin ﬁlaments. Treatment of T cells with antisense oligonucleotides to both ezrin and moesin results in dramatic loss of microvilli (11). Similarly, T cells expressing a dominant-negative ERM mutant are devoid of microvilli, whereas cells expressing a constitutively active form exhibit enhanced microvillar structures (9). Chemokine-induced microvillar loss is accompanied by dephosphorylation of ERM proteins at a regulatory C-terminal threonine (see below), an event that inhibits their linker function (9). In addition to ezrin and moesin, WASp may contribute to microvillar structure, as T cells

from patients with mutations in WASp exhibit defective microvilli (12–14). However, a recent study by Higgs and coworkers failed to ﬁnd microvillar abnormalities in WASpdeﬁcient T cells, when analyzed immediately ex vivo (5). Because WASp promotes the formation of branched actin ﬁlaments rather than the linear ﬁlaments found in microvilli, WASp may affect microvillar dynamics only indirectly.

Migration Within Tissues Once a T cell receives the appropriate cues to cross the vascular wall, it begins to migrate within tissues. Migrating T cells exhibit a www.annualreviews.org • Actin in T Cell Activation

ERM: ezrin, radixin, moesin

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“hand mirror” morphology, with a large cell body comprised of the nucleus and a thin band of cytoplasm, and a narrow trailing uropod that projects above the substrate surface (Figure 1, cell 5). The leading edge is enriched in chemokine receptors and is sensitized for antigen recognition (15, 16). The uropod contains a meshwork of cytoskeletal elements, including the microtubule organizing center (MTOC), vimentin ﬁlaments, actin ﬁlaments, ERM proteins, and the cytoskeletal linker protein plectin (17). Within tissues, T cells can be highly motile, sampling multiple APCs, or can pause to engage in more prolonged interactions. Transition between these states is controlled in part by the presence of antigen, but also involves the activation state of both the T cell and the APC, as well as the three-dimensional nature of the matrix (18). Motile T cells move much faster than most nonhematopoietic cells, averaging 10 μm/min, and reaching rates of 25 μm/min (19). T cell movement is essentially amoeboid in nature, and is driven by protrusion of actin-rich pseudopodia at the leading edge, working in concert with contractile forces at the rear of the cell (19). A fundamental feature of this mechanism is the ability to form protrusions at the leading edge, and not at the uropod. As in other cell types, it is thought that Rho GTPases control this cycle (20–26). Leading edge protrusion is controlled largely by Rac1, whereas the lack of protrusion within the uropod is controlled by Rho. Cdc42 serves as a “compass” controlling overall cell polarity. Myosins, in particular MyH9, the heavy chain of nonmuscle myosin IIA, are also required for uropod formation and crawling (19). Using video analysis, Krummel and coworkers (19) have shown that MyH9 moves in clusters toward the uropod of crawling T cells. Thus, as in other amoeboid cells, it appears that actomyosin-based contraction within the uropod generates an extrusive force that propels the bulk of the T cell cytoplasm forward. Burkhardt

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Interaction with APCs and Immunological Synapse Formation Upon interaction with an APC bearing appropriate MHC-peptide complexes, the T cell rounds up, draws in its uropod, and extends large pseudopodia and lamellipodia toward the APC. This process results in the formation of a ﬂattened, F-actin-rich interface with the APC. In parallel with these shape changes, the MTOC and associated secretory organelles reorient within the T cell cytoplasm and come to lie just beneath the plasma membrane, near the center of the APC contact site. This reorientation of T cell cytoskeletal elements was observed by several investigators in the early 1980s and was recognized as a hallmark of productive T cell engagement and a prerequisite for directed cytolysis and T cell help (27–29). The morphological transition induced by interaction with an APC is associated with several changes in cytoskeletal proteins. Myosin IIA heavy chain is phosphorylated at a site known to lead to downregulation of motor function (19). This event is thought to be a key factor in controlling uropod resorption. Indeed, the idea that myosin II activity is “switched off” upon TCR engagement is consistent with the ﬁnding that this protein is dispensable for organizing signaling molecules at the T cell–APC contact site. TCR crosslinking also leads to the transient dephosphorylation of ERM proteins, an event that is proposed to facilitate membrane protrusion and receptor clustering at the site of TCR engagement by increasing the ﬂuidity of the plasma membrane (9, 30–32). Ca2+ -dependent cleavage of actin-binding proteins and severing of actin ﬁlaments by proteins such as calpain and gelsolin probably also contribute to this process (33–35). TCR-induced “inside-out signaling” leads to conformational changes in T cell integrins, particularly LFA-1 (reviewed in 36, 37). This process, which results in high-afﬁnity binding to ligands such as ICAM-1 (intercellular adhesion molecule-1) on the APC, is controlled in part by regulated interactions of the β integrin

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cytoplasmic tail with actin-binding proteins such as ﬁlamin and talin (38–40). Once integrins have undergone this conformational change, these proteins cluster as a result of binding to multivalent ligands on the APC, resulting in strengthening of adhesion between the T cell and APC (41). This latter facet of integrin function is closely tied to the increased membrane ﬂuidity that results from the release of cortical cytoskeletal tethers. LFA-1 clustering is largely ligand-driven, although there is also evidence for the active involvement of talin in this process (40). In concert with all these changes, TCR cross-linking leads to localized activation of actin polymerizing proteins and actin-binding proteins at the site of TCR engagement, resulting in lamellipodial protrusion toward the APC and the formation of a dense actin network at the cell-cell contact site (see below). Actin responses at the T cell–APC contact site are important for the organization of signaling molecules at this site, to form the immunological synapse (IS) (42–45). IS organization and function has been an area of intense study (reviewed in 46–48). Protein segregation within the IS was initially described as forming a “supramolecular activation cluster (SMAC)” comprised of a central region (cSMAC) containing TCR and associated signaling proteins within a peripheral ring (pSMAC) containing LFA-1 and associated adhesion molecules (49). Recent studies have shown, however, that IS architecture and dynamics vary greatly depending on several variables, including the nature of the T cell and the APC, agonist strength, costimulatory interactions, and the tissue context in which the interaction is taking place (48, 50). Our understanding of IS function has evolved considerably over the past decade. Once thought to facilitate TCR signaling by bringing interacting molecules together, the c-SMAC was subsequently shown to be a site where signaling is terminated by internalization of TCR signaling complexes (51, 52). The role of the c-SMAC in downregulating T cell signaling is supported by studies in which signaling is en-

hanced under conditions where c-SMAC formation is blocked and inhibited under conditions where c-SMAC formation is induced. (51–53). Recently, an interesting study combining empirical analysis with mathematical modeling has shown that the IS probably both augments and downregulates TCR cell signaling and that parameters such as peptideMHC half-life determine the balance between these two interrelated facets of IS function (52). Video analysis of molecular movements during IS formation shows that the TCR and associated signaling molecules form microclusters around the periphery of the cell-cell contact site and converge to form the cSMAC (43, 54–56). Sustained signaling occurs in the peripheral clusters, while central clusters are destined for degradation, at least in the context of a strong signal. Microcluster formation and centripetal movement are actin-dependent processes, controlled in part by Cdc42 (42, 43, 56). Once formed, however, these signaling complexes are highly stable and are largely unaffected by actindepolymerizing agents. It will be interesting to learn how speciﬁc actin regulatory molecules work to orchestrate microcluster formation and movement, especially as this relates to parameters such as peptide-MHC half-life and T cell subset.

Immunological synapse (IS): specialized membrane domain enriched in signaling molecules formed at the T cell–APC contact site SMAC: supramolecular activation cluster Guanine nucleotide exchange factor (GEF): protein that activates small GTPases by catalyzing the exchange of GDP for GTP

CONTROL OF ACTIN DYNAMICS AT THE IMMUNOLOGICAL SYNAPSE Engagement of the TCR activates multiple actin-regulatory proteins that work in concert to drive actin polymerization at the IS. The best understood of these molecules function downstream of Vav1 and other guanine nucleotide exchange factors (GEFs), which activate the Rho GTPases Rac1 and Cdc42 at sites of TCR engagement (Figure 2). The signaling pathways linking TCR ligation to Rho GTPase activation are addressed in a later section. Here, we focus on molecules that directly inﬂuence actin dynamics at the IS. www.annualreviews.org • Actin in T Cell Activation

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CD4 TCR LFA-1

Actin

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HS 1

Costimulation (e.g., CD28)

L Lck Zap70 A T WASp Nck Slp76 WIP Vav Itk

PP2A Cofilin

Cdc42 Rac1 WAVE2 complex

Arp2/3

?

NF- B, NFAT, AP-1 IL-2

?

Dephosphoryation/ rephosphorylation

ER

M

ER

M

Scribble DLG1

ER

M

Cytoplasmic DPC proteins (e.g., RhoDGI)

Transmembrane DPC proteins (e.g., CD43)

Figure 2 Simpliﬁed model of signaling pathways linking TCR signaling to actin remodeling. Encounter of an APC induces a signaling cascade comprised of tyrosine kinases (dark green), adaptor proteins (light green), and immediate upstream actin-regulatory proteins (dark blue). The latter transduce signals to several nucleation-promoting factors (red ) that direct the polymerization of branched actin ﬁlaments at sites of TCR engagement. Engagement of costimulatory molecules leads to activation of proteins that sever actin ﬁlaments (dark brown), creating new barbed ends as substrates for ﬁlament growth. Signals emanating from the TCR also lead to transient dephosphorylation and rephosphorylation of ERM proteins (orange), which direct the localization of transmembrane and cytoplasmic proteins (light blue) to cortical actin ﬁlaments at the distal pole complex (DPC). Also localized to the DPC are proteins containing PDZ (PSD-95/Discs large/ZO-1) domains ( purple), which form a network at this site. Question marks indicate the unknown mechanisms by which actin remodeling leads to IL-2 promoter activation and other changes in gene expression.

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Arp2/3 Complex–Dependent Actin-Regulatory Proteins The initiation of actin ﬁlament formation is a kinetically unfavorable process, requiring the action of nucleation-promoting factors (NPFs) to generate a “seed” from which a ﬁlament can elongate. Many of the bestunderstood NPFs work in concert with the seven-subunit Arp2/3 complex to form new actin branches on the sides of existing actin ﬁlaments (reviewed in 57). This complex, which contains two actin-related proteins, Arp2 and Arp3, binds to the side of an existing actin ﬁlament, where Arp2 and Arp3 mimic two actin monomers as part of the core nucleus. NPFs such as WASp and WAVE2 then bind to the Arp2/3 complex and present an actin monomer, allowing elongation to take place. Using an RNAi-based strategy, the Billadeau group showed that suppression of either Arp2 or Arp3 in Jurkat T cells results in degradation of other complex components (58). Arp2/3-deﬁcient cells fail both to polymerize F-actin at the IS and to form lamellipodia in spreading assays using anti-TCR-coated coverslips. These cells generate highly dynamic F-actin-rich ﬁlopodia in response to either APCs or anti-TCR-coated surfaces, a ﬁnding that points toward additional Arp2/3 complex–independent actin regulatory pathways. Nonetheless, this study demonstrates that the Arp2/3 complex and its upstream activators are central effectors of actin polymerization at the IS. The best-characterized activator of the Arp2/3 complex is WASp, named for its causative role in Wiskott Aldrich syndrome (WAS), a severe immune deﬁciency disorder. The domain structure of WASp is shown in Figure 3. The N-terminal WASp-homology region (WH1) binds constitutively to WASp interacting protein (WIP). Disruption of this interaction results in degradation of WASp (59, 60). Most mutations associated with WAS are found in this region and interfere with WIP binding (61). WIP is almost certainly an important actin-regulatory protein in its own

right, but WIP function per se has not been tested, presumably because of the challenges associated with constructing WIP-deﬁcient cells that express normal amounts of WASp. However, evidence that WIP and WASp play nonredundant roles comes from analysis of WIP/WASp double-knockout mice; T cells lacking both proteins migrate less well than T cells from either of the single knockouts (62). WASp recruitment and activation at the IS are relatively well understood. Recruitment occurs through an interaction between the WASp proline-rich domain (PRD) and the SH3 domains of the adaptors Nck and PSTPIP (63, 64). Localization to the IS facilitates WASp contact with Cdc42-GTP, which binds to the GTPase-binding domain (GBD) (63), leading to a conformational change in WASp. In resting cells, WASp exists in a closed, inactive state in which the GBD contacts the verprolin, coﬁlin, acidic (VCA) region. Cdc42 binding releases the VCA domain, allowing the VCA to interact with the Arp2/3 complex (65). Phosphorylation of WASp at tyrosine 291 by the Src family kinase Fyn, and subsequent binding of the SH2-SH3 domain module of Src kinases to WASp, works in a synergistic manner with Cdc42 binding to induce optimal WASp activity (66–68). WASp-deﬁcient T cells from both mice and humans exhibit consistent defects in signaling pathways leading to IL-2 production, but the literature is divided about the importance of WASp for controlling actin polymerization at the IS. Although there are numerous reports that T cells from WAS patients exhibit defective actin responses (reviewed in 61), Jurkat cells depleted of WASp using RNAi polymerize actin normally (69). Moreover, T cells from two different WASp-knockout mice behave differently with respect to their ability to polymerize actin in response to TCR engagement (67, 70, 71). A possible explanation for these discrepancies is that other proteins (including the close homolog N-WASp, as well as less closely related proteins such as WAVE2 and HS1) have partially overlapping functions. Recent work from the Dustin lab www.annualreviews.org • Actin in T Cell Activation

Nucleationpromoting factor (NPF): protein that promotes the nucleation of actin ﬁlaments, either alone or by activating Arp2/3 complex Arp2/3 complex: seven subunit complex that nucleates the formation of new actin ﬁlaments on the sides of preexisting ﬁlaments Wiskott Aldrich syndrome (WAS): X-linked immunodeﬁciency disorder characterized by eczema, recurring infections, and thrombocytopenia. WAS results from mutations in WASp

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1

HS1

YY NTA

HTH

CC

PRD

Arp2/3

F-actin

F-actin

Lck

1

WASp

Lck Vav1

Y WH1

GBD

WIP

Cdc42

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502 PRD

V CA

Nck G- Arp2/3 PST-PIP Actin

Y

WAVE2

WHD

498 Basic

PRD

V CA G- Arp2/3 Actin

Abi1/2 PtIns(3,4,5)P3 IRSp53

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486 SH3

1

WIP

503

PRD WH2 WH2

WASpbinding WASp

PRD

Profilin G-actin

Nck

166

1S

Cofilin F-actin 1

Y

Ezrin

N-ERMAD

Y

C-ERMAD T 586

α-helical

FERM

PRD ABD F-actin

CD43, RhoGDI, etc. 1

Moesin

Y

N-ERMAD

C-ERMAD T 577 α-helical

FERM

ABD F-actin

CD43, RhoGDI, etc. 1

904

Dlg1

PRD

PDZ

PDZ PDZ

PDZ

Lck

Ezrin p38 MAPK

Ezrin

p38 MAPK

SH3

I3

MAGUK

Ezrin

1

1655

Scribble

LRR

PDZ

PDZ

PDZ

PDZ

Figure 3 Structure of proteins involved in regulating actin dynamics and T cell polarity. The domain structure of the indicated molecules is shown, with related regions colored similarly. Important binding partners are indicated below the domains to which they bind. Total amino acid number is shown at the upper right of each protein. Important phosphorylation sites are indicated (Y, T, S). NTA, N-terminal acidic region; HTH, helix-turn-helix repeats; CC, coiled-coil; PRD, proline-rich domain; SH3, Src homology 3; WH1, WASp homology 1; GBD, GTPase-binding domain; VCA, verprolin homology, coﬁlin homology, acidic region; WHD, WAVE homology domain; WH2, WASp homology 2; FERM, Band 4.1, ezrin, radixin, and moesin; ABD, actin-binding domain; PDZ, PSD-95/Discs large/ZO-1; I3, splice insert 3; MAGUK, membrane-associated guanylate kinase; LRR, leucine-rich repeats.

supports the idea that WASp-dependent actin polymerization is indispensable for T cell activation (44). This study shows that naive T cells contacting APCs or lipid bilayers con240

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taining MHC-peptide complexes and ICAM1 form a short-lived IS, then migrate some distance before generating a second, more stable contact. Although WASp−/− T cells form

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an initial contact in this assay, they continue to migrate and fail to re-form and maintain a long-lived IS. Another important activator of the Arp2/3 complex is the WAVE/SCAR family member WAVE2, an NPF that functions as an effector for Rac1. RNAi-mediated suppression of WAVE2 in Jurkat T cells results in loss of actin polymerization at the IS (69, 72). In addition, WAVE2 suppression perturbs conjugate formation, suggesting a role in integrin function. WAVE2 resembles WASp in that it binds the Arp2/3 complex via a C-terminal VCA domain (Figure 3), but WAVE2 lacks a GBD and does not bind Rac1 directly (73). WAVE2 exists as part of a constitutive complex containing Abi1/2, Sra1/PIR121, Hspc300, IRSp53, and HEM1 (reviewed in 37). WAVE complex components stabilize one another against degradation, and suppression of Abi1/2 or HEM-1 also results in actin defects (69, 72, 74, 75). WAVE complex components also function in WAVE2 targeting and activation. IRSp53 acts as an adaptor between Rac1 and WAVE2, and this interaction leads to the recruitment of WAVE2 to the membrane (75). Abi1/2 can mediate an interaction between Abl kinase and WAVE2, resulting in phosphorylation at Y150, which increases the actin polymerization activity of WAVE2 (76). A third activator of the Arp2/3 complex is HS1, the hematopoietic lineage–restricted homolog of the actin-binding protein cortactin (Figure 3). Like WASp and WAVE, HS1 contains an acidic region (NTA) that binds the Arp2/3 complex, and HS1 can modestly activate Arp2/3-dependent actin polymerization (Figure 3). However, HS1 also binds to F-actin directly through its helixturn-helix (HTH) repeats and coiled-coil (CC) domain (77). It is therefore thought that HS1 functions primarily to stabilize existing branched actin ﬁlaments by bridging the Arp2/3 complex with F-actin (78–80). In keeping with this idea, HS1-suppressed T cells extend unstable lamellipodia on antiTCR-coated coverslips and exhibit short-

lived actin responses at the IS (81). TCR engagement induces ZAP-70-dependent phosphorylation of HS1 at Y378 and Y397, an event that is required for HS1 function at the IS. WASp, WAVE2, and HS1 are highly modular in structure and can interact with multiple signaling molecules at the IS. The adapter properties of these proteins undoubtedly facilitate coordination of actin polymerization. For example, phosphorylation of HS1 creates binding sites for Vav1, such that HS1 contributes to Cdc42 and Rac1 activation upstream of WASp and WAVE2. It is also likely that these proteins facilitate T cell activation by coordinating other signaling molecules independent of their role in actin polymerization.

Arp2/3 Complex–Independent Actin-Regulatory Proteins In other cell types, the function of Arp2/3 complex–dependent NPFs is complemented by proteins that drive actin polymerization independently of Arp2/3 complex, as well as by proteins that cap, sever, cross-link, and bundle actin ﬁlaments. Only a few of these proteins have been analyzed in T cells. One actinsevering protein, coﬁlin, has been shown to play an important role in controlling actin responses at the IS (82). Coﬁlin is a small (19-kDa) protein that severs actin ﬁlaments, promoting depolymerization but also generating fresh barbed ends that serve as substrates for elongation factors. Because of its requirement for cell viability, coﬁlin has been difﬁcult to study. However, the Samstag group has used cell-permeant peptides that compete with actin for coﬁlin binding to approximate a loss-of-function phenotype. Cells treated with these peptides form conjugates inefﬁciently, polarize CD2 and coﬁlin to the IS poorly, and exhibit severe defects in proliferation and cytokine secretion (82). Phosphorylation at serine 3 negatively regulates coﬁlin activity by antagonizing its capacity to bind actin (83). Coﬁlin is phosphorylated in resting www.annualreviews.org • Actin in T Cell Activation

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normal human T cells, but upon accessory receptor (CD2, CD4, CD8, or CD28) ligation, the serine phosphatases PP1 and PP2a can dephosphorylate and activate it (84, 85). Regulation of coﬁlin activity is likely to be an important control point for actin dynamics at the IS because coﬁlin can either promote lamellar growth or collapse, depending on whether Arp2/3-dependent NPFs are activated in parallel. Among the other Arp2/3-independent actin-regulatory proteins that have been studied in T cells are members of two important families, formins and Ena/VASP proteins. Members of both families localize to sites of TCR engagement (86, 87); however, their role in controlling actin responses at the IS is not clear. Formins are NPFs that promote the formation of long, linear actin ﬁlaments (87). So far, two formins have been found at signiﬁcant levels in T cells, FMNL1 and Dia1 (58). Jurkat T cells suppressed for these proteins (alone or together) polymerize F-actin at the IS normally. Ena/VASP proteins bind to the barbed ends of actin ﬁlaments and promote ﬁlament growth by antagonizing the binding of capping proteins (88). The predominant Ena/VASP protein expressed in T cells is EVL. Overexpression of sequences derived from the protein ActA in Listeria monocytogenes competes for binding of EVL to other proteins. This treatment displaces EVL from sites of TCR engagement and disrupts actin responses at the IS. However, it is not clear that the effects of this construct are speciﬁc, as suppression of EVL (alone or together with the related protein VASP) has no effect on T cell spreading or actin responses at the IS (58). One possibility is that the activity of formins and Ena/VASP proteins, which can work together to generate ﬁlopodia, is masked by the broader lamellipodia generated by Arp2/3-dependent NPFs. However, even the ﬁne ﬁlopodia generated in Arp2/3-deﬁcient cells do not depend on the expression of formins or Ena/VASP proteins (58). Thus, it appears that the presence of these proteins at the IS reﬂects a role

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in processes other than control of actin polymerization. Formins are crucial for polarization of the MTOC and directed cytolysis (58), whereas EVL, which binds to the adaptor protein ADAP and the Rap1 activator RIAM, is likely to function in integrin-based adhesion (reviewed in 37).

Proteins that Affect IS Actin Dynamics by Unknown Mechanisms Among the many proteins required for efﬁcient actin polymerization at the IS are the immediate regulators of actin dynamics described above and several well-understood signaling molecules that function upstream of these proteins. However, some proteins affect actin dynamics in ways that are not yet understood. One such protein is the methyltransferase Ezh2. Better known as a nuclear protein that methylates histones on lysine residues, Ezh2 is also found in the cytoplasm of T cells in a complex containing Vav1 (89). Recently, the Tarakhovsky group has made the unexpected observation that mature T cells deﬁcient for Ezh2 completely fail to polymerize actin at the IS (89). Cytoplasmic Ezh2 possesses methyltransferase activity, but cytoplasmic substrates have not yet been identiﬁed. Indeed, it is not known if Ezh2 requires its methyltransferase activity to control actin responses; it could play an adaptor function by binding Vav1 and stabilizing its open active conformation (90). The position of Ezh2 within the TCR signaling pathway appears to be upstream of Cdc42 function, as Ezh2−/− T cells activate Cdc42 inefﬁciently, and constitutively active Cdc42 rescues TCR signaling–dependent actin polymerization in these cells (89). The large GTPase dynamin2 is also required for actin polymerization at the IS (91). Dynamin2 interacts directly with Vav1, and requires the presence of Vav1 for its localization to the IS. Although dynamin has been shown to interact with several actin-binding proteins and to affect actin dynamics in many cell types, its mechanism of action is not

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understood (92). Because dynamin plays a critical role in endocytosis, it is interesting to speculate that it functions at a point of crosstalk between actin regulatory and endocytic machinery.

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ORGANIZATION OF THE DISTAL POLE COMPLEX BY ACTIN LINKER PROTEINS In addition to organizing signaling proteins at the IS, actin and actin-binding proteins organize a second protein complex at the opposite face of the T cell, termed the distal pole complex (DPC) (93). The DPC was initially discovered based on the localization of CD43 and ERM proteins to a region that was clearly outside the IS, and in many cases distributed in a tight cap opposite the IS (30, 94). Further study showed that basic residues within the cytoplasmic tail of CD43 are required for its localization and function in T cell activation (30, 95). These residues bind to the ERM proteins ezrin and moesin (T cells Table 1

express low levels of the third family member, radixin). As detailed below, these ERM proteins bind through their N-terminal FERM (Band 4.1, ezrin, radixin, and moesin) domain to CD43 and other cargo molecules, linking them to the actin cytoskeleton in a regulated fashion. Localization of CD43 and other ERM binding proteins (e.g., RhoGDI and DLG1) to the DPC is ERM dependent because mutation of interacting residues or overexpression of a dominant-negative ERM mutant disrupts their distribution (93–95). A survey of the literature reveals that many proteins exhibit an antipodal distribution during at least some phases of the T cell response (Table 1). It is not known if ERM proteins control the localization of all of these; some may prove to lie upstream of ERM proteins in assembly of the protein network. For example, the polarity protein scribble, recently found to be required for DPC formation, T cell conjugation, and activation, is required to generate a polarized ERM protein response (96).

Distal pole complex (DPC): specialized membrane domain formed at the T cell pole distal to the site of TCR engagement DLG1: discs large; also known as SAP97

Molecules displaying patterns of localization corresponding to the DPC

DPC component

Proposed function in T cells

References

Ezrin/moesin

Linker of molecules to actin; required for localization of multiple DPC proteins and T cell activation

30, 94, 95, 97

CD43

Highly glycosylated transmembrane protein (mucin); regulator of T cell proliferation, adhesion, and migration

30, 94, 95

DLG1

PDZ domain–containing polarity protein; regulator of actin polymerization, NFAT, alternate p38 pathway

93, 98, 99

Scribble

PDZ domain–containing polarity protein; required for uropod and IS formation, migration, T cell activation

96

CD148

Tyrosine phosphatase; negative regulator of TCR-dependent NFAT activation

100

PDE4B2

Cyclic nucleotide phosphodiesterase; antagonizes PKA; associates with TCRζ and enhances IL-2 production

101

PKCζ

Atypical protein kinase C; positive regulator of NF-κB and JAK/STAT signaling

102

PtIns(3,4,5)P3

Phospholipid; required for the localization and activation of PH domain–containing proteins

103

RhoGDI

Rho GTPase guanine dissociation inhibitor; inhibits Rho GTPases; regulator of T cell development and migration

25, 94

SHP-1

Tyrosine phosphatase; negative regulator of early T cell activation signaling events

104a

EBP50

Adaptor protein; links CBP to actin through ERM proteins

105a

PTP-BL

Tyrosine phosphatase; downregulator of STAT activity and TH cell differentiation

106a

a

Localization to the DPC was ascertained by P. Cullinan, R.S. Dupree, M.H. Shaffer & J.K. Burkhardt, unpublished data.

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Although there is good evidence that the DPC is important for T cell activation, the function of this complex is still not well understood. Based on the proteins that have been identiﬁed in the DPC to date, three models have emerged. First, the DPC may modulate signaling events at the IS by serving as a sink for negative regulators of T cell activation. It is sensible that the sequestration of SHP1, CD148, PTP-BL, CD43, RhoGDI, and DLG1 away from the IS enhances T cell function because each of these molecules can antagonize events occurring at the IS (Table 1). ERM proteins also interact via EBP50 with CBP/PAG, a negative regulator of Src kinases (105), although the role of ERM proteins in regulation of CBP/PAG activity has not been established. However, some DPC proteins are not negative regulatory molecules (or play dual roles), suggesting that DPC function is more complex than simple protein sequestration. A second model is that the DPC serves as an active signaling complex, separate from the IS. In support of this idea, cross-linking CD43 with certain antibodies leads to MAP (mitogen-activated protein) kinase and NFAT (nuclear factor of activated T cells) activation (107), and coligation with TCR enhances T cell signaling (108). Cross-linking CD46 (using antibodies or by binding to measles virus) inhibits T cell activation and drives T cells to a regulatory fate (109, 110). Finally, crosslinking ICAM-2, an ERM binding protein, can activate PI3K-dependent survival signaling (111). A third model for DPC function is that it establishes overall T cell polarity, as discussed below. These models are not mutually exclusive; resolution of their relative importance will require functional analysis of individual DPC proteins.

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functions ranging from maintenance of microvilli, TCR-induced signaling events leading to cytokine production, and regulation of CD95-induced cell death (9, 30, 32, 94, 97, 112, 113). ERM proteins are present in two conformations. In the inactive form, the N-terminal FERM domain (equivalent to the N-terminal ERM-association domain, NERMAD) interacts with a C-terminal region (C-ERMAD), which encompasses the actinbinding region. This intramolecular interaction masks both the actin and cargo binding sites (Figure 3) (114, 115). In the active conformation, this intramolecular interaction is relieved, allowing linkage of cargo proteins to actin ﬁlaments. Conversion to the activated state is regulated by phosphorylation of a conserved threonine (T558 in moesin, T567 in ezrin) in the C terminus. In response to TCR engagement, active ERM proteins undergo transient dephosphorylation, temporarily relaxing their constraint of cortical protein mobility. Reactivation is then achieved by concerted binding of PtIns(4,5)P2 to the FERM domain and phosphorylation of the regulatory threonine. In parallel with rephosphorylation, ERM proteins move away from the site of TCR engagement by an unknown mechanism. The inactivation/reactivation of ERM proteins is associated with changes in cellular morphology, including the collapse of microvilli and the ﬂattening of the T cell against an APC (9, 32). So far, neither the relevant kinase nor the phosphatase in T cells has been identiﬁed. ERM dephosphorylation requires Vav1-dependent activation of Rac1, independently of Rho and Cdc42 (Figure 2) (116). ERM proteins may use this transient dephosphorylation to alter cargo binding patterns and therefore change the localization of a subset of binding proteins, but this idea has not been tested. Prolonged ERM threonine dephosphorylation is associated with unresponsiveness induced by measles virus binding to CD46 (117). In addition to on/off regulation by phosphorylation at T567, ezrin is tyrosine phosphorylated in response to cross-linking of

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TCR, CD4, or ICAM-2 (111, 118, 119). Tyrosine phosphorylation of ezrin downstream of TCR or CD4 is dependent on the Src family kinase Lck, which can phosphorylate ezrin at Y146 in vitro (118, 120). The signiﬁcance of ezrin tyrosine phosphorylation in T cells is unknown. In nonhematopoietic cells, ezrin tyrosine phosphorylation leads to activation of the PI3K/Akt pathway and enhanced cell survival (121). In T cells, ezrin phosphorylation in response to ICAM-2 cross-linking may play a similar role (111). Although the dogma in the ﬁeld has been that ezrin and moesin are functionally redundant, two of the known tyrosine phosphorylation sites in ezrin are not conserved in moesin and differences in cargo binding have been described. In lymphocytes, l-selectin and CD95/Fas bind ezrin and moesin differentially (113, 122). Thus, ezrin and moesin may play unique roles in DPC function.

The DPC in T Cell Polarity Several DPC proteins (PKCζ, DLG1, and scribble) control cell polarity in epithelial cells, neurons, and developing organisms (123). These proteins, many of which contain PDZ domains, function in complex networks (124). Analysis of polarity proteins in T cells is still in its infancy, but it is already clear that they play key roles. Scribble appears to be important for polarity per se; T cells in which scribble is suppressed using RNAi fail both to form a uropod and to segregate IS and DPC proteins in response to contact with APCs (96). DLG1 is required for TCR-induced actin polymerization and regulates p38 and NFAT activation (98, 99, 125). PKCζ, an initiator of polarity cascades in other systems, has been implicated in Rap1-induced T cell polarization and chemokineinduced migration (126), as well as NF-κB and JAK/STAT activation (102). Recently, a role for polarity proteins has been established in T cell responses in vivo. Reiner and colleagues showed that during the ﬁrst cell division after stimulation, PKCζ and scribble, as well as sig-

naling proteins such as CD3, IFN-γR, CD8, LFA-1, and Numb, segregate asymmetrically to the two daughter cells (127). This segregation leads to functionally distinct daughter cells; the “proximal” daughter takes on an effector phenotype, whereas the “distal” daughter is more memory-like. Many DPC proteins also localize to the uropod in migrating T cells (31). Both the uropod and DPC sequester proteins away from the site of active actin polymerization (leading edge or IS, respectively), reﬂecting the fundamental role of cytoskeletal architecture in dictating intrinsic cell polarity, as well as regulating signaling complexes.

INTERPLAY BETWEEN T CELL SIGNALING AND ACTIN DYNAMICS The actin cytoskeleton functions as a key intermediate in diverse signaling pathways downstream of the TCR, chemokines, death receptors, and integrins. The role of actin in integrin function is complex and has been recently reviewed elsewhere (36, 37). Here, we summarize brieﬂy the literature regarding TCR signaling pathways, and review what is known about signaling downstream of chemokines and death receptors.

TCR Signaling To and Through the Actin Cytoskeleton Signaling pathways leading from TCR engagement to actin remodeling have been reviewed elsewhere (128). Brieﬂy, the primary pathway involves signaling through Lck and ZAP-70, leading to the formation of a signaling complex at the IS containing the adaptor proteins LAT and SLP-76, the Tec family kinase Itk, and the Rho GEF Vav1 (diagrammed in Figure 2) (128). These proteins stabilize one another at sites of TCR engagement, and loss of any of these molecules results in disruption of actin polymerization at the IS. Interaction with complex components promotes the activation of Vav1 by phosphorylation and www.annualreviews.org • Actin in T Cell Activation

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phosphoinositide-dependent engagement of its PH domain (129). Vav1 then generates localized pools of active Cdc42 and Rac1, promoting actin polymerization by WASp and WAVE2, respectively (130, 131). As discussed above, other events also contribute to WASp and WAVE2 activation; however, Vav1 function is clearly critical (3, 132). Importantly, Vav1 binds to multiple actin regulatory molecules, suggesting that its central role in controlling actin dynamics involves adaptor functions as well as GEF activity. Costimulatory signaling also controls actin responses at the IS (42). A major consequence of costimulation via CD28 is the activation of PI3K. Through the production of PtIns(3,4,5)P3, PI3K can inﬂuence the localization and activation of PH domain–containing signaling molecules including Vav1 and Itk, leading to enhanced and/or sustained activation of the pathways described above (133–135). CD28 can also signal through a PI3K-Ras pathway to dephosphorylate and activate coﬁlin (136). Though less well studied, costimulatory signaling via other molecules such as CD2, CD82, CD46, and ICOS also affects TCRinduced actin responses (137–140). Thus, one way that costimulation promotes full T cell activation is by augmenting actin-dependent signaling events at the IS. Despite signiﬁcant progress in determining the upstream events linking TCR engagement to actin remodeling, our understanding of how actin contributes to downstream signaling events is largely restricted to activation of the IL-2 promoter. Disruption of the actin network perturbs the activation of each of the three key elements within the IL2 promoter (NFAT, NF-κB, and AP-1). Virtually every condition that affects actin dynamics also affects Ca2+ signaling pathways leading to NFAT activation. Treatment of T cells with CytoD affects Ca2+ ﬂux and NFAT activation, although, as noted above, the effects vary depending on the time of treatment and dose of inhibitor (2–4). Diminished Ca2+ inﬂux and inefﬁcient NFAT activation

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CRAC: calcium release–activated calcium

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have also been observed in T cells deﬁcient for Vav1, WASp, WAVE2, and HS1 (3, 69, 71, 81, 132, 141, 142). However, the requirement is not simply for actin per se, because individual actin-regulatory proteins facilitate Ca2+ signaling in distinct ways. For example, WAVE2-deﬁcient T cells exhibit defects in coupling store release to calcium release– activated calcium (CRAC) channel activation (69), whereas HS1-deﬁcient T cells exhibit a defect at the level of release from stores (81). The defect in release from stores presumably reﬂects defects in PLCγ1 activation, but this has not been demonstrated. T cells deﬁcient for Vav1 and WASp also fail to activate AP1, likely stemming from upstream defects in ERK activation (142, 143). Activation of NFκB is defective in T cells lacking Vav1 or HS1 (81, 143). The molecular basis for this defect has not been established, but it may reﬂect defects in PKCθ signaling. Additional analysis of signaling events in T cells lacking actin-regulatory proteins will be required to ﬁll in the “black box” linking actin polymerization and changes in T cell gene expression, but it is unlikely that these events represent simple, linear pathways. There is growing evidence that actin networks facilitate formation or stabilization of proteinprotein interactions to promote sustained signaling (43, 81). In addition, actin promotes the internalization of TCR signaling complexes and costimulatory molecules, an event that is thought in some cases to downregulate signaling and in others to sustain it (144, 145).

Chemokine-Induced Migration Chemoattractant signaling pathways leading to actomyosin-based motility have been studied in many cell types, but remarkably little is known about how chemokine signaling in T cells reshapes the actin cytoskeleton. Even the role of PI3 kinases, central regulators of migration in other systems, is unclear in lymphocytes (146). In T cells, chemokine receptor engagement induces activation of G protein–coupled receptors. This

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induces activation of Tec kinases (Itk/Rlk), Ilk, Vav, and CrkL (147–150) and downstream activation of small GTPases, especially Rac but also Cdc42 and Rho. The primary function for Cdc42 seems to be in the activation of WASp, which together with WIP promotes T cell migration through tissues (62). In contrast, Rac activation leads to the dephosphorylation of ERM proteins and microvillar collapse that precedes the initiation of polarization and movement. Rac activity also leads to the activation of additional actinregulatory proteins, including PAK, LIMK, and p160ROCK (24, 151, 152). The central role of Rac is highlighted by the migratory defects in cells lacking Rac2, RhoGDI, and the Rac-activating proteins DOCK2 and TIAM1 (25, 126, 153, 154). DOCK2 appears to be a particularly important upstream regulator of Rac in chemokine-activated T cells. T cells from DOCK2−/− mice exhibit profound defects in Rac activation, motility, and microvillar collapse in vitro (155), and diminished migration within lymph nodes, limited egress, and impaired homing in vivo (156, 157). Recently, coﬁlin has emerged as another central player in signaling pathways leading to T cell migration. Regulation of coﬁlin activity is maintained by the opposing action of LIMK1 and the slingshot phosphatase SSH1L. Both LIMK1 and SSH1L are required for chemokine-induced T cell migration, but loss of LIMK1 suppresses lamellipodial formation, whereas loss of SSH1L results in the generation of multiple lamellipodia (158). In chemokine-stimulated cells, SSH1L and dephospho-coﬁlin accumulate in the leading edge, consistent with the idea that SSH1L activates coﬁlin-dependent actin dynamics locally, establishing actin polarity and directional migration. Other pathways also converge on coﬁlin activity. For example, actin-interacting protein 1 (Aip1) and caspase-11 function together to promote migration by activating coﬁlin (159). T cells lacking the β-propeller protein coronin 1 have a distinct phenotype, characterized by excessive F-actin accumulation at the cortex and

drastically impaired cell movement (160). Recent work in nonhematopoietic cells shows that coronin plays a dual role, inhibiting ﬁlament nucleation by the Arp2/3 complex and recruiting SSH1L to lamellipodia, where it activates coﬁlin (161). It remains to be demonstrated whether coronin 1 targets SSH1L in response to chemokine signaling. Nonetheless, because coﬁlin can promote either actin depolymerization or new growth, depending on whether the Arp2/3 complex is activated in parallel, this axis may prove to be particularly important in controlling lamellipodial dynamics.

DISC: death-inducing signaling complex

Death Receptor Signaling Some T cell signaling pathways leading to apoptosis are highly actin dependent (162). In cells where CD95/FasL signaling induces sufﬁcient active caspase-8 to directly activate downstream caspases (Type I cells), maintenance of an intact actin cytoskeleton is required for assembly of the death-inducing signaling complex (DISC) and CD95 internalization (163). Thus, treatment of these cells with actin depolymerizing agents reduces the generation of activated caspase8 and signiﬁcantly inhibits apoptosis (164). This mechanism is mediated, in part, by interactions between CD95 and ezrin. Ezrin (but not moesin or radixin) interacts directly with CD95, and antisense suppression of ezrin or mutation of its CD95 binding site inhibits CD95-mediated apoptosis (112, 113). Ezrin is cointernalized with CD95 in Type I cells, and ezrin-suppressed B cells fail to undergo CD95 internalization and DISC formation (165). It remains unclear exactly how ezrin facilitates CD95 uptake or what distinguishes Type I from Type II cells, in which CD95 is not internalized efﬁciently. No differences in ezrin expression were observed in Type I versus Type II cells, although differences in other actin-regulatory proteins, including HS1, have been described (164). Finally, we note that under some conditions, ERM proteins also function to oppose CD95 www.annualreviews.org • Actin in T Cell Activation

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signaling. Engagement of ICAM-2, an ERM cargo protein within the DPC, leads to tyrosine phosphorylation of ezrin, thereby creating a binding site for PI3K and initiating Akt-dependent survival signaling that protects against apoptosis induced by Fas or TNF-α (111).

PERSPECTIVES

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Actin polymerization at the site of APC binding was recognized as a hallmark of T cell activation over 25 years ago (27), but for many years the cytoskeleton was thought to play a “housekeeping” role in T cell biology. Today, it is well known that regulated actin dynamics are important for T cell development and homeostasis, activation, migration, and effector function. The past few years have seen an explosion in the identiﬁ-

cation of proteins that control actin dynamics in response to environmental cues. Progress has also been made in understanding how signaling from the TCR, in particular, regulates the activity of these molecules. The challenge now is to understand how the function of these proteins is coordinated. It will be interesting to learn how the cytoskeletal response varies with respect to T cell subtype, agonist strength, and costimulatory signaling and how T cells integrate chemokine signals with TCR signals. Finally, it will be important to learn more about how actin dynamics promote T cell activation and effector function. Answers to these basic cell biological questions will provide important insights into how the assembly of signaling modules at the appropriate time and place within the cell works to “ﬁne-tune” the cellular immune response.

SUMMARY POINTS 1. Depending on the physiological context in which they ﬁnd themselves, T cells exhibit distinct cytoarchitectural features that are required to carry out specialized functions. These features facilitate tethering and rolling in the vasculature, extravasation, and migration through tissues and interactions with APCs. 2. During interaction with APCs, signaling events initiated by ligation of the TCR and costimulatory molecules lead to formation of a dense, branched actin network at the site of APC binding. This network helps to facilitate T cell activation by organizing signaling molecules into microclusters at the IS. The actin network is also important for the movement of signaling molecules to the center of the IS, where the complexes are internalized and signaling is terminated. 3. TCR engagement activates the assembly of a signaling complex containing Vav1, an important GEF for the Rho GTPases Cdc42 and Rac1. These and other events lead to the localized activation of Arp2/3 complex–dependent NPFs, including WASp, WAVE2, and HS1, which work in concert with proteins such as coﬁlin to generate F-actin ﬁlaments at the IS. 4. TCR engagement also leads to the formation of the DPC at the opposite pole of the T cell. Many proteins are brought into the DPC by binding to ezrin and moesin, which link transmembrane and cytoplasmic proteins to actin ﬁlaments in a regulated fashion. The function of the DPC is poorly understood. It is proposed to sequester negative regulators of T cell activation and serve to organize overall T cell polarity.

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5. Signaling through chemokine receptors and CD95 also affects actin dynamics. Reorganization of the actin cytoskeleton is important for downstream activation of directed cell migration and cell death, respectively. Some proteins involved in these pathways, e.g., Rac, coﬁlin, ezrin, are shared with TCR-dependent actin regulatory pathways. Other proteins, e.g., coronin, are so far known to function only in chemokine-mediated signaling.

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FUTURE ISSUES 1. Although the list of proteins known to control actin remodeling in T cells has grown considerably, future research will undoubtedly reveal other key players. Future studies must ask how these proteins work together to generate the appropriate response to speciﬁc stimuli. 2. Our understanding of how actin responses affect downstream events such as IL-2 promoter activation is still very rudimentary. Actin responses need to be better deﬁned using video microscopy and electron microscopy, and structure-function analysis of individual actin-regulatory proteins will be needed to separate adaptor functions from events that depend on actin per se. Finally, mathematical modeling and other new tools will be needed to help us envision how cytoarchitectural dynamics can affect signaling cascades. 3. The importance of actin-regulatory proteins for in vivo immune responses is highlighted by the severe immunodeﬁciency that afﬂicts patients with mutations in WASp, WIP, or Vav. Recent studies have also linked changes in actin-regulatory proteins to autoimmune disease (166–168). Finally, cytoskeletal proteins play a critical role in infection by HIV and other retroviruses (reviewed in 169). Going forward, it will be important to understand how dysregulation of actin-regulatory proteins contributes to human disease and to develop new therapeutic approaches based on modulating the function of these proteins.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS Owing to space limitations, it was impossible to cite every relevant reference. We apologize to those whose work was not included here. We thank Yair Argon, Keri Sanborn, and members of the Burkhardt laboratory for critical reading of the manuscript. This work was supported by NIH grants R01-AI065644 and R01-AI50098 to J.K.B., F31-AI071385 to E.C., and T32HD07516 to M.H.S.

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162. Celeste Morley S, Sun GP, Bierer BE. 2003. Inhibition of actin polymerization enhances commitment to and execution of apoptosis induced by withdrawal of trophic support. J. Cell. Biochem. 88:1066–76 163. Algeciras-Schimnich A, Shen L, Barnhart BC, Murmann AE, Burkhardt JK, Peter ME. 2002. Molecular ordering of the initial signaling events of CD95. Mol. Cell. Biol. 22:207– 20 164. Algeciras-Schimnich A, Peter ME. 2003. Actin dependent CD95 internalization is speciﬁc for Type I cells. FEBS Lett. 546:185–88 165. Chakrabandhu K, Herincs Z, Huault S, Dost B, Peng L, et al. 2007. Palmitoylation is required for efﬁcient Fas cell death signaling. EMBO J. 26:209–20 166. Otsuka J, Horiuchi T, Yoshizawa S, Tsukamoto H, Sawabe T, et al. 2004. Association of a four-amino acid residue insertion polymorphism of the HS1 gene with systemic lupus erythematosus: molecular and functional analysis. Arthritis Rheum. 50:871–81 167. Sawabe T, Horiuchi T, Koga R, Tsukamoto H, Kojima T, et al. 2003. Aberrant HS1 molecule in a patient with systemic lupus erythematosus. Genes Immun. 4:122–31 168. Li Y, Harada T, Juang YT, Kyttaris VC, Wang Y, et al. 2007. Phosphorylated ERM is responsible for increased T cell polarization, adhesion, and migration in patients with systemic lupus erythematosus. J. Immunol. 178:1938–47 169. Fackler OT, Krausslich HG. 2006. Interactions of human retroviruses with the host cell cytoskeleton. Curr. Opin. Microbiol. 9:409–15

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:233-259. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader Department of Molecular Genetics and Microbiology, Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-012; email: [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:261–92

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

AID, UNG, AP endonuclease, DNA Pol β, DNA-PK, XRCC4-DNA ligase IV, ATM, Mre11-Nbs1-Rad50, 53BP1, γH2AX, germline transcripts, R-loops, end joining

This article’s doi: 10.1146/annurev.immunol.26.021607.090248 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0261$20.00

Abstract Antibody class switching occurs in mature B cells in response to antigen stimulation and costimulatory signals. It occurs by a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (CH ) region genes, except Cδ]. The recombination is initiated by the B cell– speciﬁc activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions. AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting abasic sites are nicked by apurinic/apyrimidinic endonuclease (APE). AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S-S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, γH2AX, 53BP1, Mdc1, and XRCC4ligase IV. These proteins are important for faithful joining of S regions, and in their absence aberrant recombination and chromosomal translocations involving S regions occur.

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INTRODUCTION AND OVERVIEW

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CSR: class switch recombination SHM (somatic hypermutation): the process that introduces mostly single-nucleotide mutations into the variable regions of antibodies after antigen activation during infection or after immunization AID: activation-induced cytidine deaminase GC (germinal centers): highly dividing B cells undergoing SHM that form in B cell follicles in spleen and lymph nodes during a T cell–dependent immune response

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Antibody class, or isotype, is determined by the heavy chain constant (CH ) region, which is important for determining the antibody’s effector function. The CH region is bound by cell-surface receptors, e.g., Fc receptors on many cell types and poly immunoglobulin (Ig) receptors on mucosal epithelial cells, and also by complement. Different CH regions have different afﬁnities for these proteins, thus greatly inﬂuencing antibody function and determining whether antibody-antigen complexes will activate cells that help to eliminate pathogens, e.g., macrophages, NK cells, or mast cells. Also, the CH region determines whether an antibody can be transcytosed through epithelial membranes at mucosal surfaces, can diffuse into tissues, and will polymerize and thereby have a greater avidity. The CH region also inﬂuences the stability of the antibody (reviewed in 1). The membrane-bound forms of the various isotypes differ in their intracytoplasmic carboxy termini; the different termini result in varying abilities to associate at cell membranes with intracellular signaling proteins, although the biological roles of these differences are not yet understood (2–4). Ig isotype switching occurs by an intrachromosomal deletional recombination event, diagrammed in Figure 1 for the mouse H chain locus. The human H chain locus is similarly organized but not identical. Class switch recombination (CSR) occurs between switch (S) regions located upstream of each of the CH regions except Cδ and results in a change from IgM and IgD expression by naive B cells to expression of one of the downstream isotypes. IgD expression occurs by alternative transcription termination/splicing of the CμCδ genes. S regions consist of tandem repeats of short G-rich sequences (20–80 bp), which differ for each isotype, with an overall length varying from ∼1 kb to 12 kb, and CSR can occur anywhere within or near the S regions (5, 6). CSR occurs by an end-joining type of

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recombination, rather than by homologous recombination (7, 8). CSR and somatic hypermutation (SHM) are initiated by activation-induced cytidine deaminase (AID), which converts cytosines in S regions and Ig variable regions to uracils by deamination (9–14). Subsequent repair of the dU residues leads to single-strand DNA breaks (SSBs) that must be converted to double-strand breaks (DSBs) within the donor Sμ region and within an acceptor Sx region, to initiate the process of intrachromosomal DNA recombination. This review focuses mainly on the overall mechanism of CSR, which is discussed in the next section. Although there are interesting similarities and differences between CSR and SHM, we do not discuss them owing to space constraints. SHM is reviewed in another article in this volume by M.D. Scharff (15). Also, we do not extensively review all the information available about AID, as this protein is extensively discussed in the Scharff article (15) and in several other reviews (16–19). B cells undergo antibody, or Ig, class switching in vivo after immunization or infection or upon appropriate activation in culture. Engagement of the CD40 receptor on B cells by CD154 (CD40L) or, speciﬁcally for mouse B cells, the Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), provides crucial signaling for CSR. AID expression is induced in mouse splenic B cells activated to switch in culture, and also in vivo, with especially high levels detected in germinal center (GC) B cells, which are undergoing SHM and probably CSR (9, 20, 21). Most investigations into the roles of various genes in CSR examine their effects in mouse splenic B cells induced to switch in culture. This model allows one to use the numerous mouse gene knockout models and also ensures that the effects of the genes are B cell intrinsic and not due to effects on other cell types. CSR requires cell proliferation, appearing to require a minimum of two complete rounds of cell division for IgG and IgA CSR and perhaps additional rounds for IgE CSR

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Figure 1 Diagram of Ig class switch recombination (CSR) to IgA. (a) The mouse IgH locus in B cells expressing IgM and IgD (by alternative RNA transcription/processing). During CSR, activation-induced cytidine deaminase (AID) deaminates dC residues in the top and bottom strands of transcriptionally active S regions (Sμ and Sα in the diagram shown), initiating a process (described in the text) that results in double-strand DNA breaks (DSBs) in both S regions and CSR by intrachromosomal deletion (b). (c) The IgH locus after CSR to IgA. Splicing diagrams of the μ, δ mRNAs and the germline α transcript are indicated below the diagram of the locus. Similar germline transcripts are induced from unrearranged Cγ, Cε, and Cα genes, depending on the cytokine stimulation received by the B cell.

(22–25). This requirement appears to be at least partly due to the requirements for induction of AID expression (25). Transcription of AID mRNA is induced synergistically by IL4 and CD40 signaling via induction of Stat6 and NF-κB transcription factors (26). However, these signals are very rapid. Pax5 is also essential for AID mRNA transcription, and Pax5 binds to the AID promoter in LPS+IL4-treated splenic B cells (27). Most interestingly, binding of Pax5 to the AID promoter is not detected until two days after addition of the activators, suggesting that the kinetics

of Pax5 binding might be important for explaining the requirement for cell division for AID induction. Furthermore, AID function is regulated by active export from the nucleus (28–30), which might also contribute to the delay in CSR. Naive B cells have the potential to switch to any isotype, and cytokines secreted by T cells and other cells direct the isotype switch (reviewed in 7, 31, 32). Although there is more to be discovered, the predominant mechanism for regulating isotype speciﬁcity is by regulation of transcription through S regions, www.annualreviews.org • Class Switch Recombination

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and only transcriptionally active S regions undergo CSR. The regulation of isotype speciﬁcity is further discussed in the section on “Regulation of Switching.”

MECHANISM OF CSR

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Recently, the greatest progress in the ﬁeld of CSR has occurred in identifying and understanding the roles of the enzymes and proteins involved in both creating DNA breaks and recombining the S regions. This, then, is the focus of our review. In this section, we start with the enzymes that create the DSBs required for CSR and then discuss the joining mechanism and its regulation. We restrict our discussion to proteins known to contribute to CSR and do not mention proteins shown not to be involved or for which the contribution is only suggestive or might have only minor effects. Pan-Hammarstrom et al. (33) comprehensively reviewed which DNA repair proteins are involved in V(D)J, CSR, and/or SHM. Other recent reviews discuss the mechanism and regulation of CSR (18, 34–37).

Creation of DSBs for CSR A reasonably convincing model for how single-strand breaks (SSBs) are introduced into S regions and how they are converted to the DSBs required for CSR has been developed in recent years. After initiation of the process by AID, the base excision repair (BER) pathway creates the SSBs, and if the SSBs on opposite strands are sufﬁciently near, this results in a DSB. However, conversion of more distal SSBs to DSBs appears to require another DNA repair pathway, mismatch repair (MMR). The proteins and their function at each step in this process are discussed in this section. Activation-induced cytidine deaminase (AID). The ﬁnding that AID is essential for both CSR and SHM was a major breakthrough for these ﬁelds (9, 10). Although originally investigators postulated that AID is an 264

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RNA-editing enzyme, owing to its homology with the RNA-editing cytidine deaminase APOBEC-1 (9), a second major breakthrough came with the discovery that the role of AID is to initiate these processes by deamination of dC nucleotides within S regions and antibody variable regions (11–14, 38, 39). In 2000, a model was proposed by Poltoratsky et al. (40) for how deamination of dC in DNA could lead to the SSBs required for CSR, and shortly thereafter Petersen-Mahrt et al. (11) provided evidence for this model. This model is now supported by numerous studies. Using the technique of linker-ligation-mediated PCR (LM-PCR), researchers demonstrated that AID is required for generation of S region DSBs in both mouse and human B cells (21, 41, 42). Also, localization of γH2AX foci at the Ig locus during CSR is AID-dependent (43). Figure 2a presents the portion of this model that is relevant for CSR. Puriﬁed recombinant AID converts dC to dU nucleotides in single-stranded (ss) DNA and in supercoiled transcribed plasmids (12– 16, 18, 44–46). It is clear that ssDNA but not dsDNA is the AID substrate. When transcribed duplex DNA substrates are used in vitro or in Escherichia coli, the nontranscribed strand (top strand if the DNA sequence is oriented with the 5 side to the left) is preferentially targeted. This may be because the nontranscribed strand in these substrates is single-stranded at the small bubble formed by RNA polymerase, whereas the transcribed strand is transiently hydrogen bonded to RNA (12, 14, 44, 47). However, both strands can be targeted by AID in vitro in transcribed supercoiled plasmid DNA, presumably because single-strand extrusions can occur in supercoiled plasmids (45, 46). It is clear that in B cells, dC on both transcribed and nontranscribed strands is equally mutated in proportion to its distribution in V genes and S regions, indicating that AID attacks both strands (48–50). AID preferentially, but not exclusively, deaminates the underlined dC in WRC (W = A or T, R = purine, Y = pyrimidine), the

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Figure 2 Diagrams of (a) model for generation of DNA breaks, mutations, and translocations in IgS regions by AID-UNG-APE and (b) model for conversion of SSBs to DSBs by mismatch repair. (a) AID deaminates dC, resulting in dU bases, which are excised by one of the uracil DNA glycosylases, UNG. Abasic sites are cut by AP-endonuclease (APE1 and APE2) (74), creating SSBs that can spontaneously form DSBs if they are near each other on opposite DNA strands, or, if not, mismatch repair activity converts them to DSBs (see b). Alternatively, DNA Pol β can correctly repair the nick, preventing CSR, or error-prone translesion polymerases can repair the nick but introduce mutations. Finally, the DNA breaks can lead to aberrant recombinations/translocations. (b) AID is hypothesized to introduce several dU residues in S regions during one cell cycle. Some of the dU residues are excised by UNG, and some of the abasic sites are nicked by APE. The U:G mismatches that remain would be substrates for Msh2-Msh6 (100). Msh2-Msh6, along with Mlh1-Pms2, recruit Exo1 (and accessory proteins) to a nearby 5 nick, from where Exo1 begins to excise toward the mismatch (90, 91), creating a DSB with a 5 single-strand overhang, which can be ﬁlled in by DNA polymerase. Fill-in synthesis is probably performed by translesion polymerases owing to the presence of abasic sites. Alternatively, the overhang is removed by a 5 ﬂap endonuclease (Fen1) or by Exo1.

AID hotspot target (51–53), in transcribed targets in vitro (54), in oligonucleotide substrates (55), and in vivo (50). S regions contain numerous AID hotspot targets. However, experiments using puriﬁed recombinant AID and plasmid DNA substrates suggest that AID is highly processive (14, 45, 54), and in this process AID deaminates many dC nucleotides

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that are not in WRC motifs. Furthermore, studies in ung−/− msh2−/− B cells, in which the initial AID-induced lesions cannot be repaired and thus can be examined, also suggest that AID acts processively across S regions in vivo (50). The regulation of AID function is just beginning to be studied. AID is phosphorylated www.annualreviews.org • Class Switch Recombination

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by protein kinase A at S38 and T27, and alanine mutations at either of these sites nearly eliminate the ability of AID to initiate CSR when retrovirally transduced into aid−/− splenic B cells (56, 57, 58). Phosphorylation by protein kinase A is required for binding of AID by replication protein A, which enables AID to deaminate transcribed duplex DNA. Replication protein A appears to increase the interaction of AID with DNA. Another very interesting but not understood discovery about AID is that the C-terminal 10 amino acids of AID are required for CSR but not for SHM (59, 60). The C terminus may be required for interaction with a protein essential for CSR but not for SHM.

UNG. Removal of the dU residues by enzymes within the BER pathway is required to introduce the DNA breaks necessary for CSR (11, 21, 38). BER consists of highly active ubiquitous DNA repair pathways for repairing oxidized and deaminated bases, which are generated more than 104 times per cell per day by oxidation, especially during inﬂammation, and by spontaneous hydrolysis (61). There are four mammalian uracil DNA glycosylases in the BER pathway (i.e., enzymes that excise dU bases), UNG, SMUG1, MBD4, and TDG. CSR is reduced by 95% in B cells from UNGdeﬁcient mice and in patients that have deleterious mutations in UNG (21, 38, 62). Furthermore, S region DSBs are also greatly reduced in ung−/− mouse splenic B cells induced to undergo CSR (21). Thus, it is reasonable to conclude that UNG is the uracil DNA glycosylase that excises the dU residue created by AID activity. Two other uracil glycosylases (MBD4 and SMUG1) appear to have no role in CSR (38, 63). However, when SMUG1 is greatly overexpressed by retroviral transduction, it can support a limited amount of CSR in ung−/− cells, although in the presence of UNG, SMUG1 actually inhibits CSR (64). These data suggest that UNG may be speciﬁcally recruited to sites of AID activity and perhaps has a unique property that makes it the 266

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preferred uracil DNA glycosylase for generation of the DNA breaks required for CSR. Apurinic/apyrimidinic endonuclease (AP endonuclease/APE). The BER enzyme that repairs the abasic sites left by UNG activity is APE, which incises the phosphate backbone of DNA at abasic sites, producing SSBs (61). In mammals, there are three enzymes with AP endonuclease activity, APE1 and APE2, which share homology (65), and a very different, recently discovered enzyme, PALF/APLF/XIP1 (66–68). The main AP endonuclease known to be involved in BER is APE1. APE1 endonuclease activity is essential for early embryonic development and for viability of human cell lines (69, 70). Much less is known about APE2, which is encoded on the X chromosome. APE2-deﬁcient mice show a slight growth defect and have a twofold reduction of white blood cells in the periphery, mainly affecting T and B cells (71). Enzyme assays using abasic site-containing oligonucleotide substrates showed that puriﬁed recombinant human APE2 has weaker AP endonuclease activity than APE1 (65, 72, 73). Examination of APE1- and/or APE2deﬁcient mouse splenic B cells activated to switch in culture demonstrated that both of these enzymes contribute to CSR (74). Because APE1 is an essential gene, the investigators used ape1+/− mice, which have DNA repair defects (75, 76), although the phenotype was probably less severe than if ape1−/− mice could have been studied. Splenic B cells from mice deﬁcient for APE2 and heterozygous for APE1 switch 60%–80% as well as wild-type B cells, depending on the isotype, and have very few S region DSBs, almost as few as in aid−/− cells, which have approximately 10% of wildtype (77). These cells divide in culture as well as wild-type cells, so the reduced CSR is not due to poor proliferation (74). Although CSR is modestly reduced, the much greater reduction in Sμ DSBs suggests that Sμ DSBs are not limiting for CSR. In wild-type cells, AID-dependent blunt and staggered DSBs in the Sμ region occur

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preferentially at G:C bp and at AID hotspot targets, with p values for blunt DSBs ≤ 0.002 relative to random (21, 74). This indicates that SSBs and DSBs occur at the dC nucleotides that are targeted by AID, as predicted by the DNA-deamination model (Figure 2a). In aid−/− or ung−/− or APE-deﬁcient cells, DSBs do not speciﬁcally occur at AID hotspot targets, although B cells deﬁcient in only one of the APEs maintain some speciﬁcity for AID targets (21, 74). These data suggest that APE1 and APE2 both serve as endonucleases to incise abasic sites introduced by AID and UNG. However, whether the third AP endonuclease PALF/APLF/XIP-1 also has a role is unknown. DNA polymerase β (Pol β). In the canonical BER pathway, the single-nucleotide gap generated by the action of UNG and APE is ﬁlled in by DNA polymerase β (Pol β) (78, 79). A multiprotein complex that can perform BER and that contains UNG2 (nuclear form of UNG), APE1, Pol β, replicative DNA polymerases δ and ε, XRCC1, and DNA ligase I has been isolated from both proliferating and growth-arrested HeLa cells and also from human peripheral blood lymphocytes (80, 81). Furthermore, physical interactions among BER enzymes increase repair efﬁciency (82–84). These ﬁndings suggest that BER will proceed to completion once it is initiated by UNG. Therefore, Pol β activity could reduce SSBs and therefore reduce CSR. Hence, an intriguing question arises as to how S region breaks are spared from faithful repair. One appealing hypothesis is that BER components downstream of UNG and APE might be downregulated in cells undergoing CSR or speciﬁcally prevented from accessing S region lesions. As Pol β is recruited by APE1 (78), the levels of Pol β or its activity may be inhibited during CSR, or APE2 may not recruit Pol β, which could explain why APE2 is used for CSR. Alternatively, the introduction of numerous S region lesions may overwhelm the BER machinery, although BER activity is not inhibited during CSR. A recent

report supports the second alternative (85). Pol β levels were increased in nuclear extracts from mouse splenic B cells induced to undergo CSR, and chromatin immunoprecipitation showed that Pol β associates speciﬁcally with the Sμ region, but not with Cμ or Cα genes, in switching B cells. Although Pol β–deﬁcient mice die in utero, one can obtain polβ −/− splenic B cells by transfer of polβ −/− fetal liver cells into irradiated wild-type mice. Using polβ −/− and polβ +/+ splenic B cells obtained by fetal liver transfer, investigators found that polβ −/− cells actually show modestly increased CSR (1.5to 1.7-fold) to a subset of isotypes (IgG2a, IgG2b, and IgG3) relative to polβ +/+ controls. Furthermore, LM-PCR experiments showed that polβ −/− cells have a two- to threefold increase in Sμ and Sγ3 DSBs and a twofold increase in mutations in the germline (GL) Sμ and recombined Sμ segments. These data indicate that Pol β indeed functions to accurately repair AID-instigated SSBs but cannot repair them all, and the authors hypothesize that Pol β only inhibits switching when SSBs are limiting (85). If B cells were to downregulate BER during CSR, this could be dangerous, given the great amount of reactive oxygen species produced during B cell activation and proliferation (86). Therefore, it is plausible that instead a mechanism is adopted that endows S regions with such numerous AID targets that the ability of BER to repair them is overwhelmed, rather than abrogating overall BER ability and thus jeopardizing the integrity of the B cell genome. Consistent with this hypothesis is the ﬁnding that, in ung−/− msh2−/− B cells, AID introduces many more lesions into the Sμ region than result in mutations in wild-type cells, most likely because they are correctly repaired in wildtype cells (50). Furthermore, recent experiments show that artiﬁcially introduced I-SceI sites in Sμ and Sγ1 regions mediate CSR to IgG1. These experiments suggest that only a single DSB in the donor and acceptor S region is sufﬁcient for CSR (87). Introduction of www.annualreviews.org • Class Switch Recombination

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MMR (mismatch repair): a ubiquitous repair pathway that corrects DNA synthesis errors, including mismatches and deletions/insertions

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numerous dU residues increases the likelihood of DSBs occurring in the donor and acceptor S regions simultaneously. In conclusion, Pol β may function normally during CSR to repair AID-initiated DNA lesions, but the numerous AID lesions overwhelm it, and thus some breaks remain unrepaired. Role of mismatch repair in CSR: to convert SSBs to DSBs. A second repair pathway, mismatch repair (MMR), contributes to CSR but is not essential. The major role of MMR in all cells is to correct misincorporated nucleotides during DNA synthesis (88). This process involves recognition of the mismatch by a heterodimer of Msh2-Msh6 (for nucleotide substitutions and small loops) or Msh2-Msh3 (for larger loops), followed by recruitment of the Mlh1-Pms2 heterodimer (88). The combined heterotetramer recruits replication factor C, the processivity factor proliferating cell nuclear antigen (PCNA), and Exonuclease1 (Exo1) to a nearby nick, and together they excise the single-strand segment containing the mutated nucleotide (89, 90). The excised single-strand patch can be hundreds of nucleotides long in vitro, but the length in vivo is unknown. MMR speciﬁcally repairs the newly synthesized DNA strand, probably because of its predilection to excise and resynthesize the nicked DNA strand (91). In mice that lack one of the MMR genes (Msh2, Msh6, Mlh1, Pms2, or Exo1), CSR is reduced by two- to sevenfold, depending on the gene and the Ig isotype (92–98). The most attractive model for the role of MMR during CSR is to convert SSBs that are not near each other on the opposite DNA strands to DSBs (77, 99). If the SSBs that are introduced by AID-UNG-APE are near each other on opposite DNA strands, they can spontaneously form a DSB, but if not, the SSBs do not destabilize the duplex and are simply repaired. As S regions are large and the breaks appear to occur anywhere within S regions (5, 6, 21), it seems unlikely that the SSBs would be sufﬁciently proximal to form a DSB most of the time. MMR could conStavnezer

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vert these distal SSBs to the DSBs that are required for CSR. Figure 2b presents a model for how MMR could do this (99). Msh2-Msh6 can recognize and bind U:G mismatches created by AID activity (100). Mlh1-Pms2 would be recruited and Exo1 would excise from the nearest 5 SSB created by AID-UNG-APE activity toward the mismatched dU:dG. Exo1 is hypothesized to continue past the mismatch until it reaches a SSB on the other strand, thus creating a DSB. Several additional experimental results support this model. First, B cells in which the tandem repeats of Sμ have been deleted (SμTR−/− ), which thus have very few AID hotspot targets, only show a ∼twofold reduction in CSR (101). However, in these B cells, CSR is nearly ablated in the absence of Msh2 or Mlh1 (77, 102; J. Eccleston, C.E. Schrader, J. Stavnezer & E. Selsing, manuscript in preparation). Second, IgG2a, the isotype with the fewest AID hotspot targets in its S region, is the isotype most dependent upon MMR (93). Third, the great majority of S-S junctions in msh2−/− B cells occurs within the Sμ tandem repeat region, whereas in wild-type cells they can also occur upstream of Sμ, where the AID hotspot targets are infrequent (92). Fourth, the S-S junctions differ between wild-type and MMR-deﬁcient B cells as to the lengths of microhomology between the donor (Sμ) and acceptor (downstream) S regions. This suggests that MMR is involved in end processing from the sites of the SSBs, resulting in different lengths of single-stranded tails that can participate in homology search during S-S recombination (94, 95, 98, 103). Fifth and most importantly, LMPCR experiments show that MMR-deﬁcient B cells have fewer S region DSBs than do wildtype B cells (77). End processing and mutations. After DSB formation, 5 or 3 single-strand overhangs remain. These tails must be either excised or ﬁlled in to create blunt, or nearly blunt, DSBs appropriate for an end-joining recombination with the other S region. The

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structure-speciﬁc endonuclease ERCC1XPF excises 3 single-strand tails at the junction with dsDNA and has a minor role in CSR (104). MMR normally recruits PCNA and replicative DNA polymerase for ﬁll-in synthesis of 5 overhangs (88). However, replicative Pol cannot replicate past an abasic site, resulting in the recruitment of error-prone translesion Pols. Msh2-Msh6 recruit the translesion Pol η (100), which is the most important translesion Pol for introducing mutations at A:T bps into V region genes and into both unrecombined (GL) Sμ and recombined S-S junction regions during CSR (105, 106). There are numerous mutations at both A:T and G:C bp in the regions surrounding S-S junctions (107, 108, 145). The mutations at G:C bp could be due to ﬁll-in DNA synthesis across dU nucleotides by replicative Pols or across abasic sites, probably by the translesion DNA Pol θ (109–111). Additionally, not all mutations in S regions are likely to be due to ﬁll-in DNA synthesis after creation of DSBs. It is likely that sometimes the excision by Exo1 does not lead to DSBs, but instead the single-strand patch is simply repaired by translesion Pols. Also, sometimes DSBs will form that do not successfully synapse with acceptor downstream S regions and that result in internal Sμ deletions rather than S-S recombination (112, 113).

Joining of Donor and Acceptor S Regions After formation of the DSBs in the donor and acceptor S regions, the S regions are recombined using ubiquitous proteins that perform nonhomologous end-joining (NHEJ) in all cell types. These proteins are observed within a complex visible by in situ immunoﬂuorescence in cells that have been treated with gamma radiation to induce DSBs, which is consistent with the fact that these proteins are involved in repair of DSBs after gamma radiation as well as repair of DSBs generated during CSR. Many of these proteins are also

involved in V(D)J recombination during lymphocyte development. S-S recombination occurs by an endjoining type of recombination. DNA DSBs can be induced by ionizing radiation or during repair of oxidative damage or replication. Numerous ubiquitous repair proteins exist that rapidly repair DSBs in all cells, many of which are also involved in CSR. DSBs produced during DNA replication or during G2 phase of the cell cycle are generally repaired by homologous recombination, as there is a chromosomal homolog that can be copied. However, S region DSBs in B cells induced during CSR are generated and resolved during G1 phase (77), and S regions lack sufﬁcient homology to undergo homologous recombination. Four proteins known to be essential for NHEJ, Ku70, Ku80, and the two-protein ligase complex XRCC4-ligase IV, are very important for CSR (113–120). Ku70-Ku80 binds to the DNA ends and serves as a tool belt for the end-joining reaction by recruiting enzymes that effect the recombination. Ku70Ku80 improves the binding of XRCC4-ligase IV to DNA ends (121–123). Although Kudeﬁcient cells apoptose upon induction of CSR, Reina-San-Martin et al. (113) showed by CFSE staining that, at each cell division, CSR in the few viable ku80−/− cells is nearly ablated. These investigators also increased cell viability by expressing Bcl2 from a transgene in ku80−/− cells and again showed that CSR is nearly ablated. XRCC4 or ligase IV deﬁciency is incompatible with life owing to problems during brain development, although human patients with hypomorphic mutations have been described, and mice with deﬁciencies have been created (120, 124, 125). Mice entirely lacking XRCC4 were produced by mating xrcc4+/− mice with p53-deﬁcient mice, allowing survival of xrcc4−/− mice, which have ∼25% of normal levels of CSR, indicating that XRCC4 is important but not essential for CSR (125). Also, humans with mutations in ligase IV have fewer peripheral blood cells that have www.annualreviews.org • Class Switch Recombination

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ATM (ataxia telangiectasia mutated): a phosphoinositol 3-kinase-like kinase, which is mutated in the human syndrome Ataxia telangiectasia

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undergone CSR than normal controls (119). Examination of the S-S junctions in the xrcc4−/− mouse B cells and in the human patients demonstrated that the S-S junctions are aberrant, as they have greatly increased lengths of junctional microhomology (119, 125). Numerous studies have shown that SS junctions in wild-type mice or normal individuals show very little microhomology, or identity, between the donor Sμ and acceptor S regions at the junction, usually 0 or 1 bp of identity (5), consistent with recombination by NHEJ. In contrast, in the xrcc4−/− mice and human ligase IV hypomorphs, many junctions have up to 10 bp or more of identity. Taken together, the data indicate that CSR occurs by NHEJ but can also occur by an alternative type of end-joining reaction that favors the use of microhomologies. XRCC4-ligase IV in the presence of Ku70-Ku80 ligates incompatible ends, consistent with the lack of microhomology at S-S junctions in wild-type cells (126). It is unknown, however, whether in wild-type cells the alternative pathway is actually used. A fundamental unanswered question regarding the joining process is whether the donor and acceptor S regions are in close proximity before AID attacks the donor (Sμ) or acceptor (downstream) S region. It is attractive to expect that they are preassociated in order to increase the likelihood of correct S-S recombination. This association could be directed to the correct S region by GL transcription. Another reasonable possibility is that the association occurs immediately after AID attacks Sμ. In situ studies suggest that the Ig loci form loops, apparently bringing the V and J genes in proximity prior to V(D)J recombination (127, 128). Mre11-Rad50-Nbs1. Accumulating evidence indicates that this complex of three proteins (MRN complex) travels along the DNA duplex scanning for DNA breaks and that MRN binds DSBs within seconds of their formation (129, 130). However, owing to its great abundance, Ku is likely to bind

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DSBs even faster during CSR (see the section below, Ku70-Ku80-DNA-PKcs). When a DSB is encountered, the conformation of Mre11 and Rad50 changes, resulting in unwinding the DNA ends at the break. Mre11 is a globular protein associated with the DNA, whereas Rad50 forms a long coiled-coil that at its middle abruptly reverses direction, forming a loop with a zinc hook at its apex. The hooks from two Rad50 molecules associate homotypically, and this is thought to be important for holding two DNA duplexes together at the DSB (131, 132). Once bound to a DSB, the kinase ataxia telangiectasia mutated (ATM) binds the complex via Nbs1, becomes activated, and phosphorylates several substrates, among which are Nbs1, 53BP1, p53, Chk2, and H2AX, causing a further accumulation of MRN and several other repair proteins and also activating cell-cycle checkpoints (133– 135) (Figure 3). Thus, MRN is upstream of a cascade of events that function to sense the DSB, resulting in repair by end joining or by homologous recombination. Null mutations in any component of MRN are lethal, and hypomorphic mutations result in aberrant chromosomes and translocations (136) and the disease syndromes Nijmegen breakage syndrome (Nbs-1 mutations) and ataxia-telangiectasia-like disorder (Mre11 mutations), characterized by increased sensitivity to ionizing radiation and other DSB-inducing agents (137, 138). Two groups have shown that CSR is reduced two- to threefold in cultured splenic B cells from mice in which the Nbs1 gene is inactivated by a conditional mutation (136, 139). The reduced CSR was analyzed in cells stained with CFSE, measuring switched cells at each cell division, as cell viability and proliferation are reduced in the mutant cells. Because some Nbs1 remains in the cells, these are most likely underestimates of the importance of Nbs1 for CSR. When wildtype mouse splenic B cells are treated with switch inducers in culture, the Sμ region undergoes rare translocations with the c-myc

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DNA-PKcs H4-K20me2 H2AX

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P ATM

Cell-cycle regulation, apoptosis Figure 3 Proteins that bind to DSBs that are involved in CSR. The evidence supporting this diagram is discussed in the text. DNA-PK, which consists of Ku70-Ku80 and DNA-PKcs, most likely binds DSB ends ﬁrst. Mre11-Rad50-Nbs1 (MRN) probably binds next, perhaps helping to hold the DNA ends together, and MRN recruits and activates ATM (ataxia telangiectasia mutated), which phosphorylates H2AX, 53BP1, and Mdc1, resulting in accumulation of large amounts of these proteins and MRN around the DSB sites. After the DNA ends are brought sufﬁciently near by activities of these other proteins, DNA-PK can hold the DNA ends in the correct position for recombination. These proteins are all important for correct S-S recombination, and they inhibit aberrant recombinations and translocations. ATM also phosphorylates proteins that regulate the cell cycle and induce apoptosis, but nothing is known about how important this is during CSR.

gene, which is located on a different chromosome (140–142). This translocation is AIDdependent and, most interestingly, occurs six times more frequently in Nbs1-hypomorph B cells than in wild-type B cells (140, 143). Patients with hypomorphic mutations in Nbs1 or Mre11 also have a lower percentage of peripheral blood lymphocytes that have undergone CSR, as assayed by detection of Sμ-Sα junctions in these cells (33, 144, 145). Further evidence for the participation of Nbs1 in CSR is the demonstration by immunocytochemistry-fluorescence in situ hybridization (immuno-FISH) that Nbs1 foci colocalize with the IgH loci, but not with Igκ loci, in wild-type, but not aid−/− , splenic B cells induced to undergo CSR (43). The ﬁnding of increased translocations with c-myc in MRN-deﬁcient B cells suggests that the MRN

complex is involved in organizing efﬁcient and accurate S-S recombination.

Ataxia telangiectasia mutated (ATM). ATM is a ser/thr protein kinase, a member of the phosphoinositol 3-kinase-like kinase (PIKK) family, which includes DNA-PKcs and ATR and which is involved in cell-cycle control and DNA damage responses. Upon activation by MRN, ATM accumulates at repair foci, orchestrates repair of the DSB, and initiates a cell-cycle checkpoint until repair is complete (146, 147). Recently, investigators demonstrated that V(D)J recombination is abnormal and that DSBs resulting from incomplete V(D)J recombination during lymphocyte development are maintained over several cell generations in atm−/− mice (148–150). www.annualreviews.org • Class Switch Recombination

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RAGs (recombinationactivating genes): initiate and are required for V(D)J recombination

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Thus, both repair and the cell-cycle checkpoints are compromised, as atm−/− lymphocytes continue to replicate despite chromosomal breaks (142, 143). CSR is reduced about threefold at each cell division cycle in cultured atm−/− splenic B cells relative to wild-type B cells (151, 152). Also, lower titers of switched antibodies are detected in sera after immunization. However, GL transcripts are expressed at normal levels. In B cells induced to switch in culture, the Sμ region undergoes translocations with the c-myc gene eight times more frequently in atm−/− cells than in wild-type cells (143). Thus, it seems likely that when atm−/− B cells are activated to switch, DSBs are generated as usual, are maintained longer than usual, and do not undergo normal Sμ-Sx recombination; this can result in aberrant recombinations with other chromosomes. Ataxia telangiectasia patients often have IgA and IgG deﬁciencies and have peripheral blood lymphocytes with fewer Sμ-Sα junctions than normal individuals, similar to patients with mutated Nbs1 or Mre11 and consistent with reduced CSR (33, 144). Thus, during CSR, ATM likely organizes the repair complex and perhaps halts the cell cycle, and it contributes to the correct positioning of DSBs together during the long-range interaction required for accurate S-S recombination. In the absence of ATM, prolonged duration of unrepaired DSBs and aberrant recombination events result in translocations. 53BP1. 53BP1 was ﬁrst discovered in a yeast two-hybrid screen as a protein that binds p53 (153). 53BP1 is a transcriptional coactivator for p53, binding through its tandem Tudor domains to p53 dimethylated at lysine 370 (154). However, this is not its only function. 53BP1 accumulates at DSBs within 2 min after ionizing radiation treatment (155). Its initial recruitment to DSBs does not depend on any other known protein, including Nbs1, ATM, or DNA-PK (156), although its subsequent accumulation in foci does depend on γH2AX and Mdc1 (157–159). 53BP1 func-

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tions to induce phosphorylation of ATM and ATR (160), and thus it might also increase MRN activity and MRN’s accumulation at DSBs. Strikingly, CSR is reduced about 90% in cultured 53bp1−/− splenic B cells relative to wild-type B cells, which is not due to decreased cell proliferation nor to reduced GL transcripts. Serum IgG, IgE, and IgA isotypes are reduced even more, although IgM levels are normal (161, 162). The S-S junctions are normal. 53BP1-deﬁcient cells do not have a dramatic increase in general chromosome instability, unlike atm−/− and h2ax−/− cells, but a much higher proportion of the chromosomal aberrancies in 53bp1−/− cells involve the IgH locus, suggesting that 53BP1 has a special role at this locus (142, 159). Another hint about the role of 53BP1 in CSR comes from the ﬁnding that there is a threefold increase in internal deletions within S regions in IgM hybridomas produced from 53bp1−/− B cells induced to undergo CSR in culture in comparison to IgM hybridomas from wildtype mice (163). Interestingly, these deletions are not increased in atm−/− or h2ax−/− B cells, despite the general increase in unrepaired DSBs in the genome. These results suggest that 53BP1 might also be important for bringing together, or synapsing, the donor Sμ and downstream S regions (159, 161). How 53BP1 performs this role, however, is completely unknown. This role would be consistent with the lack of a role for 53BP1 during V(D)J joining because the RAG complex itself possesses synaptic activity (161, 162). Mammalian 53BP1 binds dimethyl-lysine 20 of histone H4 (H4-K20-me2), but not mono- or trimethyl K20, via its Tudor domains (164). Most interestingly, 53BP1 with single amino acid mutations within the Tudor domains that prevent H4-K20me2-binding and RNAi-mediated knockdown of PrSet7/Set8, the histone methyltransferase that monomethylates K20 (a prerequisite for the dimethylation), decrease 53BP1 foci (164). Association of 53BP1 with

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irradiation-induced foci is disrupted by RNase A treatment, suggesting that there may be an RNA component involved in its binding (155). Taken together, H4-K20-me2 may be important for recruiting 53BP1 to chromatin, perhaps to the IgH locus, although this modiﬁcation would probably need to be present before induction of DSBs during CSR because 53BP1 binding to DSBs is so rapid. The GL RNA transcribed from S regions may help 53BP1 recruitment. This hypothesis requires that the H4-K20-me2 mark is associated with actively transcribed regions on the IgH locus during G1 phase, which would differ from its distribution on bulk chromatin. γH2AX (phosphorylated form of H2AX). H2AX is a variant of histone H2A, representing about 15% of the cellular pool of H2A. It is randomly incorporated into nucleosomes (165). Within seconds after formation of a DSB induced by ionizing radiation or by a restriction enzyme, the extended C-terminal tail of H2AX is phosphorylated by a PIKK kinase, most frequently ATM (166, 167), and this phosphorylation spreads over a region estimated to span up to a megabase surrounding the break (147, 168). In fact, the peak accumulation of γH2AX (phosphorylated H2AX) is not directly at the DSB but instead located at 8–10 kb on either side in mammals (168). ATM also phosphorylates 53BP1, Nbs1, and Mdc1, which then all bind the phosphorylated tail of γH2AX, which serves as a docking site for these proteins. This phosphorylation results in a rapid assembly of these factors, plus Mre11, Rad50, and Brca1, into a large multiprotein complex. γH2AX is required for the accumulation of these proteins into foci near DSBs (169, 170). However, mice lacking H2AX can still repair DSBs, although with lower efﬁciency, and they can still induce cellcycle checkpoints (169), probably because the initial assembly of repair proteins, including MRN, ATM, and 53BP1, does not depend on γH2AX (157). CSR is markedly reduced in H2AXdeﬁcient mice. In vitro CSR to IgG3 and IgG1

is reduced to ∼25%–30% of wild-type, and this is not due to defective cell proliferation. The antigen-speciﬁc IgG1 response to immunization is reduced to about 30% of wild-type mice, and nonimmune serum levels of IgG1, IgG3, and IgA are all reduced to 15%–50% of wild-type (43, 113, 142, 169). Similar to cells deﬁcient in either ATM, 53BP1, or Mdc1 or cells having Mre11 or Nbs1 hypomorphic mutations, h2ax−/− B cells show numerous chromosome breaks and aberrant recombination events (142, 143, 169, 171). In h2ax−/− B cells induced to switch in culture, there is a greater than tenfold increase in AID-dependent chromosome breaks within the IgH locus relative to wild-type cells, resulting in separation of the V genes and 3 end of the CH genes and translocations in metaphase chromosome spreads (142). The breaks occur on both chromatids, indicating that they occur prior to S phase, consistent with evidence that S region DSBs are observed in G1 phase (77). Also, foci containing γH2AX (and Nbs1) colocalize with the IgH locus in mouse splenic B cells induced to switch, in wild-type cells in the G1/early S phase of the cell cycle, but not in aid−/− cells, as detected by immuno-FISH (43). Taken together, these data suggest that γH2AX, like MRN, Mdc1, ATM, and 53BP1, is involved in holding AID-initiated DSBs in a structure that promotes accurate synapsis between Sμ and the downstream acceptor S region and also prevents recombinations with DSBs on other chromosomes.

Mdc1 (mediator of DNA damage checkpoint protein 1). Mdc1 is a mediator protein that is recruited to DSBs by binding to Nbs1 and is phosphorylated by ATM (172, 173). Mdc1 subsequently recruits ATM to γH2AX and is required for accumulation of γH2AX at DSBs (167, 173). Without Mdc1, the initial recruitment by MRN of ATM and γH2AX occurs, but the complexes are unstable and do not form the large repair foci observed in wildtype cells. CSR in cultured mdc1−/− B cells is www.annualreviews.org • Class Switch Recombination

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only mildly reduced, to about 50%–70% of wild-type B cells (167).

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Ku70-Ku80, DNA-PKcs. The proteins discussed above are all involved in repairing DSBs both by end joining and by homologous recombination. However, Ku70 and Ku80 are only involved in end joining and appear to prevent the use of homologies during recombination. Ku70-Ku80 binds DNA ends at DSBs and telomeres and mediates synapsis of the two DNA ends, positioning the ends to allow end processing and direct end-to-end joining (174, 175). Ku70-Ku80 forms a ring with a broad base that encircles DNA and can only dissociate at an end (176). After binding, Ku slides away from the ends, allowing the catalytic subunit, the kinase DNA-PKcs, to bind to each end (177). Ku binds the nuclear matrix, and this binding might localize the DSBs and telomeres to the matrix (178). DNA-PKcs is transphosphorylated by the other DNA-PKcs bound at the other end of the DSB and also phosphorylates Ku70, Ku80, and XRCC4 (175, 179). Thus, DNA-PK appears to be acting both as an activator and scaffold during the actual ligation event. DNA-PKcs has several transautophosphorylation sites, and mutation studies demonstrate that their phosphorylation regulates the accessibility of the DNA ends to end-processing activities required for recombination (179). All three components of the DNA-PK holocomplex are involved in CSR, as they are essential for NHEJ, although Ku70 and Ku80 are much more important than DNA-PKcs. All three proteins are essential for V(D)J recombination, which occurs by NHEJ, so mice lacking any of these proteins do not develop B cells, unless they are supplied with transgenic (Tg) recombined heavy (H) and light (L) chain genes. Ku70- and Ku80-deﬁcient B cells reconstituted with a Tg L chain gene and a H chain gene knocked into the endogenous locus do not have switched isotypes in their serum nor do they undergo CSR when induced to switch in culture, although they 274

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have normal levels of GL transcripts (113, 117, 118). Unlike wild-type cells, internal Sμ deletions do not occur in ku80−/− cells (113). These data suggest that Ku80-deﬁcient B cells that sustain DNA breaks owing to AID activity cannot recombine these DSBs, even by internal Sμ deletions, and therefore die. This would also explain why cells with mutations in the unrecombined Sμ segment are not observed in ku80−/− cells (113). Although the investigators did not demonstrate that ku80−/− cells express AID, this is highly likely. It is puzzling, however, that transition mutations that result from dU bases being replicated prior to UNG and APE activity, which would not lead to DNA breaks, were not observed in the GL Sμ. Perhaps Sμ regions with dU bases also have UNG-APE induced DNA breaks, resulting in death of these Ku-deﬁcient cells. It is surprising that Ku is essential for CSR, although XRCC4 is not, given the evidence that CSR can occur by an alternative endjoining pathway. This suggests that Ku might have another function and perhaps even function in the alternative pathway. Also, Ku appears to be more important than MRN for CSR. Ku is one of the most abundant nuclear proteins (∼4 × 105 molecules per cell) and has a very high afﬁnity for DNA ends (5 × 10−10 ) (174). Thus, it might bind prior to MRN. Perhaps MRN is more important for recruiting additional proteins involved in chromatin accessibility and perhaps for cellcycle regulation, whereas Ku is bound at the ends, positioning them precisely for recombination and for end processing by nucleases and polymerases and for recruiting XRCC4DNA ligase-IV (121, 122). It is interesting that Ku is required for V(D)J recombination, but MRN is not, suggesting that the synaptic activity of RAG cannot replace the role of Ku but can replace MRN. By contrast, DNA-PKcs is not essential for CSR, and its importance is controversial, with different results obtained by three different groups. DNA-PKcs deletion was reported to eliminate CSR in cultured B cells to every isotype except IgG1 (180, 181). Two

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groups (182, 183) studied B cells from scid mice, which have a deletion in the DNAPKcs gene resulting in loss of the C-terminal 83 amino acids, in barely detectable protein levels, and in no detectable kinase activity. These two groups found a smaller reduction in CSR. It was reduced to 25%–50% of wildtype for all isotypes in the study by Cook et al. (183), but CSR occurred at 50%–100% of wild-type, depending on the isotype, in the Bosma et al. (182) study. There was no difference in proliferation between the wild-type and DNA-PKcs-deﬁcient B cells induced to switch, but there was more cell death in the mutant cell cultures (183). It is difﬁcult to reconcile the different results among the three groups, except that Manis et al. (180, 181) studied mice with no DNA-PKcs, whereas the Bosma (182) and Cook (183) groups studied scid mice, suggesting that the tiny amount of protein present that lacks kinase activity has some function, perhaps as a scaffolding protein. The differences in CSR between the scid mice studied by the two groups might be due to different amounts of back-crossing, as B cells from different mouse strains have different abilities to switch (184). The role of DNA-PKcs in V(D)J recombination appears to be to stimulate the hairpin cutting activity of Artemis (185), which is consistent with its importance for joining the coding ends but not the signal ends. As CSR does not involve hairpin ends, and Artemis appears to have no role in CSR (181), DNAPKcs must have another role in CSR. This may involve its ability to regulate end processing, which may help recombination at some S-S junctions and possibly involves its ability to phosphorylate other proteins (174).

REGULATION OF SWITCHING Germline (GL) Transcripts As described above, CD40 and/or TLR stimulation provides essential activation for B cells to undergo CSR. Additionally, cytokines produced by T helper cells and dendritic cells de-

termine the isotype to which B cells will switch by inducing transcription from GL promoters located upstream, i.e., 5 , to each acceptor S region. The resulting GL transcripts do not encode proteins and are therefore also referred to as sterile RNAs. Figure 1 shows the transcription unit and splicing diagram for an example, GL α RNA. The exon located 5 to the S region is called the I exon. Each GL transcript has a similar transcription and splicing pattern (31). There is also a similar GL Sμ RNA, initiating near the μ intron enhancer. Gene-targeting experiments in which a single I exon and/or promoter for a speciﬁc GL transcript is deleted showed that GL transcription of the acceptor S region is required for CSR to that isotype and that the GL transcription only functions in cis, i.e., not on the other chromosome (186, 187). Surprisingly, deletion of the GL γ1 exon splice donor also prevents IgG1 CSR, although there is no known role for the spliced transcript (31, 188, 189). The role of splicing is an intriguing unanswered question. GL promoters have cytokine-responsive elements within them. GL γ1 and promoters, which are induced by IL-4, have binding sites for the IL-4-induced transcriptional activator Stat6. Several promoters have binding sites for NF-κB, which is induced in response to both CD40 and TLR signaling. GL γ2b and α promoters have binding sites for Smad and Runx, two factors induced by TGF-β, which induces CSR to IgG2b and IgA. A thorough review of GL transcriptional regulation was recently published (32). The function of GL transcription appears to be to direct AID to a speciﬁc S region and to make the S region a suitable substrate for AID. There are at least three possible roles for GL transcription, which are not mutually exclusive. First, the substrate for AID is ssDNA, and the act of transcription creates short regions of ssDNA at the transcription bubble. In addition, RNA transcribed from S regions is G-rich and therefore can form an RNA-DNA hybrid (R-loop) with the bottom C-rich DNA strand. This hybrid formation leaves the top www.annualreviews.org • Class Switch Recombination

R-loops: RNA-DNA hybrids that form cotranscriptionally if the DNA is highly G-rich, which results in extensive single-strand regions on the G-rich DNA strand

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G-rich DNA strand single-stranded over long stretches (190, 191), thereby making the top strand a target for AID. However, as AID appears to target both top and bottom strands equally in vivo (50), this makes the importance of R-loops unclear. Furthermore, when the Xenopus Sμ region, which is A:T rich and cannot form R-loops, is substituted into the Sγ1 locus by gene targeting, CSR is reduced only about twofold relative to a Sγ1 segment of the same length (192, 193). However, as R-loops form at both the Sμ and acceptor S regions normally, the effect of both R-loops might be to increase CSR by fourfold, which should be physiologically signiﬁcant. Evidence suggests that R-loops at the Sμ region begin upstream of the tandem repeats, at a particular sequence GGGGCTGGG, which is within a zone that has a high content of G (50%) (190), and, interestingly, S-S junctions often involve sequences 5 to Sμ, although many also occur 5 to this sequence (5, 102). Mice with a targeted deletion of the Sμ tandem repeats retain this sequence and have R-loops and extensive single-strand regions on the nontranscribed strand, consistent with their modest reduction in CSR efﬁciency (101, 190). A likely explanation for why both the top and bottom strands are equally targeted during SHM of Ig V regions comes from the recent demonstration by Ronai et al. (194) that in human B cell lines undergoing SHM both the top and bottom VH region strands have single-strand patches averaging ∼11 nucleotides in length. These single-strand patches were detected by treating ﬁxed, permeabilized nuclei with sodium bisulﬁte, which deaminates dC bases within ssDNA. This group also showed that in these cell lines V regions are transcribed in both directions, and the length of the patches are consistent with being caused by the bubble that forms at the site of transcription. However, no one has shown that S regions are transcribed in both directions, and thus it is not known if this explains why the top and bottom S region strands are equally targeted by AID. Ronai et al. (194) found that if they ﬁrst depro-

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teinized the V region DNA they could not detect the single-strand patches, and thus far experiments to detect single-strand patches in S regions have always used deproteinized DNA. Thus, undetected single-strand patches may exist on both strands at S regions. However, the putative antisense transcripts would not be G-rich and therefore should not form R-loops. The second likely role for GL transcription is to recruit AID. AID coimmunoprecipitates with RNA polymerase II in splenic B cells undergoing CSR (195). This hypothesis is also supported by the ﬁnding that AID-induced dU lesions are found in a region beginning ∼150 bp 3 to the GL RNA initiation site and extending over several kb downstream, with more mutations near the 5 end of the S regions and fewer at the 3 ends (50). Thus, investigators have proposed that AID is recruited to RNA Pol II either at the initiation phase of transcription or when it switches to the elongation phase at ∼150 bp 3 to the initiation site (18, 19, 49). AID may leave the transcription complex stochastically as it progresses through the S region. A third possible role is that transcription can alter histone modiﬁcation of the transcribed region, which might make the DNA more accessible to AID (196–198). Transcription clearly does alter chromatin accessibility, but whether the histone modiﬁcations affect AID binding has not yet been shown.

Roles of IgH Intron Enhancer and 3 Enhancers There are two enhancer regions in the IgH locus. The μ intron enhancer is located 3 to JH 4 and just 5 to the Sμ region and is essential during B cell development for normal V(D)J recombination. Consistent with the fact that the Iμ promoter lies within the enhancer core, deletion of the enhancer core reduces CSR on that allele by about twofold (199). As neither GL Sμ transcription nor DJ transcription is eliminated, this might explain why CSR, albeit at a reduced frequency, still occurs. There

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are matrix attachment regions located 5 and 3 to the core enhancer, but this same study found no role for the matrix attachment regions in CSR. A second set of enhancers is located 3 to the Cα gene in mouse and 3 to each of the two Cα genes in human. The mouse 3 IgH enhancers are spread over 30 kb and contain four DNase hypersensitive regions, termed (from 5 to 3 ) hs3A, hs1,2, hs3B, and hs4. Although the entire 30-kb enhancer region has not been successfully deleted, various segments have been deleted by gene targeting using Cre-mediated deletion after insertion of loxP sites. Deletion of hs3A or hs1,2 has no effect on CSR (200); however, combined deletion of hs3B and hs4 greatly reduces CSR to all isotypes except IgG1 (201). Neither hs3B nor hs4 has been individually deleted. The hs3Bhs4 deletion eliminates GL transcripts (except for GLγ1 RNA). Thus far, the only known role for hs3B-hs4 during CSR is to enhance GL transcription. The levels of Ig μ mRNA are also reduced in these mice. Further evidence that the role of the 3 IgH enhancer is to stimulate GL transcription was provided by transient transfection experiments in which a DNA segment containing all four human 3 DNase-hypersensitive sites was found to stimulate human GL γ3 RNA promoter activity (202). There is some evidence from transgene experiments that the region just 5 to Sγ1 has enhancer activity for GL γ1 RNA, perhaps explaining the independence of IgG1 and GLγ1 transcripts from hs3B-hs4 (203, 204). As the hs3B-hs4 enhancers are located far downstream from the GL promoters, their ability to stimulate GL transcription likely involves formation of a loop between the 3 IgH enhancers and GL promoters (32). This might involve complex formation between transcription-activating factors bound to the enhancers and promoters, as has been demonstrated between the T cell receptor Dβ promoter and the 3 T cell receptor β enhancer (205). This complex would most likely recruit histone acetylases and other chromatin modiﬁers to increase accessibility of the promot-

ers to RNA polymerase and also position the donor and acceptor S regions near each other. Direct evidence for the existence of loops between the 3 IgH enhancers and the DNA segments containing the promoters for speciﬁc GL transcripts in splenic B cells under conditions that induce CSR to that speciﬁc isotype has recently been obtained using the chromosome conformation capture technique (206). Most interestingly, the loops depend on the hs3B-hs4 enhancer segment and are reduced in aid−/− cells. It will be very interesting and important to determine which sequences and proteins are involved.

Regulation of Isotype Specificity by S Regions Although GL transcription is clearly essential for CSR, a few reports suggest that isotype speciﬁcity is also regulated by the S region sequence. The evidence for this was obtained by using transiently transfected plasmid switch substrates (207). The acceptor S region isotype determines whether the plasmid will switch in particular B cell lines or in splenic B cells activated under conditions that induce CSR on the chromosome to speciﬁc isotypes. For example, plasmids containing an acceptor Sγ1 or Sγ3 sequence will not switch in two different B cell lines that switch on their endogenous chromosome to IgA, but not to IgG1 or IgG3. However, plasmids with acceptor Sα sequences will undergo Sμ-Sα recombination in these cell lines. Likewise, a plasmid with the Sγ1 acceptor S region will undergo Sμ-Sγ1 recombination in splenic B cells treated with LPS+IL-4, which induces IgG1 CSR on the chromosome, but not if the B cells are treated with LPS alone, which induces IgG3 but not IgG1 CSR. Likewise, the plasmid with an Sγ3 acceptor S region will undergo CSR in B cells induced with LPS, but not if the cells are treated with LPS+IL4. Kenter et al. (208) also showed that mutations of 3 bp within a repeat unit of the Sγ1 sequence to the sequence found in Sγ3 allowed the plasmid to switch in the absence www.annualreviews.org • Class Switch Recombination

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of IL-4, further supporting the hypothesis that isotype speciﬁcity is also controlled by switch sequences. The element that was mutated is part of a NF-κB binding motif, which therefore might be involved in determining the isotype speciﬁcity. It was not determined whether the mutations affected transcription across the plasmid S regions, although there are no Ig isotype–speciﬁc promoters in these plasmids, so this is an unlikely explanation. Another example of isotype speciﬁc regulation by a S region comes from the ﬁnding that plasmid Sμ-Sα recombination in cell lines and splenic B cells can be stimulated threefold and tenfold, respectively, by the histone methyltransferase Suv39h1, which trimethylates histone H3 on lysine 9. Suv39h1 does not stimulate switch recombination in plasmids with any other acceptor S region (209). Mice deﬁcient in Suv39h1 show a 50% reduction in chromosomal CSR to IgA, but to no other isotype. This reduction is not accompanied by a reduction in GL α transcripts, suggesting that the Sα sequence itself is responding to Suv39h1. The Suv39h1 target in these switching cells is unknown. Furthermore, the K9-trimethyl mark is repressive and is usually found on heterochromatin associated with centromeres, making the stimulatory role of Suv39h1 on IgA switching even more puzzling. Taken together, these two sets of results suggest that isotype speciﬁcity is not regulated solely by GL transcription but is also regulated by S region sequences by an unknown mechanism.

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ISOTYPE SWITCHING OCCURS PRIOR TO GERMINAL CENTER (GC) FORMATION, AND PERHAPS ALSO IN GCs The GC provides a unique environment that allows rapid proliferation of cells despite sustaining DNA damage initiated by AID activity. Bcl-6 is upregulated in GC cells and is required for GC formation (210, 211). Ranuncolo et al. (212) suggest that GC centroblasts are uniquely able to withstand the DNA 278

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damage caused by AID because of the repression of the ATR damage-sensing pathway by Bcl-6. Protection from cell death in GC cells had previously been thought to be due to the downregulation of p53 by Bcl-6, as demonstrated in Ramos cells (213), but Ranuncolo et al. (212) found that primary human centroblasts express p53 and that its expression is not affected by downregulation or inhibition of Bcl-6. Although both isotype-switched cells and high levels of AID are found in GCs, class switching clearly can occur very early after antigen exposure, prior to GC formation. By adoptive transfer of B cell receptor Tg B cells and carrier-speciﬁc Tg CD4 T cells into normal recipients, Pape et al. (214) were able to visualize very early stages in the antibody response, tracking the B cells with anti-idiotype antibody. Tg+ isotype-switched B cells (IgG2a+ ) appeared in splenic B cell follicles as early as two days after immunization with cognate antigen and peaked on days 3 and 4, prior to formation of GCs. The B cells had divided at least three times and appeared to have migrated away from the Tg T cells. Their appearance was antigenand T cell–dependent. By day 4, progeny of the Tg+ IgG2a+ follicular B cells could be found in the outer edges of the periarteriolar lymphoid sheath near the red pulp, in the marginal zone, and in pre-GCs. By day 10, GCs contained PNA+ IgG2a+ Tg B cells that showed evidence of many cell divisions. IgM+ Tg B cells were abundant in follicles and the marginal zone at this time but were not in GCs. As only IgG2a+ Tg B cells were found in GCs, this suggests that CSR does not occur in GCs in this model, but instead occurs prior to GC formation. In another model, normal mice infected with attenuated Salmonella typhimurium also showed very rapid (day 4) Tdependent IgG2a isotype switching, occurring much before GC formation (215). Also, AID is expressed in large proliferating B cells in the extrafollicular areas of human tonsil and lymph node (216). However, Kolar et al. (217) have recently suggested that a population that

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expresses IgD+ CD38− CD23− FSChi CD71+ in human tonsil may be the initial GC cell to express AID. V region mutation frequency places these cells between naive and GC cells. This ﬁnding is consistent with previous assumptions that CSR also occurs in GCs. CSR can also occur independently of T cell help. B cell activating factor (BAFF) can synergize with IL-4 to induce AID expression in CD40−/− B cells in culture (218). Near normal levels of gut IgA were detected in CD40−/− mice despite a lack of GCs (219). The site where this T-independent IgA switching occurred was not identiﬁed in this study, although the authors excluded the gutassociated lymphoid tissue (GALT), the lamina propria, and the peritoneal cavity. Mucosal epithelial cells lining the crypts of human tonsils can support class switching through the production of BAFF and IL-10 upon TLR stimulation. These epithelial cells also secrete thymic stromal lymphopoietin (TSLP), which stimulates production of BAFF by dendritic cells (220, 221). Furthermore, CSR is detectable in pre-B and immature B cells isolated from bone marrow, as assayed by AID expression, detection of transcripts from excised DNA circles owing to S-S recombination, and detection of postswitch (Iμ-Cx) transcripts (222, 223). AID expression, circle transcripts, and postswitch transcripts were also observed in developing B cells from nude mice, further indicating T cell independence. The expression of AID in both wild-type and nude mice depends on B cell receptor signaling

through Bruton’s agammaglobulinemia tyrosine kinase (BTK) in immature B cells and on TLR signaling in both pre-B and immature B cells (222).

REMAINING QUESTIONS Numerous interesting questions remain in the ﬁeld of CSR. For example, how does AID target the V and S regions speciﬁcally? CSR and SHM do not appear to occur simultaneously in a cell. Why not? What determines whether a cell will undergo CSR or instead undergo SHM? What proteins does AID interact with? Most interestingly, what is the role of the C terminus of AID? Why is UNG required for CSR, i.e., why do other uracil DNA glycosylases not substitute? Why is APE2 used to create SSBs in S regions, in addition to APE1? Is another AP endonuclease also involved? How is synapsis of two distal S regions achieved? What is the contribution of S region sequence to isotype speciﬁcity? What is the role of 53BP1? Does 53BP1 bind specifically to the IgH locus? If so, does it require a speciﬁc histone modiﬁcation, and is this modiﬁcation speciﬁc to the IgH locus? How is this regulated? Does the binding require GL transcripts? Why is Ku70-Ku80 essential for CSR but XRCC4-ligase IV is not? Is the ability of Ku to bind nuclear matrix important for CSR? Many very interesting questions are approachable with current techniques and will be addressed in the near future, thus promising much excitement for this ﬁeld in the coming years.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We thank Drs. Katheryn Meek for helpful information on DNA-PK and Amy Kenter for her manuscript in press. We acknowledge support by NIH to J.S. (RO1AI23283, RO1 AI63026, R21AI62738) and to C.E.S. (RO1 AI65639) and a postdoctoral fellowship from the Cancer Research Institute to J.E.J.G. www.annualreviews.org • Class Switch Recombination

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184. Kaminski DA, Stavnezer J. 2007. Antibody class switching differs among SJL, C57BL/6 and 129 mice. Int. Immunol. 19:545–56 185. Ma Y, Pannicke U, Schwarz K, Lieber MR. 2002. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108:781–94 186. Jung S, Rajewsky K, Radbruch A. 1993. Shutdown of class switch recombination by deletion of a switch region control element. Science 259:984–87 187. Bottaro A, Lansford R, Xu L, Zhang J, Rothman P, Alt F. 1994. I region transcription (per se) promotes basal IgE class switch recombination but additional factors regulate the efﬁciency of the process. EMBO J. 13:665–74 188. Lorenz M, Jung S, Radbruch A. 1995. Switch transcripts in immunoglobulin class switching. Science 267:1825–28 189. Hein K, Lorenz MG, Siebenkotten G, Petry K, Christine R, Radbruch A. 1998. Processing of switch transcripts is required for targeting of antibody class switch recombination. J. Exp. Med. 188:2369–74 190. Huang FT, Yu K, Balter BB, Selsing E, Oruc Z, et al. 2007. Sequence-dependence of chromosomal R-loops at the immunoglobulin heavy chain Sμ class switch region. Mol. Cell. Biol. 27:5921–32 191. Yu K, Chedin F, Hsieh CL, Wilson TE, Lieber MR. 2003. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4:442–51 192. Zarrin AA, Alt FW, Chaudhuri J, Stokes N, Kaushal D, et al. 2004. An evolutionarily conserved target motif for immunoglobulin class-switch recombination. Nat. Immunol. 5:1275–81 193. Zarrin AA, Tian M, Wang J, Borjeson T, Alt FW. 2005. Inﬂuence of switch region length on immunoglobulin class switch recombination. Proc. Natl. Acad. Sci. USA 102:2466–70 194. Ronai D, Iglesias-Ussel MD, Fan M, Li Z, Martin A, Scharff MD. 2007. Detection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutation. J. Exp. Med. 204:181–90 195. Nambu Y, Sugai M, Gonda H, Lee CG, Katakai T, et al. 2003. Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 302:2137– 40 196. Workman JL. 2006. Nucleosome displacement in transcription. Genes Dev. 20:2009–17 197. Woo CJ, Martin A, Scharff MD. 2003. Induction of somatic hypermutation is associated with modiﬁcations in immunoglobulin variable region chromatin. Immunity 19:479–89 198. Wang L, Whang N, Wuerffel R, Kenter AL. 2006. AID-dependent histone acetylation is detected in immunoglobulin S regions. J. Exp. Med. 203:215–26 199. Sakai E, Bottaro A, Alt FW. 1999. The Ig heavy chain intronic enhancer core region is necessary and sufﬁcient to promote efﬁcient class switch recombination. Int. Immunol. 11:1709–13 200. Manis JP, van der Stoep N, Tian M, Ferrini R, Davidson L, et al. 1998. Class switching in B cells lacking 3 immunoglobulin heavy chain enhancers. J. Exp. Med. 188:1421– 31 201. Pinaud E, Khamlichi AA, Le Morvan C, Drouet M, Nalesso V, et al. 2001. Localization of the 3 IgH locus elements that effect long-distance regulation of class switch recombination. Immunity 15:187–99 202. Pan Q, Petit-Frere C, Stavnezer J, Hammarstrom L. 2000. Regulation of the promoter for human immunoglobulin γ3 germ-line transcription and its interaction with the 3 α enhancer. Eur. J. Immunol. 30:1019–29

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203. Cunningham K, Ackerly H, Alt F, Dunnick W. 1998. Potential regulatory elements for germline transcription in or near murine Sγ1. Int. Immunol. 10:527–36 204. Adams K, Ackerly H, Cunningham K, Dunnick W. 2000. A DNase I hypersensitive site near the murine γ1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation. Int. Immunol. 12:1705–13 205. Oestreich KJ, Cobb RM, Pierce S, Chen J, Ferrier P, Oltz EM. 2006. Regulation of TCRβ gene assembly by a promoter/enhancer holocomplex. Immunity 24:381–91 206. Wuerffel R, Wang L, Grigera F, Manis J, Selsing E, et al. 2007. S/S synapsis during class switch recombination is promoted by distantly located transcriptional elements and activation-induced deaminase. Immunity. 27:711–22 207. Shanmugam A, Shi M-J, Yauch L, Stavnezer J, Kenter AL. 2000. Evidence for class speciﬁc factors in immunoglobulin isotype switching. J. Exp. Med. 191:1365–80 208. Kenter AL, Wuerffel R, Dominguez C, Shanmugam A, Zhang H. 2004. Mapping of a functional recombination motif that deﬁnes isotype speciﬁcity for μ->γ3 switch recombination implicates NF-κB p50 as the isotype-speciﬁc switching factor. J. Exp. Med. 199:617–27 209. Bradley SP, Kaminski DA, Peters AH, Jenuwein T, Stavnezer J. 2006. The histone methyltransferase Suv39h1 increases class switch recombination speciﬁcally to IgA. J. Immunol. 177:1179–88 210. Dent AL, Shaffer AL, Yu X, Allman D, Staudt LM. 1997. Control of inﬂammation, cytokine expression, and germinal center formation by BCL-6. Science 276:589–92 211. Ye BH, Cattoretti G, Shen Q, Zhang J, Hawe N, et al. 1997. The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inﬂammation. Nat. Genet. 16:161–70 212. Ranuncolo SM, Polo JM, Dierov J, Singer M, Kuo T, et al. 2007. Bcl-6 mediates the germinal center B cell phenotype and lymphomagenesis through transcriptional repression of the DNA-damage sensor ATR. Nat. Immunol. 8:705–14 213. Phan RT, Dalla-Favera R. 2004. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature 432:635–39 214. Pape KA, Kouskoff V, Nemazee D, Tang HL, Cyster JG, et al. 2003. Visualization of the genesis and fate of isotype-switched B cells during a primary immune response. J. Exp. Med. 197:1677–87 215. Cunningham AF, Gaspal F, Serre K, Mohr E, Henderson IR, et al. 2007. Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection. J. Immunol. 178:6200–7 216. Cattoretti G, Buttner M, Shaknovich R, Kremmer E, Alobeid B, Niedobitek G. 2006. Nuclear and cytoplasmic AID in extrafollicular and germinal center B cells. Blood 107:3967– 75 217. Kolar GR, Mehta D, Pelayo R, Capra JD. 2007. A novel human B cell subpopulation representing the initial germinal center population to express AID. Blood 109:2545–52 218. Castigli E, Wilson SA, Scott S, Dedeoglu F, Xu S, et al. 2005. TACI and BAFF-R mediate isotype switching in B cells. J. Exp. Med. 201:35–39 219. Bergqvist P, Gardby E, Stensson A, Bemark M, Lycke NY. 2006. Gut IgA class switch recombination in the absence of CD40 does not occur in the lamina propria and is independent of germinal centers. J. Immunol. 177:7772–83 220. Xu W, He B, Chiu A, Chadburn A, Shan M, et al. 2007. Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI. Nat. Immunol. 8:294–303 www.annualreviews.org • Class Switch Recombination

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221. He B, Xu W, Santini PA, Polydorides AD, Chiu A, et al. 2007. Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 26:812–26 222. Han JH, Akira S, Calame K, Beutler B, Selsing E, Imanishi-Kari T. 2007. Class switch recombination and somatic hypermutation in early mouse B cells are mediated by B cell and Toll-like receptors. Immunity 27:64–75 223. Ueda Y, Liao D, Yang K, Patel A, Kelsoe G. 2007. T-independent activation-induced cytidine deaminase expression, class-switch recombination, and antibody production by immature/transitional 1 B cells. J. Immunol. 178:3593–601

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:261-292. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph,1 Jordi Ochando,2 and Santiago Partida-S´anchez3 1

Department of Gene and Cell Medicine, Immunology Institute, Mount Sinai School of Medicine, New York, New York 10029; email: [email protected]

2

Unidad de Immunolog´ıa de Trasplantes, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, 28220 Madrid, Spain

3

Department of Pediatrics, The Research Institute at Nationwide Children’s Hospital and Ohio State University College of Medicine, Columbus, Ohio 43205

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Key Words

First published online as a Review in Advance on November 28, 2007

chemotaxis, lymphatic vessel, postcapillary venule, lymph node

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090254 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0293$20.00

Abstract The ability of dendritic cells (DCs) to initiate and orchestrate immune responses is a consequence of their localization within tissues and their specialized capacity for mobilization. The migration of a given DC subset is typiﬁed by a restricted capacity for recirculation, contrasting markedly with T cells. Routes of DC migration into lymph nodes differ notably for distinct DC subsets. Here, we compare the distinct migratory patterns of plasmacytoid DCs (pDCs), CD8α+ DCs, Langerhans cells, and conventional myeloid DCs and discuss how the highly regulated patterns of DC migration in vivo may affect their roles in immunity. Finally, to gain a more molecular appreciation of the specialized migratory properties of DCs, we review the signaling cascades that govern the process of DC migration.

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INTRODUCTION

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Dendritic cells (DCs) are highly efﬁcient antigen-presenting cells that are central to the induction and regulation of most adaptive immune responses. Their specialized capacities for acquiring, processing, retaining, and ﬁnally presenting peptides on major histocompatibility complex (MHC) molecules are critical properties that account in part for their superior role in antigen presentation (1). Unlike other antigen-presenting cells, DCs are specialized for homing efﬁciently to the T cell zones of lymphoid organs for optimal interactions with T lymphocytes. Their migratory capacity distinguishes them from macrophages. For example, alveolar macrophages outnumber DCs by 100-fold in the airways, yet DCs that migrate from the airways to lung-draining lymph nodes vastly outnumber macrophages that migrate through lymph (2). The distinct migratory properties that lead to DCs being present where macrophages clearly are not found underlie a critical difference in the distinct immune-priming capacity of these two cell types. For example, even in instances when macrophages and DCs bear viral antigens for presentation, productive presentation to CD8+ T cells is limited to DCs, seemingly in part because macrophages are not appropriately localized (3). Many DCs in lymph nodes mobilize, with antigen in tow, from peripheral tissues that drain to downstream lymph nodes via a network of lymphatic vessels (4), though some antigens may travel to the lymph node freely through lymph and then gain access to DCs therein. Molecules smaller than ≈70 kDa that travel within lymph are ﬁltered from the subcapsular sinus into conduits that limit, but do not prevent, access of lymph node DCs to these antigens (5–7). Thus, some DCs may pick up antigens directly in lymph nodes. Regardless of whether DCs pick up antigen before or after entering lymphoid organs, the pathways and mechanisms that govern how DCs migrate to lymphoid and nonlym-

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phoid tissues ﬁgure importantly in immune responses. Thus, we discuss herein how different subpopulations of DCs get to where they are going and how the migratory routes that they take affect their role in immune responses. In many instances, molecular details behind the routes of trafﬁcking we discuss are unknown. After discussing DC migratory behavior in the context of DC subtypes and the anatomy of trafﬁcking at the level of the whole organism, we take a step back and view the DC more generically, but with more molecular detail: We consider the signaling cascades that occur during DC mobilization, as these signals unite many DC subtypes around the potent capacity for migration in general.

DENDRITIC CELL MIGRATION AND LYMPHOID ORGANS: TRAFFICKING FROM SKIN TO LYMPH NODES AS A PARADIGM Given that the genesis of immune responses occurs largely in secondary lymphoid organs, the spleen and lymph nodes, the ﬁrst consideration with regard to DC trafﬁcking is to ask how DCs seed these organs. The spleen is one of the most widely studied sources of DCs in mice, but the mechanisms by which DC precursors access the spleen have not been explored in detail. The anatomic properties of the spleen preclude a role for lymphatics as a means of DC delivery (8). Instead, DC precursors, continuously replenished from blood precursors (9), necessarily arrive to the spleen by a hematogenous route and likely initially gain access to the marginal zone as they develop into “conventional” populations of immature DCs: CD8α− and CD8α+ DC subpopulations (10). DCs do not arise from monocytes in resting spleen, but instead derive from other blood precursors that are difﬁcult to detect in the circulation owing to low frequency. Consequently, entry of DC precursors into spleen has not been characterized in detail, but there is evidence that they largely reside as immature DCs in the marginal zone.

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During maturation/terminal activation, DCs mobilize from the marginal zone to the T cell zone of the splenic white pulp, as they upregulate CCR7 and become responsive to CCR7 ligands CCL19 and CCL21 therein (11–13). In the absence of these signals, DCs are found scattered in the spleen in regions outside of the white pulp or in the marginal zone (12, 13).

Dendritic Cells in Lymph Nodes: Arrival by Afferent Lymphatics As in the spleen, the subsets of DCs in lymph nodes also include CD8α− and CD8α+ DCs. CD8α− DCs in lymph nodes are not absolute counterparts to splenic CD8α− DCs, in part because lymph node CD8α− DCs include DCs that travel through afferent lymph from upstream peripheral organs. CD8α− DCs in spleen and lymph nodes have different rates of turnover (14). At least some CD8α− DCs within peripheral organs can arise from monocytes (15), whereas splenic DCs cannot (15, 16); thus, some DCs in lymph nodes, but not spleen, are expected to be monocyte derived. The classical view regarding how lymph nodes are populated with DCs suggests that they emigrate there from tissues through afferent lymph constitutively (4). Lymph output of DCs in the steady state is substantial and is further augmented during inﬂammatory conditions (17, 18). The potent effects of inﬂammation in inducing DC migration are related to the fact that inﬂammatory cues will trigger maturation of DCs and concomitant upregulation of CCR7, the chemokine receptor centrally required for DC trafﬁcking to lymph nodes (19, 20). The functional advantage of trafﬁcking through afferent lymph is that it permits DCs to acquire and process antigen within peripheral organs; some antigen may be cell associated and unable to travel freely through lymph to reach the T cell zone of lymph nodes for optimal interactions with T cells.

Lymph Node–Homing Langerhans Cells: Seemingly Slower and Fewer than Expected Langerhans cells, the DCs present within epidermis, have for many years served as a paradigm for the general study of DC biology (21). Yet, as we have learned over the past few years, many aspects of their life cycle do not comply with the paradigmatic view previously developed. First, they are not continuously replenished by migratory blood precursors as anticipated, but rather renew through local proliferation under homeostatic conditions (22). Furthermore, older, seemingly established models regarding the mobilization of DCs from the epidermis to lymph nodes in response to activating stimuli are not entirely accurate, as described below. A popular assay to study DC migration involves the epicutaneous application of ﬂuorescent contact sensitizers (23) that were once widely thought primarily to label Langerhans cells within the epidermis. However, two recent developments allowed the contribution of Langerhans cells to contact sensitization and the overall migratory DC population after epicutaneous ﬂuorochrome application to be directly and deﬁnitively assessed: (a) the identiﬁcation of the Langerhans cell–restricted (but not entirely speciﬁc) marker Langerin (24) and (b) the generation of transgenic mice in which the Langerin promoter drives GFP expression (25), such that Langerhans cells are largely distinguished from other sources of DCs. The astounding surprise resulting from the application of these new tools was that peak arrival of Langerhans cells in the lymph node is slow (Figure 1), reaching a maximum three to four days after application of sensitizer (14, 25) when immune responses are already well under way. The vast majority of contact antigen–bearing DCs are Langerin− , and they arrive within the lymph node abundantly during the ﬁrst two days after sensitization. The late entry of Langerhans cells to the lymph node, along with additional recent evidence that Langerhans cells may not

www.annualreviews.org • Migration of Dendritic Cell Subsets

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“dDC” Day 1 LC Day 3 pDC

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CD8+ DC

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Afferent lymphatic

Rare entry into efferent lymph

Venule (HEV)

Rare return to blood Venule in nonlymphoid organ

pDC

CD8+ DC

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Figure 1 Anatomical routes of dendritic cell (DC) trafﬁcking. Many DCs that seed peripheral organs enter lymph nodes through afferent lymph. In skin, apparent dermal DCs migrate to lymph nodes much faster than epidermal Langerhans cells after contact sensitization. These different types of DCs emigrate to distinct areas of the lymph node. Only a few DCs enter the blood either through reentry into venules found within peripheral tissues or through escape into efferent lymph. CD8α+ DCs are often referred to as lymph node–resident DCs, and they are believed to enter the lymph node through high endothelial venules (HEVs), rather than afferent lymphatics. However, their true trafﬁcking patterns remain to be determined. By contrast, plasmacytoid DCs (pDCs) are known to enter inﬂamed lymph nodes through HEVs; unlike other DCs, they appear incapable of mobilizing from peripheral organs into afferent lymph. 296

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be essential for immune priming to at least some antigens that enter skin (25–27), raises the possibility that Langerhans cells in particular fulﬁll an immunoregulatory role upon their tardy arrival to the lymph node. This revised concept raises many new questions in the ﬁeld. From the perspective of migration, it will be important to determine whether Langerhans cells are sometimes more rapidly and robustly mobilized to lymph nodes than observed thus far in response to contact sensitizers. Studies using TNF-α, rather than contact sensitizers, should be useful to address this question. Within 1 h, local, exogenous TNF-α dramatically reduces the density of Langerhans cells from the epidermis, presumably as a result of robust migration to lymph nodes (28, 29), whereas a far more modest reduction in Langerhans cell density within epidermis is seen one day after contact sensitization (30). The rapid and robust mobilization of Langerhans cells induced by exogenous TNF-α might lead to a correspondingly rapid and robust accumulation of Langerhans cells in the T cell zone of lymph nodes. If so, then it would be important to determine if Langerhans cells participate more centrally in immune priming under conditions where they are more efﬁciently mobilized, as compared with their apparently dispensable role in contact sensitivity.

Dermal Dendritic Cells Take Center Stage The failure of Langerhans cells to account for the migratory skin DCs after contact sensitization has turned renewed attention to dermal DCs (25), because the ready explanation for the origin of the Langerin− migratory DC population was that these were dermal DCs. However, it remains to be determined how many of the DCs mobilized by application of contact sensitizer are dermalresident DCs. Hypothetically, the migratory DC population could also include blood DC precursors like monocytes that enter the skin, acquire labeled antigen/sensitizer as they ma-

ture, and then rapidly mobilize to the lymph node.

Distinct Localization of Dendritic Cell Subtypes Within Lymph Nodes Upon arrival of so-called dermal DCs and Langerhans cells to the lymph node through lymphatics, the two populations assume distinctive positions within the lymph node. At all times examined, Langerin+ DCs mobilized to the deep paracortex of the T cell zone, but dermal DCs remained nearer B cell follicles (25). This distinct localization likely permits these different DC populations to carry out nonoverlapping roles in immune responses. The molecular events that account for the differing distribution of DC subtypes within the lymph nodes are unknown. One regulatory event affecting DC localization may relate to how strongly particular DCs bind to conduits in the lymph node. Compared with more mature DCs, immature DCs are anticipated to have a stronger capacity to bind to conduits (6). Thus, the phenotype and relative maturation status of a given DC type upon entry into the lymph node may dictate its positioning therein.

HEMATOGENOUS ENTRY INTO LYMPH NODES Although accumulation of DCs in lymph nodes by way of afferent lymphatics is the most well-established route of entry, the presence of DCs in lymph nodes does not depend entirely on input of DCs from afferent lymph. Some DCs or their DC precursors can enter lymph nodes through the same hematogenous route that naive lymphocytes use to access lymph nodes: passage across the specialized high endothelial venules (HEVs) within lymph nodes. DCs thought to use this route into the lymph node include CD8α+ DCs or their precursors (14), plasmacytoid DCs (pDCs) (31, 32), and CCR2hi monocytes (33) (Figure 1). For example, some monocytes that enter through the HEV during the www.annualreviews.org • Migration of Dendritic Cell Subsets

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course of Leishmania infection may develop into DCs within the lymph node, although routes of trafﬁcking for these monocytes that later became DCs were not directly explored (34).

CD8α+ DCs: From Blood to Lymph Node

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The idea that CD8α+ DCs enter lymph nodes through the blood, by crossing HEVs, rather than through afferent lymph is widely circulated. The concept bears substantial consequences on the events that initiate immune responses. CD8α+ DCs possess superior capacity for cross-presentation of antigen to CD8+ T cells, but if these DCs do not pick up antigens in peripheral organs to transport them through lymph, then their access to antigen for cross-priming may depend on antigen transfer from other DCs that do trafﬁc through lymph (35, 36). Consequently, some DCs, such as monocyte-derived DCs that mediate cross-presentation (37), may mainly function to deliver, but not present, antigen to lymph node T cells, at least in the context of certain infections or conditions. We now review the evidence that CD8α+ DCs enter lymph nodes only through a blood route, requiring passage across HEVs. Organs that feed lymph nodes through lymphatic drainage do not contain DCs with a CD8α+ phenotype. When CD8α+ DCs are adoptively transferred into skin, they do not migrate to downstream LNs via afferent lymphatics (38), in contrast to their CD8α− counterparts. Moreover, the rate of labeling with bromo-deoxyuridine is observed to be more rapid in CD8α+ DCs from skin-draining lymph nodes than the rate in dermal and epidermal DCs that accumulate in the same lymph nodes, suggesting that CD8α+ DCs probably did not arise from these skin-derived populations (14). In addition, CD8α+ DCs in lymph nodes never seem to acquire tracers like epicutaneously applied FITC that mark other DCs that originated within peripheral organs (35, 36), but there is precedence for 298

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the possibility that the tracers tested may not be accessible to all DCs within a given peripheral organ (39). Finally, researchers have argued that the population of CD8α+ DCs in the spleen must by nature of the organ come from a blood route, so this is likely true of the lymph node CD8α+ DCs as well (21). However, entry of DCs into the spleen from the blood is not regulated by passage across an HEV (8), so there is little basis for suggesting that the pathways of entry into the two lymphoid organs would be similar. Attempts to demonstrate directly that CD8α+ DCs traverse HEV to enter lymph nodes have so far failed. Speciﬁcally, Reinhardt and colleagues created knockin mice that expressed eYFP from the p40 IL-12/23 subunit locus. CD8α+ DCs robustly produce IL-12 upon activation, so eYFP+ CD8α+ DCs were readily detected in this model (40). Following lipopolysaccharide injection or Leishmania monocytogenes infection, the number of eYFP+ CD8α+ DCs in lymph nodes was greatly elevated. Migration seemed required to elevate the number of CD8α+ DCs in lymph nodes because their increase was blocked with pertussis toxin. However, the increased number of CD8α+ DCs was surprisingly unaffected by neutralizing mAb to CD62L (40), a selectin typically required for leukocyte passage across HEV. Thus, these data failed to support a model in which CD8α+ DCs enter the lymph node via the bloodstream/HEV route, at least during inﬂammation. Therefore, at present, the concept that CD8α+ DCs enter lymph nodes only through a blood route, rather than through lymph, is supported only by indirect evidence, as attempts to provide direct evidence have not succeeded. Without this pattern of trafﬁcking established, some of the strength behind the idea that CD8α+ DCs must acquire antigen through transfer from DCs that migrated through lymph dissipates because the CD8α+ DCs may be among the lymph-migrating DC population. Of course, antigen transfer can also take place between two DCs that emigrate into the lymph node

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from lymphatics. Identiﬁcation of the elusive circulating precursor for CD8α+ lymph node DCs would permit a direct analysis of the migratory routes and mechanisms by which this population of DCs seeds lymph nodes.

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Plasmacytoid Dendritic Cells: Definitive Use of a Hematogenous Route to the Lymph Node In contrast to CD8α+ DCs, pDCs have been experimentally established to have the capacity to enter lymph nodes through HEV (Figure 1). In humans, markers for pDCs include BDCA-2, BDCA-4, and IL-3 receptor (41, 42). pDCs, in contrast to myeloid DCs and monocytes in humans, lack expression of CD11c. In mice, pDCs are identiﬁed as CD11clo cells that coexpress B220, Gr-1, and 120G8 (42, 43) and selectively express siglec-H (44, 45). Functionally, in both species, they are well known as principal producers of type I interferon and hence are often alternatively called interferon-producing cells (IPCs). Soon after the discovery of this cell type, Cella and colleagues reported that IPCs accumulated in human inﬂamed lymph nodes just beneath HEVs, suggesting that they may enter lymph nodes through this route (31). Formal demonstration of this trafﬁcking pattern was carried out in mouse models by intravital microscopy (32). Noninﬂamed lymph nodes support adhesion but not transmigration of pDCs, whereas inﬂamed lymph nodes permit robust emigration of pDCs across the HEV in a manner that utilizes L-selectin (CD62L) and E-selectin (CD62E) during attachment and rolling, and involves β1 and β2 integrins for ﬁrm attachment to the endothelium (32). Accordingly, pDCs are reduced in lymph nodes of CD62L-deﬁcient mice (46). Contrary to expectations that CXCR3 would be a requisite chemokine receptor for pDC migration across HEVs (31), CCR5 but not CXCR3 was found to have a critical role (32).

In humans, chemokine-like receptor 1 (CMKLR1), also called ChemR23, is expressed by pDCs and appears to mediate their recruitment across HEVs (47, 48), which facilitate recruitment by displaying chemerin, the ligand for this receptor. In mice, CMKLR1 is not expressed by pDCs and may instead participate in macrophage recruitment (48).

MYELOID AND PLASMACYTOID DENDRITIC CELL RECRUITMENT AND INTERPLAY IN PERIPHERAL ORGANS Myeloid Blood Dendritic Cells and Monocytes As with lymphoid organs, the seeding of peripheral organs with DCs is a topic of immunological relevance because DCs in peripheral organs are the ﬁrst to interact with most pathogens. However, it is not possible to discuss some DC precursors or subsets in depth with regard to trafﬁcking or function because so little is known about them. In particular, CD14− BDCA-1+ /CD1c+ and CD14− BDCA-3+ DCs (41), which may be very relevant human DC precursors in the blood, have no established destinations in vivo. Some studies have operated under the assumption that BDCA-1+ DCs within human tissues arise from BDCA-1+ DCs in blood, but this point has not been ﬁrmly established and seems unlikely given that BDCA1+ myeloid DCs in blood are CD14− , whereas some BDCA-1+ cells in lung are CD14+ (49). Moreover, the murine counterparts to these cells are also unknown. Indeed, among potential DC precursors, only pDCs and monocytes have been studied in sufﬁcient detail for researchers to begin to develop a picture of their full life cycle. Monocytes can serve as precursors for DCs in the periphery during various conditions of inﬂammation (10) or in the absence of inﬂammation (15). Monocytes may thus serve as www.annualreviews.org • Migration of Dendritic Cell Subsets

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precursors for many of the myeloid peripheral tissue DCs that ultimately emigrate through afferent lymph, although they play a small role in becoming splenic DCs (15). The signals and molecular events involved in recruitment of monocytes into tissues have been recently reviewed in detail (50).

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Compared with the difﬁculty of discerning whether myeloid DCs in peripheral organs are derived from monocytes or myeloid blood DCs or are otherwise macrophages, pDCs are easy to trace by staining for their particular signature markers within tissues. pDCs can apparently enter normal organs at very low levels in humans (49, 51, 52) and mice (53, 54). However, they are far more numerous in diseased or inﬂamed tissues. For example, in humans and mice, they have been described in skin inﬂammatory diseases (47, 55), in allergic diseases of the airway (52), and in mouse allografts (56). pDCs also home to the small intestine of mice in the steady state, with increased recruitment during inﬂammation, by a mechanism that relies on CCR9 (54). By contrast, CCR9 is not required for pDC entry into lymph nodes or other organs. Thus, there may be speciﬁc requirements for pDC migration into peripheral organs that are distinct from mechanisms used by pDCs to enter lymphoid organs. However, some receptors may be used by pDCs to trafﬁc to multiple tissues. For example, CKLR1/ChemR23 appears to mediate human pDC migration into lymph nodes and inﬂamed skin (47).

Exit of Plasmacytoid Dendritic Cells from Peripheral Organs The classical paradigm that activated DCs become induced to enter lymphatic vessels from tissues (Figure 1) prompts the possibility that pDCs within tissues will enter lymph nodes through not only HEVs (as discussed 300

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above), but also afferent lymphatics. Surprisingly, this predicted pattern of trafﬁcking has not been upheld experimentally. On the contrary, afferent lymph is devoid of pDCs under homeostatic and inﬂammatory conditions (57). Researchers drew this conclusion in a rat model (57) using a well-established technique to sample pseudoafferent lymph directly (17). The possibility that pDCs do not enter afferent lymph vessels has pivotal implications for the role of tissue pDCs in presenting antigen that they may acquire within tissues. Without the capacity to home to a T cell–rich environment, pDCs that localize to peripheral tissues may not be there primarily to acquire antigen for the purposes of antigen presentation. They may instead mainly support the antigen-presentation function of myeloid DCs. For instance, the activation of pDCs within the intestine in turn enhances mobilization of myeloid DCs during intestinal inﬂammation (47). On the other hand, limited lymphatic access may not affect the capacity of pDCs to bring tissue-derived antigens to lymph nodes. Following allograft transplantation, pDCs acquire MHC II antigen-containing peptide derived from the allograft, and these pDCs are found in the blood and accumulate in lymph nodes by a CD62L-dependent mechanism (56). Putting these data together with the observations described above generates an unexpected picture in which pDCs have access to lymph nodes through only a hematogenous route, in striking contrast to the trafﬁcking patterns of conventional DCs.

CAN MATURE DENDRITIC CELLS RETURN TO THE BLOOD AND ENTER OTHER ORGANS? The possibility that pDCs carry antigen from an allograft or spleen to lymph nodes via a hematogenous route raises the broader issue of whether DCs derived from a peripheral organ or lymph node can generally mobilize back into blood and ultimately home

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to another organ. Mechanisms to reenter the bloodstream would include either (a) direct migration across vascular endothelium in the ablumenal-to-lumenal direction, or (b) failure of DCs to be trapped in lymph nodes such that they emerge into efferent lymph that accesses the bloodstream through the thoracic duct. Either route of entry into the bloodstream could have a major impact on immunity and inﬂammation. For example, such trafﬁcking might facilitate the spread of infectious microbes from tissue to tissue, carried by DCs serving as Trojan horses. Indirect evidence for this possibility exists (58, 59). In addition, presentation of antigen at distal sites may become possible. A fraction of DCs is known to enter efferent lymph at a low frequency (4, 60, 61). Until recently, researchers had only indirect evidence for the possibility that immune responses are mediated by DCs that pass through the lymph node into efferent lymph or return to blood directly (4). In addition to gaining access to the bloodstream, DCs would need to express the right subset of molecules to home into other organs, but mature DCs have downregulated many chemokine receptors and adhesion molecules that are generally used for leukocyte homing into other organs. In mice whose lymphotoxin β receptor pathway has been manipulated so that the mice lack skin-draining lymph nodes, but possess mesenteric lymph nodes (62), endogenous skin DCs are not ﬁltered by skindraining lymph nodes and therefore enter the efferent lymph and blood unimpeded. After accessing blood, these DCs fail to accumulate in the spleen but instead enter in mesenteric lymph nodes (62), implying that, against expectations, homing mechanisms are in place for matured skin DCs to enter mesenteric lymph nodes. The molecular nature of this homing pathway is unknown. Other recent publications, discussed below, have elaborated on whether and with what consequences DCs leave one tissue, traverse blood, and enter another tissue to modulate immunity. Bone marrow–derived

DCs injected into the rear footpad of mice migrate exclusively to popliteal lymph nodes as expected. However, coinjecting these mice with vitamin D3 (63) appears to cause the transferred DCs to home to mesenteric lymph node and Peyer’s patches (63), allowing induction of mucosal immunity. Homing of adoptively transferred splenic DCs from the footpad to the bone marrow, where central memory T cells expand, has also been described (60). This migration occurred at a very low frequency. Nonetheless, on the basis of the frequency of labeled DCs found in the bone marrow, researchers developed the argument that the migration most likely involved direct reentry into the bloodstream by the transferred DCs rather than passage through efferent lymph (60). Both of these studies used adoptive transfer models and did not probe endogenous DCs. When large numbers of endogenous skin DCs were labeled by epicutaneous administration of a ﬂuorescent sensitizer, labeled endogenous DCs were not recovered in the bone marrow (61), suggesting that the bone marrow is an extremely minor destination for endogenous DCs, at least those from skin.

Dendritic Cell Migration to the Thymus If antigen-bearing DCs that return to blood subsequently emigrate into the thymus, they may be a source of self-antigen for the induction of central tolerance (64). This issue assumes that regulation by autoimmune regulator (AIRE) of the expression of tissuerestricted antigens by thymic epithelium (65) leaves gaps in the repertoire of self-antigens that are presented to thymocytes during thymic selection and that antigen-bearing DCs that enter from the periphery might be able to present antigen to ﬁll in those gaps (64). When millions of labeled crude splenic cells, mainly CD11c+ cells, were injected directly into the bloodstream as a model designed to mimic DC reentry into blood, bypassing the tremendous hurdle of www.annualreviews.org • Migration of Dendritic Cell Subsets

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leaving a peripheral organ, a few labeled DCs entered the thymus by a mechanism that required P-selectin and α4β1 integrin (66). Studies in parabiotic mice conﬁrmed that blood DCs or their precursors can enter the thymus (66). However, neither of these approaches addresses the most interesting and relevant question of whether DCs that might have picked up antigen in one tissue reenter the blood and home to thymus for antigen presentation. Further experiments in the same study were designed to probe whether endogenous, FITC antigen-bearing DCs can leave a tissue like skin, gain access to blood, and then home to the thymus. In these experiments, mice were treated epicutaneously with FITC. Subsequently, FITC+ CD11c+ DCs were found in the thymus, although at a frequency so low that fewer than 10 scattered events said to correspond with the labeled cells were typically detected by FACS (66). Moreover, the protocol of delivering FITC as antigen to the skin was unusual; the skin was tape-stripped prior to FITC application to remove the stratum corneum that largely absorbs most of the applied FITC (30), and a very large volume of FITC was applied. The investigators in the study also found FITC+ DCs in the spleen using this protocol, in contrast to others who have used epicutaneous FITC via a more standard protocol (61, 62). As a result, the use of FITC painting in conjunction with removal of the stratum corneum may be ﬂawed, likely because it leads to systemic spread of the FITC label that may not require any DC transport from the skin. In any case, given the probability that only a very small number of DCs mobilize from tissues to thymus (66) and that only a very small fraction of those DCs bears significant self-antigens from tissues that may not be expressed in the thymus (antigens acquired by DC engulfment of apoptotic parenchymal cells), DC trafﬁcking to the thymus from other tissues, even if it occurs, is most likely irrelevant for presentation of self-antigens and clonal deletion of lymphocytes.

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Anatomic Restrictions that May Affect Migration Routes from Tissue to Blood Versus Lymph In the context of transplantation, pDCs, conventional DCs, and cells closely resembling DCs can be found to leave tissues and reenter the bloodstream (56, 67–70). These observations may reveal a pattern of trafﬁcking that is always ongoing but difﬁcult to observe. Alternatively, the transplantation may have an impact on DC trafﬁcking. In particular, because lymphatic vessels sometimes reanastomose slowly after transplantation, migratory cells may instead mobilize via atypical or normally less-favored routes. Indeed, accumulation of DCs or their precursors in tissues that for any reason have restricted access to lymphatics may provoke nonstandard routes of emigration. For example, priming of immune responses by DCs within lymph nodes is redirected to the spleen when emigration to lymph nodes is hindered by genetic deﬁciency in CCR7 ligands that normally mediate DC mobilization to lymph nodes (71). An example of naturally restricted access to lymphatics may be found in atherosclerosis. Early in this disease, monocyte-derived cells that can develop into macrophages or DCs accumulate beneath arterial endothelium in a position lumenal to the internal elastic lamina (72). Mononuclear phagocytes with a “veiled” morphological appearance akin to DCs have been observed to return across arterial endothelium in the ablumenal-to-lumenal direction (73), allowing their escape from atherosclerotic plaques by reentry into the bloodstream. Within atherosclerotic lesions, monocyte-derived cells that differentiate into would-be lymph-homing DCs are expected to have limited capacity to mobilize toward the lymphatic vessels, which are located in the periadventitial space of the artery, because they are separated from those lymphatics by the imposing arterial media lined with smooth muscle cells and elastic laminae. As mentioned, the internal elastic lamina provides a ﬂoor for developing atherosclerotic plaques,

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and this lamina acts as a barrier to transport of large molecules. After traversing the arterial endothelium, low-density lipoproteins, for example, travel beneath and laterally along the endothelium rather than passing through the elastic lamina (74). Likewise, the elastic laminae may impede cellular passage and possibly even perceptions of chemotactic signals that would support movement toward the periadventitia. Consistent with the possibility that cellular passage into and across the media is very limited are observations that herpes virus–infected smooth muscle cells within elastic arteries are inaccessible to clearance by phagocytes and lymphocytes (75). In the absence of an open pathway to lymphatics and without a physical barrier between them and the ablumenal surface of the endothelium, “veiled” mononuclear phagocytes that may be akin to DCs from atherosclerotic plaques return to blood (73, 77) using what is likely an alternative, secondary route.

SIGNALING CASCADES THAT GOVERN MIGRATION OF DENDRITIC CELLS Migratory cells use highly conserved mechanisms to sense and respond to small changes in the concentration of chemoattractants in their external milieu (78). Over the past few years, many key intracellular pathways and signaling molecules downstream of chemokine receptors in DCs have begun to be identiﬁed, and some signal transduction models have been proposed (79). Yet a global uniﬁed model encompassing the diversity of chemokine receptors, their complex downstream signaling cascades, and the remarkable heterogeneity of their biological effects is not achievable at this time. Most of the research on signaling during DC migration has been carried out using monocyte- or bone marrow–derived DCs. These DCs differ substantially from some DC subsets described above, but they do provide a valuable tool for researchers to develop a generic model of the proximal signals that govern DC motility and migration.

Before activation, conventional myeloid DCs found in peripheral organs can express CCR1, CCR2, CCR5, CCR6, CXCR1, CXCR2 and CXCR4, with these expression patterns differing somewhat among DC subsets. Upon stimulation by pathogenderived antigens and/or endogenously generated danger-associated signaling molecules, activated DCs acquire a migratory phenotype associated with the upregulation of CCR7 and express receptors linked with DC maturation, including CD40, CD80, CD86, and MHC class II. As mentioned above, the G protein–coupled receptor CCR7 is the dominant mediator of DC mobilization to the T cell compartment of lymphoid organs (19). CCR7 expression alone, however, is not sufﬁcient to confer mobilization to the T cell zone of lymph nodes and spleen; additional DC receptors are required to positively regulate CCR7 function (79).

CCR7-Mediated Migration: A “Multimodule” Model DC signaling through CCR7 initiates and orchestrates a vast repertoire of molecular events that control chemotaxis and several other biological functions in these cells (reviewed in Reference 79). The CCR7 polyfunctional signal transduction apparatus exempliﬁes the complexity of the molecular events initiated upon ligation of most chemokine receptors. Various nonoverlapping signaling modules, rather than a single pathway, appear to regulate distinct DC migratory responses, such as chemotaxis or migratory speed, induced by the chemokine ligands for CCR7, CCL19, and CCL21. Similar to all G protein–coupled receptors, CCR7 interacts with G proteins to transmit intracellular signals. A classical G protein– dependent mechanism involves the use of the alpha (α) subunit and the release of the beta and gamma dimer (βγ) of the heterotrimeric G protein (80, 81) (Figure 2). Four families of α subunits are known: Gαs , Gαq , Gαi , and Gα12 . Each family is composed of several www.annualreviews.org • Migration of Dendritic Cell Subsets

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Figure 2 Various signaling pathway modules regulate distinct DC migratory responses induced by chemokines through CCR7. Upon agonist binding, trimeric G proteins are uncoupled, and a series of signal transduction events ensue that result in cell activation followed by enhanced motility. At least two nonoverlapping independent signaling modules are activated upon CCR7 ligation. One module is initiated by Gα12 activation, resulting in the release of the free βγ subunit, which in turn activates downstream effectors including Rho GTPases, the proline-rich tyrosine kinase 2 (Pyk2) and the actin-binding protein coﬁlin. This module may regulate the basal migratory speed of DCs, but not DC chemotaxis. Conversely, another signaling module engages upon Gαi stimulation, leading to mitogen-activated protein kinase (MAPK) activation, including p38, ERK1/2, and JNK. In addition, free βγ activates phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), and Akt-NF-κB pathways, which are important for activation and survival of the cells. Moreover, phospholipase Cβ2 (PLCβ2) breaks down plasma membrane lipids into diacylglycerol (DAG) and the calcium second messenger, inositol trisphosphate (IP3), which in turn induces the intracellular release of calcium ions (Ca2+ ) from IP3-gated stores on the endoplasmic reticulum (ER). This second module may regulate chemotaxis in most cellular systems. A third module appears to be required for CCR7-induced DC chemotaxis, and it would be sparked by sequential Gαi and Gαq activation. The free Gαq or βγ subunit would then activate PLCβ1,3 favoring the generation of IP3 and consequently intracellular Ca2+ release. The ectoenzyme CD38 may function as a coadjuvant of this module by converting NAD+ to cyclic ADP-ribose (cADPR), which may act at ryanodine receptors to expand release of Ca2+ from intracellular stores. This capacitates a calcium channel on the cell membrane, resulting in a sustained inﬂux of extracellular Ca2+ required for DC migration.

members, thereby contributing to diversity in chemokine receptor–mediated signaling (82). Chemotaxis is thought to be regulated primarily through the Gαi subfamily, which contains three subunits, Gαi2 , β, and γ. The activation of Gαi , induced by the binding of GTP 304

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to Gαi2 and the release of free βγ, initiates the chemokine receptor signaling cascade (78) (Figure 2). The free βγ subunit subsequently activates downstream effectors such as PI3K which regulate the Akt pathway that plays a pivotal

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role in regulating chemotaxis in leukocytes, including DCs (83, 84). However, alternative pathways may also regulate chemotaxis in a PI3K/Akt-independent manner (85–88). Overall, it seems that PI3K/Akt and the transcription factor nuclear factor κB (NFκB) regulate CCR7-dependent DC survival, whereas PI3K/Akt-independent signaling pathways may be preferred, but not required, during chemokine receptor engagement in DCs (87, 88). In this model, the Gαi -proteinderived βγ subunit preferentially triggers signal transduction via mitogen-activated protein kinase (MAPK) family members p38, ERK1/2, and JNK (84, 87, 89), whereas Cdc42 and Rac, but not RhoA GTPases, coordinate actin polarization, cytoskeletal rearrangement, cell polarity, and ultimately motility (84, 87, 89). It is difﬁcult to dissect the true importance of each kinase toward a speciﬁc biological endpoint because MAPKs play important roles in many DC functions, including maturation, migration, cell proliferation, cytokine production, and survival. The literature points to a critical role for MAPKs during the upstream events that lead to DC migration (90–93). Activity of the p38 MAPK regulates IL-12p40 production in macrophages and DCs (94, 95). Homodimers of IL-12p40 facilitate DC migration from the lung to the draining lymph nodes after Mycobacterium tuberculosis infection (96), and IL-12p40 also supports migration of adoptively transferred DCs to draining lymph nodes (40). Phosphorylation of p38 MAPK is required for DC maturation and the synthesis of cytokines (97–100), but DCs are at least partially competent to chemotax when p38 is inhibited (84, 88), suggesting that p38 MAPK in particular may support DC migration through production of cytokines that in turn participate in migration. When p38 MAPK is blocked, another MAPK may take over. Riol-Blanco and colleagues (87) recently proposed an integrated signaling module to regulate chemotaxis in CCR7-stimulated DCs. This pathway is ignited by Gαi -mediated activation of either

p38 or ERK1/2, and this activation is thought to be upstream of JNK. Interestingly, inhibition of all these kinases does not completely blunt CCR7-dependent chemotaxis (87, 88), suggesting that additional unidentiﬁed molecules or signaling pathways may also regulate chemotaxis of DCs. Although the pertussis toxin–sensitive Gαi subunit is known to be the nascent component regulating various independent signaling/functional arms during DC chemotaxis (101, 102), chemokine receptor coupling to other Gα subunit families has not been thoroughly examined. By using Gα subunit– deﬁcient mice, we recently identiﬁed a novel alternative chemokine receptor signaling module that requires Gαi as well as Gαq for the in vitro chemotaxis of bone marrow– derived DCs to the ligands of CXCR4 and CCR7 (103). Gαq -coupled pathways directly activate PLCβ1 and PLCβ3, resulting in the generation of diacylglycerol and the subsequent activation of protein kinase C. This in turn leads to PI3K-dependent inositol trisphosphate production and calcium mobilization (81, 104). Immature or mature DCs from Gαq −/− mice have signiﬁcantly reduced calcium ﬂuxes in response to chemokine receptor stimulation and are unable to migrate to inﬂammatory sites or lymph nodes in vivo (103), indicating that signaling modules dependent on Gαi and Gαq are critical for DC migration. Strikingly, T cells, which also use CCR7 to migrate to CCR7 ligands, did not require Gαq , indicating unexpectedly that distinct signaling pathways are used by DCs and T cells to respond to the same chemokines (103). In addition to the Gαq pathway, another Gαi -independent signaling module via Rho/Pyk2/coﬁlin has been reported (87). Like the Gαq pathway, this signaling module is engaged by a pertussis toxin–insensitive class of G proteins, presumably G12 /G13 . However, unlike the Gαq pathway, this module was dispensable for chemotaxis and instead was found to control DC migratory speed (79, 87, 105) (Figure 2). Further understanding of www.annualreviews.org • Migration of Dendritic Cell Subsets

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the different signaling networks downstream of chemokine receptors and identiﬁcation of the individual signaling components that regulate each pathway are needed. This knowledge may lead to improved DC vaccination protocols or application in other therapeutic settings where modulation of DC migration may be desirable.

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Signaling Through Cell Surface Enzymes and Nonchemokine Dendritic Cell Receptors A myriad of maturation and nonchemokine chemotactic factors, which can be rapidly produced within inﬂamed or damaged tissue, plays a central role in stimulating recruitment of DCs and preparing them for subsequent mobilization to lymph nodes (106–109). Immature DCs express several chemotactic receptors for nonchemokine agonists (110, 111). Several of these receptors promote DC activation and are upregulated by endogenous maturation signals such as IL-1, TNF-α, and CD40L or exogenous microbial products that activate Toll-like receptors (TLRs) (112– 114). Most of the aforementioned stimuli trigger downstream signaling pathways that activate NF-κB and induce proinﬂammatory gene expression (115, 116). Inﬂammatory and cellular stress–linked signals expand the array of signaling mechanisms that can directly affect DC migration. For example, the activating surface receptor TREM-2, which induces DC maturation by a mechanism independent of NF-κB, critically regulates CCR7-dependent DC chemotaxis (117, 118). TREM-2 associates with DAP12, an ITAM-containing adaptor molecule that also regulates DC migration (119, 120). Intriguingly, TREM-2-mediated upregulation of CCR7 does not correlate with a greater capacity of DCs for chemotaxis, consistent with the notion that CCR7 responses require additional extracellular signals (118). It is not yet understood how the TREM/DAP12 pathway may converge with other intracellular signals downstream of CCR7. Knockdown of 306

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the transmembrane adapter protein NTAL, a novel component of the TREM-1/DAP12 pathway, yields signiﬁcantly reduced calcium inﬂux (121), supporting the idea that NTAL may mediate calcium mobilization that is required for DC chemotaxis to a variety of chemokines and nonchemokine stimuli (122– 125). Chemoattractant-triggered DC chemotaxis is thought to be controlled, at least in part, by cADPR and ADPR calciummobilizing metabolites (126, 127) (Figure 2), both catalytic products of the nucleotidemetabolizing ectoenzyme CD38 and the NAD+ substrate. CD38-deﬁcient neutrophils, monocytes, and DCs show impaired calcium and chemotactic responses to a variety of chemokines in vitro and in vivo (128, 129). CD38-derived metabolites have a broad impact on chemokine receptor–mediated signaling in myeloid cells, presumably by inducing calcium ﬂuxes conducted through the cADPR- and ADPR-activated plasma membrane channel TRPM2 (130), a member of the transient receptor potential TRP channel superfamily (131, 132). TRPM2 is a nonselective cation channel widely expressed by leukocytes, including DCs (130, 133, 134). Other DC receptors that induce calcium mobilization may also rely on a CD38-generated calcium second messenger to transmit signals that promote migration. Inﬂammatory signals and bacterial products that induce DC maturation and CCR7 expression often induce upregulation of CD38 (128, 135–137). By contrast, activation of monocytes through TLR2, 4, and 5 induces CD38 downregulation (135), which is possibly linked to the observation that monocyte-derived cells can become impaired in their migration to lymph nodes by bacterial signals (138). TLR signaling triggers a cascade of events in DCs that includes modiﬁed chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein–coupled receptors (112, 139–141). These changes may often

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synergistically support migration of DCs, but crosstalk between TLR-mediated signaling pathways in DCs may sometimes result in antagonistic effects. For example, engagement of TLR3 or TLR4 on monocyte-derived DCs markedly increases expression of regulator of G protein signaling 1 (RGS1) protein (101), which has been well documented as accelerating the intrinsic GTPase activity linked with Gαi subunits. Thus, increased levels of RSG1 inhibit DC migration to lymph nodes (101). How pathogen-derived signals, including but not limited to signals through TLRs, regulate DC migration needs more study. Clearly, some pathogens use mechanisms to arrest DC migration to protect themselves from the generation of potent adaptive immune responses. Schistosomal parasites produce prostaglandin D2 to inhibit DC migration to lymph nodes after an experimental infection through skin (142), and microbial antigens derived from Borrelia garinii, the causative agent of chronic Lyme disease, signiﬁcantly downregulate CD38 and CCR7 expression in DCs, thereby hampering their migratory capacity (143).

Signals that Modulate Migration as Part of a Tissue Damage Response Under conditions of cell stress, inﬂammation, or tissue insult, ordinary metabolites that are otherwise maintained intracellularly may be released to the extracellular milieu. Such molecules may then target receptors present on the surface of DCs to modulate their maturation and/or migration. One cytokine-like molecule released in necrotic tissues or by some cells in response to TLR signals is high mobility group box 1 (HMGB1), which may regulate DC migration (144). Another class of metabolites released in injured tissues includes purine nucleotides, such as NAD, ATP, ADP, ADPR, and AMP (Figure 2). DCs express a variety of purine sensors on their surface; some, like CD38, work as ectoenzymes metabolizing purine nucleotides, whereas others are a class of

plasma membrane receptors ligated by extracellular nucleotides (145, 146). Two main types of purinoreceptors, P2YR and P2XR, have been identiﬁed. P2YRs are a family of G protein–coupled receptors that are abundantly expressed on DCs. Upon activation, they induce generation of inositol trisphosphate, release of calcium, and stimulation of adenylate cyclase via Gαi or Gαq proteins. Selective P2YR agonists (e.g., ATP, ADP, and UTP) are potent chemotactic stimuli for immature, but not mature, DCs (125, 147). P2YR-dependent, downstream signals mobilize calcium and couple to pertussis toxin–sensitive G proteins (125). Thus, purinoreceptors and NAD+ -ases such as CD38 may function in a concerted manner during inﬂammation or tissue damage to regulate DC recruitment and emigration. Given both that these signals may couple with CCR7 to optimize migration of DCs and that regulating the availability of extracellular nucleotides may be feasible pharmacologically, these pathways merit further attention in future research.

SUMMARY AND FUTURE DIRECTIONS As discussed herein, it is clear that trafﬁcking patterns of DCs are highly regulated and differ greatly among different types of DCs (Figure 1). The variation in trafﬁcking patterns sets the stage for the many roles different DC subsets play in immunity. For example, the slow arrival and distinct localization of Langerhans cells to lymph nodes in response to contact sensitizers naturally limits their importance in inducing immunity to sensitizers and suggests that they play a different role in the ensuing immune response than do the earlier arriving DCs. In some cases, the immunologic signiﬁcance behind a particular pattern or restriction in trafﬁcking seems obvious. For example, the retention of most DCs within the lymph node through limited circulation into efferent lymph concentrates antigen within www.annualreviews.org • Migration of Dendritic Cell Subsets

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the lymph node, thereby likely increasing the relative density of particular peptide-MHC complexes for improved antigen presentation to a cognate population of T cells. Furthermore, the relative sequestration of activated DCs in the lymphatic system, permitting only rare return of mature DCs to blood, may protect the host. Indeed, it has been argued that the passage of highly activated, cytokine-producing and procoagulant-prone macrophages and DCs in the circulation during Ebola virus infection contributes pivotally to the deadly nature of this pathogen (148). However, especially under conditions when DCs fail to access lymphatics appropriately, they can gain at least limited access to blood and go on to mediate immune responses in distal lymphoid organs. The migratory patterns of pDCs are in almost every way different from those of conventional DCs, as we discuss in this review (Figure 1). Any immunological advantage that may be conferred by the fact that pDCs take distinct routes to the lymph node that differ so greatly from the routes of conventional DCs remains unknown. It is unclear, in particular, if there is an underlying immunological advantage in their apparent failure to move from tissues to lymph nodes through lymphatics. Why do they enter lymph nodes primarily through HEVs? Leaving the tissue of transplant immunity aside, entry of pDCs through HEVs when viruses or other antigens enter lymph nodes through afferent lymph seems to put pDCs at a disadvantage in accessing relevant antigen for presen-

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tation. Their trafﬁcking pattern seemingly ﬁts best with the concept that in many instances pDCs support antigen presentation mediated by conventional DCs, rather than present themselves. Alternatively, the entry of pDCs into the lymph node through HEVs may set up an important backup system. Upon entry through HEVs, pDCs could acquire viral particles that may have escaped capture by conventional DCs and that may be freely available in lymph ﬂuid. They may then process and present these antigens, such that their participation in presenting viral antigen in the lymph node may be a “second-tier” level of defense and depend on how well conventional DCs and other cells in tissues and the lymphatic system have managed to contain the virus. Obviously, we still have much to learn regarding how DC migration is regulated, why it is regulated the way it is, and what happens when normal migration and trafﬁcking patterns are disrupted. In addition, much work remains to be done to explain how signaling pathways work together to coordinate DC migration. Recent ﬁndings discussed herein indicate that DCs employ distinct signaling cascades to respond to chemokines like CCR7 ligands, raising the possibility that DC migration could be manipulated by means that will not alter T cell migration. Ultimately, the goal is to integrate our understanding of the signals that regulate DC migration and the molecular changes within tissues where DC migration occurs with our knowledge of the overall trafﬁcking patterns of different DC subsets in vivo.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We thank Dr. Claudia Jakubzick for critical reading of the manuscript. G.J.R. is supported in part by NIH grants and an Established Investigator Award from the American Heart Associa´ y Ciencia, Spain (reference SAF2007tion, J.O. by a grant from the Ministerio de Educacion 63579), and S.P-S. by CCRI-175404. 308

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130. Partida-Sanchez S, Gasser A, Fliegert R, Siebrands CC, Dammermann W, et al. 2007. Chemotaxis of mouse bone marrow neutrophils and dendritic cells is controlled by ADPribose, the major product generated by the CD38 enzyme reaction. J. Immunol. 179:7827– 39 131. Fliegert R, Gasser A, Guse AH. 2007. Regulation of calcium signalling by adenine-based second messengers. Biochem. Soc. Trans. 35:109–14 132. Perraud AL, Schmitz C, Scharenberg AM. 2003. TRPM2 Ca2+ permeable cation channels: from gene to biological function. Cell Calcium 33:519–31 133. Massullo P, Sumoza-Toledo A, Bhagat H, Partida-Sanchez S. 2006. TRPM channels, calcium and redox sensors during innate immune responses. Semin. Cell Dev. Biol. 17:654– 66 134. Fonfria E, Murdock PR, Cusdin FS, Benham CD, Kelsell RE, McNulty S. 2006. Tissue distribution proﬁles of the human TRPM cation channel family. J. Recept. Signal. Transduct. Res. 26:159–78 135. Farina C, Theil D, Semlinger B, Hohlfeld R, Meinl E. 2004. Distinct responses of monocytes to Toll-like receptor ligands and inﬂammatory cytokines. Int. Immunol. 16:799–809 136. Iqbal J, Zaidi M. 2007. CD38 is required for priming by TNF-α: a mechanism for extracellular coordination of cell fate. Am. J. Physiol. Renal Physiol. 292:F1283–90 137. Guerrero-Plata A, Casola A, Suarez G, Yu X, Spetch L, et al. 2006. Differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus. Am. J. Respir. Cell Mol. Biol. 34:320–29 138. Rotta G, Edwards EW, Sangaletti S, Bennett C, Ronzoni S, et al. 2003. Lipopolysaccharide or whole bacteria block the conversion of inﬂammatory monocytes into dendritic cells in vivo. J. Exp. Med. 198:1253–63 139. Watts C, Zaru R, Prescott AR, Wallin RP, West MA. 2007. Proximal effects of Toll-like receptor activation in dendritic cells. Curr. Opin. Immunol. 19:73–78 140. Pasare C, Medzhitov R. 2005. Toll-like receptors: linking innate and adaptive immunity. Adv. Exp. Med. Biol. 560:11–18 141. Krutzik SR, Tan B, Li H, Ochoa MT, Liu PT, et al. 2005. TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells. Nat. Med. 11:653–60 142. Angeli V, Staumont D, Charbonnier AS, Hammad H, Gosset P, et al. 2004. Activation of the D prostanoid receptor 1 regulates immune and skin allergic responses. J. Immunol. 172:3822–29 143. Hartiala P, Hytonen J, Pelkonen J, Kimppa K, West A, et al. 2007. Transcriptional response of human dendritic cells to Borrelia garinii–defective CD38 and CCR7 expression detected. J. Leukoc. Biol. 82:33–43 144. Dumitriu IE, Bianchi ME, Bacci M, Manfredi AA, Rovere-Querini P. 2007. The secretion of HMGB1 is required for the migration of maturing dendritic cells. J. Leukoc. Biol. 81:84– 91 145. la Sala A, Ferrari D, Di Virgilio F, Idzko M, Norgauer J, Girolomoni G. 2003. Alerting and tuning the immune response by extracellular nucleotides. J. Leukoc. Biol. 73:339–43 146. Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, et al. 2001. Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood 97:587–600 147. la Sala A, Sebastiani S, Ferrari D, Di Virgilio F, Idzko M, et al. 2002. Dendritic cells exposed to extracellular adenosine triphosphate acquire the migratory properties of mature cells and show a reduced capacity to attract type 1 T lymphocytes. Blood 99:1715–22 148. Bray M, Geisbert TW. 2005. Ebola virus: the role of macrophages and dendritic cells in the pathogenesis of Ebola hemorrhagic fever. Int. J. Biochem. Cell Biol. 37:1560–66

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:293-316. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu1 and Warner C. Greene1,2 1

Gladstone Institute of Virology and Immunology, San Francisco, California 94158

2

Departments of Medicine and of Microbiology and Immunology, University of California, San Francisco, California 94143; email: [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:317–53

Key Words

First published online as a Review in Advance on December 3, 2007

Alu, APOBEC3G, HIV-1, LINE-1, retrotransposition, Vif

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090350 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0317$20.00

Abstract All retroviruses, including HIV-1, display species-speciﬁc patterns of infection. The impaired growth of these retroviruses in foreign and sometimes even in their natural hosts often stems from the action of potent host-encoded “viral restriction factors” that form important protective components of the innate immune system. The discovery of APOBEC3G and related cytidine deaminases as one class of host restriction factors and of the action of HIV-1 Vif as a speciﬁc APOBEC3G antagonist have stimulated intense scientiﬁc interest. This Vif-APOBEC3G axis now forms a very attractive target for development of an entirely new class of anti-HIV drugs. In this review, we summarize current understanding of the mechanism of action of the APOBEC3 family of enzymes, their intriguing regulation within cells, the impact of these enzymes on viral evolution and disease progression, and their roles in controlling not only the replication of exogenous retroviruses but also the retrotransposition of endogenous retroelements.

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DISCOVERY OF APOBEC3G AS AN ANTI-RETROVIRAL FACTOR

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APOBEC: apolipoprotein B mRNA-editing enzyme, catalytic polypeptide

The HIV-1 Vif Phenotype

APOBEC proteins: a family of DNA or RNA cytidine deaminases, enzymes that have the hallmark deaminase motif (Cys/His)Xaa-Glu-Xaa23∼28 Pro-Cys-Xaa2∼4 -Cys Retroviruses: enveloped RNA viruses with positive-sense single-stranded RNA genomes that undergo reverse transcription and integration as obligatory steps in their life cycle Human immunodeficiency virus (HIV): a retrovirus with two types (1 and 2) belonging to the lentivirus family that causes AIDS Virion infectivity factor (Vif ): an HIV-1 accessory protein essential for efﬁcient viral replication in primary CD4 T cells and macrophages

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The identiﬁcation of human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, or hA3G) and related cytidine deaminases as potent innate inhibitors of a wide range of exogenous retroviruses emerged from studies of the HIV-1 protein Vif (virion infectivity factor). HIV-1 is a member of the primate lentivirus family of retroviruses. In contrast to simple retroviruses, which encode only Gag, Pol, and Env, HIV-1 encodes six additional auxiliary proteins (Tat, Rev, Nef, Vif, Vpr, and Vpu) that play pivotal roles in orchestrating the pathogenic interplay of HIV-1 with its human host. Vif itself, a basic 23-kDa phosphoprotein, is expressed late in the retroviral life cycle and is conserved among all of the primate lentiviruses, except for equine infectious anemia virus. Viruses lacking a functional vif gene (vif ) fail to mount a spreading infection in “nonpermissive” cells, which include biologically relevant primary CD4 T cells and macrophages (Figure 1). Vif is required for viral spread in cultures of some T cell leukemia lines, such as H9, HuT78, and MT2. Conversely, many “permissive” T cell lines (e.g., Jurkat and SupT1) and nonhematopoietic cell

lines (e.g., HeLa, 293T, and COS) fully support HIV spread in the absence of Vif (1– 3). Despite the early recognition of permissive and nonpermissive cell types supporting or not supporting the spread of vif HIV-1 respectively, the molecular basis for these cellular differences remained elusive for many years. Similarly, the mechanism by which Vif allowed wild-type HIV-1 to spread readily in cultures of nonpermissive cells was unclear.

Identifying the Vif-Sensitive Anti-HIV Factor Produced by Nonpermissive Cells Of note, nonpermissive cells support the normal production of vif HIV-1 virions, but these virions are unable to productively infect the next target cell (1–3). These ﬁndings suggest that Vif either overcomes the effects of a negative factor produced in nonpermissive cells or, alternatively, that permissive cells express a Vif-like host factor that positively inﬂuences virion infectivity. This issue was resolved in experiments in which heterokaryons formed between permissive and nonpermissive cells were infected with vif HIV-1 (4, 5). Progeny virions from these heterokaryons proved noninfectious, promoting the argument that nonpermissive cells express an inhibitory factor whose function is somehow defeated by Vif (Figure 1). This

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 Nonpermissive cells produce an anti-HIV factor that is neutralized by Vif. (a) The HIV-1 Vif phenotype. Viruses lacking a functional vif gene (vif ) fail to mount a spreading infection in “nonpermissive” cells, which include biologically relevant primary CD4 T cells and macrophages. Conversely, many “permissive” T cell lines (e.g., Jurkat and SupT1) and nonhematopoietic cell lines (e.g., HeLa, 293T, and COS) fully support HIV spread in the absence of Vif. Note that permissive and nonpermissive cells produce the same amount of progeny virions, but virions derived from nonpermissive cells are unable to infect the next target cell. (b) Nonpermissivity is dominant in heterokaryons formed between permissive and nonpermissive cells. Nonpermissive T cells (HuT78 or CEM-SS) were initially infected with HIV-1vifenv viruses pseudotyped with VSV-G. Heterokaryons were formed by incubation of these nonpermissive cells with permissive cells expressing HIV Env. The presence of CD4/CXCR4 receptors on the nonpermissive cells mediated fusion and heterokaryon formation. When progeny virions (HIV-1vif ) were tested for their ability to infect the next target cell, no spread was detected. However, in the presence of Vif, effective spread occurred. These ﬁndings indicated that nonpermissive cells produce an inhibitor of HIV infectious spread and that the action of this inhibitor is circumvented by Vif. This host inhibitory factor was subsequently identiﬁed as APOBEC3G. Chiu

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Vif-sensitive inhibitory factor remained uncharacterized until Sheehy et al. used subtractive hybridization techniques with two near-isogenic cell lines, CEM-S (permissive) and CEM-SS (nonpermissive) (6). One clone identiﬁed in this screen was hA3G (initially named CEM15), a known cytidine deami-

nase. Strikingly, permissive cells lacked hA3G expression, and the introduction of hA3G cDNA into permissive cells proved sufﬁcient to convert these cells into nonpermissive hosts. Additionally, virions budding from nonpermissive cells were shown to contain hA3G, providing an explanation for how viral

a Nonpermissive cell

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Cytidine deamination: the conversion of a cytidine to uridine by the hydrolytic substitution of an amine group

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replication might be altered in the next set of target cells. The mechanism by which Vif overcomes the effects of hA3G remained unexplained.

Functional Clues from Related Enzymes: APOBEC1 and Activation-Induced Deaminase

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Human A3G is a member of a much larger family of tissue-restricted cytidine deaminases (7) that exhibit RNA editing and/or DNA mutating activity (8, 9). In humans, the APOBEC family of enzymes includes activation-induced deaminase (AID), APOBEC1, APOBEC2, APOBEC3A–H, and APOBEC4 (7, 10– 12). Their active sites are characterized by the presence of a conserved zinc-binding motif (Cys/His)-Xaa-Glu-Xaa23∼28 -Pro-CysXaa2∼4 -Cys, a key glutamate involved in the proton shuttling that occurs during deamination, and two critical aromatic residues involved in nucleic acid substrate binding (7). These enzymes mediate hydrolytic deamination at the C4 position of the C (or dC) base, converting C to U (or dC to dU). These changes are often referred to as RNA or DNA editing (Figure 2). Two well-studied members of this enzyme family are APOBEC1 and AID, which are located in tandem on human chromosome 12 (13, 14). Initially identiﬁed in 1993, APOBEC1 is primarily expressed in gastrointestinal tissue (8), where it is the central component of

an RNA editosome complex mediating the deamination of cytosine-6666 in apolipoprotein B mRNA (15). This action of APOBEC1 converts a glutamine at this position to an inframe stop codon, resulting in the expression of a truncated version of the apoB protein (8). These two forms of apoB protein serve different functions. The longer apoB100 protein mediates the transport of endogenously produced cholesterol and triglycerides, while the shorter apoB48 regulates the absorption and transport of exogenous dietary lipids. Of note, forced expression of APOBEC1 as a transgene in the livers of mice consistently leads to hepatic dysplasia and hepatocellular carcinoma, possibly owing to promiscuous RNA editing of tumor suppressors or oncogenes (16). Such activity likely reﬂects the formation of nonphysiological RNA editosomes whose normally exquisite substrate speciﬁcity is lost. APOBEC1 appears to be the only member of the APOBEC superfamily of enzymes whose principal nucleic acid substrate is RNA rather than DNA. In contrast to APOBEC1’s role in lipid metabolism, AID is required for the normal evolution of the humoral immune response. AID is selectively expressed in germinal center B cells (17), where it catalyzes dC-to-dU deamination at the DNA level and thus promotes immunoglobulin-gene diversiﬁcation via somatic hypermutation and class switch recombination (18). These events are critically required for the natural maturation of

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 2 A family of APOBEC3 cytidine deaminases. The APOBEC3 gene locus containing seven related but distinct APOBEC3 genes is located on chromosome 22 in humans. This multigene family stands in sharp contrast to the single APOBEC3 gene present on syntenic chromosome 15 in mice. This genetic expansion reﬂects tandem duplication and unequal crossover resulting in a head to tail arrangement of the human APOBEC3 genes. Four genes (hA3B, hA3DE, hA3F, and hA3G) exhibit additional duplications of the cytidine deaminase domain (CD), as well as the intervening linker and pseudoactive domains. The length of these double domain enzymes varies somewhat as indicated, although each of the CDs are similar in size. The active sites of these enzymes catalyze hydrolytic deamination at the C4 position of 2 -deoxycytidine requiring the presence of cysteine (Cys) residues coordinating a single zinc ion and a key glutamate involved in proton shuttling. In this reaction, 2 -deoxycytidine is converted to 2 -deoxyuridine as a result of the removal of an amine group in the presence of water. 320

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Mouse chromosome 15 mA3

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Reverse transcription: the process of copying RNA into DNA, mediated by a reverse transcriptase enzyme

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the antibody response. Uracil DNA glycosylase, a DNA repair enzyme that removes uracil from single- and double-stranded DNA, is required for all AID-dependent processes (19). Mutations within the AID are linked to the Hyper-IgM syndrome, as well as aberrant activity linked with the development of various large B cell lymphomas and non-Hodgkin’s lymphomas (20). Like transgenic expression of APOBEC1, the constitutive and ubiquitous transgenic expression of AID in mice uniformly results in the development of various cancers, speciﬁcally T cell lymphomas, micro-adenomas, and lung adenocarcinomas (21). This link of APOBEC1 and AID to cancer emphasizes how tight intracellular regulation of these cytidine deaminases is likely required to avoid harmful transforming events within cells. When expressed in Escherichia coli, hA3G, AID, and even APOBEC1 catalyze the deamination of dC residues in single-stranded DNA (9, 22), suggesting that single-stranded DNA is the favored substrate for hA3G. This ﬁnding provides a ready explanation for how viral replication might be blocked in the next target cell via the inherent deaminase activity of hA3G acting on the nascent reverse transcribed cDNAs of the virus.

MULTIFACETED ANTIVIRAL ACTIONS OF APOBEC3G Retroviruses as a Target for the Inherent Deaminase Activity of APOBEC3G In the absence of Vif, hA3G is effectively incorporated into budding HIV-1 virions (23, 24). This encapsidation reaction involves interaction with the nucleocapsid region of the HIV-1 Gag polyprotein (25–27). This interaction is further strengthened by A3G’s propensity to bind single-stranded nucleic acids, particularly viral RNA at the plasma membrane site of virion budding (28–34). The incorporation of only 7 ± 4 molecules of hA3G into vif HIV virions produced from 322

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human peripheral blood mononuclear cells appears sufﬁcient to inhibit HIV-1 replication during the next round of infection (35). The virion-incorporated hA3G is bound to the viral core (32) and thus is effectively introduced into the target cell as a result of virion fusion. The enzyme then mediates extensive dC-todU mutations of the minus-strand viral DNA formed during reverse transcription (36, 37). These mutated viral minus-strand DNAs containing excessive dUs may then be destroyed by the action of two virion-associated DNA base repair enzymes, uracil DNA glycosylase and apurinic-apyrimidinic endonuclease (38, 39), although uracil DNA glycosylase 2 appears dispensable for this antiviral action of hA3G (40, 41). A few viral minus strands appear to survive this attack and serve as templates for plus-strand synthesis, where the dU promotes dA misincorporation. The resultant dG-to-dA mutations, which can exceed 10% of all dG residues, likely further negate HIV1 replication by altering viral open reading frames and introducing inappropriate translation termination codons (42–45) (Figure 3). HIV reverse transcription is initiated by extension from the tRNALys3 primer that anneals to the primer binding site of the viral genomic RNA generating minus-strand strong-stop DNA. This minus-strand strongstop DNA is translocated to the 3 end of the genome and further extended to complete the minus-strand cDNA. As the minusstrand cDNA is synthesized, the RNA template is degraded by the RNase H activity of the reverse transcriptase, exposing the singlestranded minus-strand DNA. Two RNase H– resistant RNA polypurine tracts (PPT), cPPT and 3 PPT, remain associated with the minusstrand cDNA to serve as initiation sites for subsequent plus-strand cDNA synthesis. As such, hA3G’s single-stranded DNA speciﬁcity (36, 37) accounts for two highly polarized (5 -to-3 ) mutational gradients within the viral genome, each with maxima just 5 to the cPPT and the 3 PPT. These gradients are in remarkable agreement with the time these regions of the minus-strand DNA

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Figure 3 Multifaceted antiviral actions of virion-incorporated hA3G. In the absence of Vif, hA3G present in the cytoplasm of virus-producing cells is effectively incorporated into budding virions and thus is carried forward into the next target cells, where it is available to act as a potent inhibitor of HIV-1 replication. These results explain how nonpermissive cells can produce normal levels of progeny virions, but the virions emanating from nonpermissive cells fail to replicate effectively in the next target cells. These inhibitory effects involve both deaminase-independent and deaminase-dependent antiviral actions of hA3G. Human A3G bound to HIV-1 RNA may physically impede reverse transcriptase movement on the viral RNA template, resulting in a deaminase-independent decrease in the production of early reverse transcripts (1). However, this inhibition is frequently incomplete, and minus-strand viral DNA is generated. This single-stranded DNA forms the target for hA3G’s deaminase-dependent antiviral attack. Human A3G mediates extensive deamination of deoxycytidine residues in the minus-stranded viral DNA. This action effectively halts HIV-1 replication either due to the accumulation of dG-to-dA hypermutations in the subsequently synthesized plus-strand DNA of the virus or because the uracil-containing minus-strand DNA is destroyed by the combined actions of uracil DNA glycosylase and apurinic-apyrimidinic endonuclease (2). Additionally, diminished chromosomal integration of the double-stranded viral DNA required for provirus formation may occur owing to defects in tRNALys3 primer cleavage leading to the formation of viral DNA with aberrant ends (3).

remain single-stranded, awaiting the commencement of plus-strand synthesis (36, 46). These gradients could also result from a processive directional attack leveled by hA3G on its single-stranded cDNA substrate (47). Human A3G binds randomly to single-stranded DNA, then jumps and/or slides processively for at least 100 nucleotides to deaminate target motifs. This intrinsic “slide-and-jump” cat-

alytic activity of hA3G exhibits a clear bias favoring 5 dCs over 3 dCs. Thus deamination occurs predominantly 3 to 5 on the minus-strand viral DNA without requiring hydrolysis of a nucleotide cofactor (47). Rarely, hA3G also modiﬁes the viral plusstrand in the 5 U3 region and primer-binding site (36). Of note, the 5 U3 region becomes brieﬂy single-stranded when displaced

www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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Simian immunodeficiency virus (SIV): a collection of related retroviruses belonging to the lentivirus family that infect many nonhuman primates

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MLV: murine leukemia virus HTLV: human T cell leukemia virus Hepatitis B virus (HBV): a DNA virus belonging to the hepadnavirus family that replicates via an RNA intermediate and requires reverse transcription

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from the minus-strand during plus-strand synthesis; the primer-binding site in the plus-strand similarly becomes single-stranded when RNase H degrades the tRNALys3 primer bound at this site. These ﬁndings strongly support the notion that single-stranded DNA is the physiological substrate of hA3G. The editing activity of hA3G exerts broad antiretroviral effects, blocking the replication of HIV-1 and other lentiviruses, including simian immunodeﬁciency virus (SIV) and equine infectious anemia virus, and even distantly related retroviruses, such as murine leukemia virus (MLV) and foamy viruses (23, 42, 44, 48–50). The common use of DNA deamination by hA3G and AID emphasizes an intriguing biological strategy whereby both the innate and adaptive immune defenses commonly exploit DNA editing for defense of the host.

Deaminase-Independent Antiviral Activity of APOBEC3G In contrast to APOBEC1 and AID, which have single catalytic domains, hA3G contains two cytidine deaminase domains (CDs), as well as duplicated intervening linker and pseudoactive domains (7) (Figure 2). Despite their homology, these two CDs display quite distinct functional properties; the N-terminal CD1 mediates RNA binding and virion encapcidation (51–53), whereas the C-terminal CD2 confers deoxycytidine deaminase activity (51–54) and sequence speciﬁcity for modiﬁcation of the single-stranded DNA substrate. A3G preferentially deaminates 5 -CC dinucleotides at the 3 dC (42, 44, 45, 55–57). The ensuing degradation of the dU-containing viral minus-strands and the massive dG-to-dA hypermutation in the surviving viral plusstrand represent important events that centrally contribute to the antiviral properties of hA3G. Additionally, the accumulation of dUs in minus-strand DNA may lead to decreased plus-strand synthesis by HIV-1 reverse transcriptase owing to aberrant initiation at the PPT sites (58).

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Despite this clear link between the deoxycytidine deaminase activity of hA3G and its antiviral activity, emerging evidence suggests that A3G exerts additional antiviral effects independent of its enzymatic activity. Specifically, hA3G analogs containing disabling mutations in the C-terminal CD continue to substantially reduce the infectivity of vif HIV-1 virions. The RNA binding activity of the N-terminal CD appears critically involved in this nonenzymatic form of inhibition, which likely involves physical impairment of reverse transcriptase activity (52, 59). Additionally, the presence of hA3G in vif HIV-1 virions results in a 50% or greater reduction in the ability of tRNALys3 primers to initiate reverse transcription (60). Whether this inhibition reﬂects decreased tRNALys3 annealing to viral RNA or an altered conﬁguration of the tRNALys3 /viral RNA hybrid in the presence of hA3G remains unclear. Finally, hA3G may also cause defects in tRNALys3 cleavage during plus-strand DNA transfer, leading to the formation of aberrant viral DNA ends that could interfere with subsequent chromosomal integration of the double-stranded viral DNA required for provirus formation (41) (Figure 3). Of note, the N-terminal linker region of hA3G has been implicated as a docking site for the C-terminal domain of HIV-1 integrase. The association of hA3G with components of the preintegration complex (PIC) such as integrase might interfere with the structural integrity of the PIC and consequently inhibit nuclear import of the PIC, thereby further impairing the successful integration of the viral DNA (61). Human A3G also mediates deaminaseindependent antiviral activity against human T cell leukemia virus type-1 (HTLV-1), although the effects are more modest than those occurring with HIV-1. Hypermutations are also not abundant in most of the HTLV-1 viral DNA (62, 63). Furthermore, hA3G also blocks the replication of hepatitis B virus (HBV) in a manner that does not require deamination. The HBV life cycle involves an obligatory reverse transcription

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step during which pregenomic RNA intermediates within nucleocapsids in the cytoplasm of virus-producer cells are converted to DNA before being packaged into budding virions. Human A3G appears to inhibit viral pregenome packaging, thereby destabilizing the reverse transcription complex and preventing HBV DNA accumulation in virions (64, 65). Only limited dG-to-dA mutations can be detected in HBV DNA, and these effects are only observed in selected hepatoma cell lines (65–67). All these studies support the notion that A3G’s antiviral activity is far more complex than previously thought. However, the precise mechanisms underlying these deaminase-independent antiviral actions of hA3G remain incompletely characterized.

Nuances of Intracellular Regulation of APOBEC3G Reveal a Novel Postentry Antiviral Activity A central question highly relevant to normal cell function is how the potentially promiscuous mutagenic activity of the hA3G enzymes is controlled. One possibility was immediately suggested by subcellular localization studies, which indicated that hA3G is chieﬂy cytoplasmic and does not appear to shuttle like APOBEC1 and AID. Rather hA3G is strongly retained in the cytoplasm (12, 42, 68–71). However, such localization is unlikely to represent the sole mechanism for limiting promiscuous editing of genomic DNA, especially because nuclear and cytoplasmic components are admixed in dividing cells after breakdown of the nuclear membrane. Indeed, subsequent studies revealed that endogenous hA3G expressed in the cytoplasm of H9 T cell lines and mitogen-activated CD4 T cells is assembled in 5–15 MDa high-molecularmass (HMM) ribonucleoprotein (RNP) complexes and that the deoxycytidine deaminase activity of hA3G is greatly inhibited in these complexes. Interestingly, these HMM hA3G complexes can be artiﬁcially converted to an enzymatically active low-molecular-mass (LMM) form by treatment with RNase A, sug-

gesting that RNA components play an important role in the assembly of HMM A3G complexes (72). This discovery of different forms of hA3G ultimately provided pivotal information bearing on the question of why resting CD4 T cells in lymphoid tissue are permissive to HIV1 infection, whereas CD4 T cells circulating in the peripheral blood are not, even though A3G is expressed in both cell types. The answer is that hA3G is expressed in two very different forms in these two populations of CD4 T cells. Circulating resting CD4 T cells are distinguished by the presence of LMM hA3G (72) and are refractory to HIV-1 infection at least in part because of an early postentry block during or soon after the reverse transcription step (73, 74). In sharp contrast, in lymphoid tissue–resident resting CD4 T cells, which display increased permissiveness to HIV-1 infection (75), hA3G is predominantly found in the HMM complexes (76). The formation of these complexes reﬂects, at least in part, the action of various cytokines, including IL-2 and IL-15, that are present in the lymphoid tissue microenvironment. These cytokines induce the assembly of HMM A3G complexes (76) (Figure 4). When RNA interference is used to knock down expression of the LMM hA3G present in peripheral blood– derived resting CD4 T lymphocytes, these cells are rendered permissive to HIV infection. This ﬁnding provides direct evidence that LMM hA3G functions as a potent postentry restriction factor that inhibits the replication of incoming HIV-1 virions (72). LMM hA3G can be converted to an HMM complex when CD4 T cells are activated with various mitogens (anti-CD3/CD28 and phorbol myristate acetate) and cytokines (IL-2, IL-7, and IL-15) (72, 77). The assembly of HMM A3G complexes appears to require entry into the G1b phase of the cell cycle (72), a stage characterized by RNA synthesis (78). These events result in HMM A3G complex assembly thereby removing the LMM hA3G postentry block and creating a favorable environment for productive HIV-1 infection www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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Figure 4 APOBEC3G also functions as a potent postentry restriction factor for HIV-1 in resting CD4 T cells present in peripheral blood. Human A3G exists exclusively in LMM forms in peripheral blood–derived resting CD4 T cells. LMM hA3G functions as a postentry restriction factor blocking the replication of incoming HIV-1 viral particles. Of note, this action does not strictly depend on editing but rather appears to involve signiﬁcant delays in the accumulation of viral reverse transcripts. Strikingly and in sharp contrast to their cellular counterparts isolated from peripheral blood, resting CD4 T cells residing in human lymphoid tissues are permissive to HIV-1 infection. In these cells, hA3G is predominantly detected in HMM complexes and as such is unable to exert the postentry restricting activity characteristic of LMM hA3G. Induction of HMM hA3G complex assembly in these tissue-derived resting CD4 T cells appears to reﬂect the action of various cytokines including IL-2 and IL-15, which are present in this lymphoid tissue microenvironment. Additional inductive signals may occur as a result of cell-cell contacts in these tissues. However, though sufﬁcient to induce HMM hA3G complex assembly, these signals do not induce full-ﬂedged lymphocyte activation.

(72, 77). Of note, the action of LMM A3G is not antagonized by Vif because virions contain little or no Vif (79), and the virus has not progressed far enough through its life cycle to produce new Vif. Thus, this postentry restricting activity of LMM A3G is equally effective against wild-type HIV-1 and vif HIV-1 virions. Vif springs into action much later in the viral replication cycle when its synthesis is triggered by Rev, and it prevents hA3G incorporation into progeny virions budding from productively infected cellular hosts. 326

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The identiﬁcation of LMM forms of hA3G in primary resting CD4 T cells isolated from peripheral blood provided the ﬁrst evidence that hA3G can exert antiviral effects in target cells independently of its prior incorporation into virions. Interestingly, this HIV-1 postentry replication block by cellular LMM hA3G does not depend strictly on editing. Instead, it involves signiﬁcant delays in the accumulation of late reverse transcription products. Over 90% of the reverse transcripts that slowly form in infected blood-derived resting CD4 T cells contain no evidence of

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dG-to-dA hypermutation, arguing for an antiviral action of hA3G independent of cytidine deamination (72). However, the possibility that many transcripts are in fact hypermutated, but rapidly degraded, cannot be completely ruled out. The status of HMM hA3G complex formation also correlates well with the susceptibility or resistance of cells within the monocyte lineage to infection with HIV-1 (72, 80– 82). Speciﬁcally, freshly isolated monocytes are highly refractory to infection in vitro (83– 85) and are distinguished by the presence of LMM hA3G (72). Differentiation of these cells into macrophages promotes the HMM hA3G complex assembly (72), a transition that correlates with a sharp increase in permissiveness to HIV-1 infection (83–85). HMM hA3G complexes also are observed in the CD16+ subset of monocytes, which are more permissive than other monocytes. These cells may form a reservoir for viral persistence during antiretroviral therapy (80). Immature dendritic cells contain low levels of hA3G that is assembled into HMM complexes. Maturation of these cells is associated with a sharp increase in hA3G expression and the additional expression of LMM hA3G forms (77, 81). Mature dendritic cells are less permissive than immature dendritic cells to HIV infection (81).

DIVERSE RETROVIRAL STRATEGIES FOR CIRCUMVENTING APOBEC3G ANTIVIRAL ACTIVITY The Vif-APOBEC3G Paradigm Human A3G poses a signiﬁcant threat to the successful replication and spread of HIV-1. One of the nine gene products of HIV1, Vif, is speciﬁcally utilized to counter this threat. Vif binds to hA3G and promotes its accelerated degradation by the 26S proteasome. The resulting depletion of the intracellular stores of hA3G prevents effective virion encapsidation of hA3G and ensures high infectivity of the progeny virions

(Figure 5). Vif uses multiple protein interaction regions to orchestrate proteasomemediated degradation of hA3G. The Nterminal region of Vif binds to the N-terminal region of hA3G (amino acids 54–124) (68, 86–88). The SLQ(Y/F)LA motif (amino acids 144–150) of Vif, which resembles a conserved sequence in the BC-box of the suppressors of cytokine signaling (SOCS) proteins, binds to Elongin C (87, 89–92). Finally, a novel zincbinding motif, His-Xaa5 -Cys-Xaa17−18 -CysX3−5 -His (HCCH, amino acids 108–139), interacts with Cullin5 (93–95). Through these interactions, Vif effectively recruits an active ubiquitin ligase (E3) complex composed of Elongin C, Elongin B, Cullin5, Nedd8, and Rbx1 (89, 92), which mediates polyubiquitylation of hA3G—a posttranslational modiﬁcation that targets proteins for destruction by the proteasome (24, 86, 87, 96). Mutation of the BC-box domain (SLQ motif) or the HCCH motif in Vif or overexpression of Cullin5 mutants that fail to engage Nedd8 or Rbx1 all result in a loss of hA3G polyubiquitylation and the preservation of hA3G’s antiviral activity (89, 91–93). Two additional Vif domains, the central hydrophilic EWRKKR domain (amino acids 88–93) and the prolinerich PPLP domain (amino acids 161–164), are important respectively for enhancing steadystate levels of Vif and for interaction with tyrosine kinases (97, 98). Mutation of these domains also compromises Vif activity. In addition to accelerating proteasomal degradation of hA3G, Vif partially impairs the translation of hA3G mRNA (24), although the mechanism remains undeﬁned. Nevertheless, the combined effects of accelerated degradation and diminished synthesis result in a striking depletion of intracellular hA3G and thus forfeiture of its antiviral activity (23, 24) (Figure 5). Additional mechanistic insights have emerged from interspecies studies. HIV-1 and HIV-2 represent the products of independent cross-species (zoonotic) transmission events of related but distinct lentiviruses (SIVs) that naturally infect nonhuman www.annualreviews.org • The APOBEC3 Cytidine Deaminases

Zoonotic transmissions: The cross-species transfer of an infectious agent from one animal species to another that is not regarded as a natural host for the pathogen

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primates in sub-Saharan Africa (99). One lentivirus, SIVcpz, present in chimpanzees (Pan troglodytes) in western equatorial Africa represents the precursor of pandemic HIV-1. Similarly, SIVsm from sooty mangabeys (Cercocebus atys) is the immediate source of HIV-2. Unlike HIV-1, HIV-2 infection remains largely restricted to western Africa.

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Other lentiviruses, such as SIV from African green monkeys (SIVagm), have not been transmitted to humans. Additionally, rhesus macaques strongly resist experimental infection with HIV-1, reﬂecting the action of TRIM5α, a second and distinct type of postentry restriction factor (100). Such species-speciﬁc barriers to infection not only

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oppose zoonotic infections but provide an important resource for better understanding virus-host interactions. In contrast to the broad antiviral action of A3G, Vif’s counterattack occurs in a highly species-speciﬁc manner (23). For example, Vif from SIVagm effectively triggers the degradation of African green monkey A3G but fails to neutralize either human or chimpanzee A3G. Similarly, HIV-1 Vif cannot induce degradation of African green monkey or rhesus macaque A3G. These species-speciﬁc limitations in Vif activity likely form an important barrier that minimizes successful zoonotic transmission of many primate lentiviruses. Of note, the fact that the Vif gene products of SIVsm and SIVcpz successfully degrade hA3G (23, 101, 102) provides a compelling explanation for how these viruses were able to spawn the HIV-1 and HIV-2 epidemics in humans. Species speciﬁcity is governed by a single amino acid at position 128 in A3G (aspartic acid in human and lysine in African green monkey) (101, 103–105) and amino acids 14–17 in Vif (106). Introduction of a D128K substitution in hA3G renders this protein sensitive to SIVagm Vif and resistant to HIV-1 Vif (101, 103–105). Corresponding mutations introduced into Vif similarly alter its species-speciﬁc effects. For example, replacement of D14 RMR17 in HIV-

1 Vif with SERQ or SEMQ, the equivalent residues in SIVagm Vif, allows functional interactions of HIV-1 Vif with rhesus macaque A3G and African green monkey A3G, as well as D128K-A3G. This loss of species restriction is likely linked to overall increase in the negative charge of amino acids 14–17 in HIV1 Vif that promotes effective interaction with the positively charged lysine present at residue 128 in African green monkey and rhesus A3G (106). Although Vif-induced degradation of hA3G plays an important role in overcoming the antiviral effects of hA3G, nondegradative mechanisms of Vif action have also been proposed. One study indicates that Vif can enhance virion infectivity even under conditions where it only moderately reduces steadystate levels of hA3G. Further, an S144A mutation in Vif that prevents phosphorylation at this site leads to progeny virions with poor infectivity. Nevertheless, this mutant effectively depletes hA3G (90). Additional complexity in Vif’s mechanism of action is highlighted by a recent report showing that Vif inhibits virion encapsidation and the antiviral activity of a hA3G variant, C97A, which is intrinsically resistant to degradation (107). Possible alternative mechanisms of Vif action include the physical exclusion of hA3G from sites of viral assembly and budding or inhibition of A3G encapsidation by competing for binding

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 5 The interplay of Vif and APOBEC3G. (a) Neutralization of hA3G in virus-producing cells by HIV Vif. Vif defeats the antiviral activity of hA3G principally by both binding to hA3G and recruiting an E3 ligase complex that mediates polyubiquitylation (Ubn) of hA3G. This posttranslational modiﬁcation of hA3G promotes its accelerated degradation in 26S proteasomes (1). Vif also partially impairs the translation of hA3G mRNA (2). These dual effects of Vif effectively deplete hA3G in the virus-producing cells; thus, hA3G is not available for incorporation into virions budding from these cells. Other auxiliary functions of Vif have been proposed, including physical exclusion of hA3G from virion encapsidation in the absence of degradation, perhaps owing to sequestration of hA3G away from the sites of viral assembly/budding (3). (b) Model of the Vif-Cul5-Elongin BC complex. Vif employs multiple protein interaction domains to orchestrate hA3G degradation. The N-terminal region of Vif has been implicated in its binding to an N-terminal region of hA3G (amino acids 54–124). The SLQ(Y/F)LA motif (amino acids 144–150) of Vif mediates binding to the Elongin C component of the E3 ligase complex. Finally, a novel zinc-binding motif (HCCH, amino acids 108–139) within Vif containing two conserved cysteines mediates a second interaction with the Cullin5 component. The Cul5-Vif E3 ubiquitin ligase binds hA3G and brings it into close proximity to the E2 ubiquitin-conjugating enzyme. www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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to viral components like the nucleocapsid component of Gag or viral genomic RNA, which play central roles in A3G incorporation into virions (23, 107, 108) (Figure 5). Additionally, both Vif and a second viral accessory protein, Vpr, induce G2 cell-cycle arrest, which may play a role in the T cell cytopathicity induced by HIV infection (109, 110). These ﬁndings suggest that Vif may play a larger biological role beyond its function as a hA3G antagonist. Indeed, due to the limited size of the HIV genome, many viral proteins perform multiple functions, thereby achieving remarkable genetic economy.

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Viron Exclusion of APOBEC3G as a Common Strategy Although lentiviral Vif efﬁciently fends off A3G’s attack at least in part through proteasome-mediated degradation of the enzyme, other retroviruses have evolved rather different counterstrikes. These different strategies unambiguously highlight the importance of virion exclusion as a way to circumvent the antiviral activity of A3G. Primate foamy viruses and a distantly related feline foamy virus resist the inhibitory effects of hA3G through the action of the accessory protein Bet. Bet binds to hA3G and prevents its packaging into virus particles, but these effects do not involve degradation of hA3G in virionproducing cells (48, 49). HTLV-1 utilizes a very different strategy. Within the C-terminal region of its nucleocapsid domain, HTLV-1 encodes a peptide motif that impairs hA3G from interacting with viral genomic RNA and thus inhibits hA3G packaging into budding virions (111). This motif is highly conserved among the primate T cell leukemia viruses but is absent in all other retroviral nucleocapsid proteins. MLV replicates effectively in murine APOBEC3 (mA3)-expressing cells and survives because its Gag protein has been evolutionarily selected neither to bind nor to package the cognate mA3 enzyme. However, MLV infectivity is effectively restricted by hA3G, which continues to bind to MLV Gag (112– 330

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114). Similarly, Mason-Pﬁzer monkey virus (MPMV) escapes inhibition by A3G in its natural host, the rhesus macaque, by failing to incorporate rhesus A3G into budding virions. However, this virus is highly susceptible to inhibition by mA3, which efﬁciently forms a complex with MPMV Gag and is effectively encapsidated into virions (115). The ﬁnding that two essentially unrelated retroviruses (MLV and MPMV) employ similar strategies to escape inhibition by APOBEC3 proteins in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles represents a general mechanism employed by simple mammalian retroviruses. Although the speciﬁc virion exclusion of cognate APOBEC3 protein has been attributed to an inability to bind MLV Gag, MLV may also utilize additional strategies to prevent A3G encapsidation. Speciﬁcally, MLV viral RNA also blocks the binding of mA3 to Gag, contributing to the exclusion of mA3 from MLV virions (114). Finally, even if mA3 is successfully packaged, this antiviral enzyme may be inactivated by cleavage by the MLV protease (114).

RIBONUCLEOPROTEIN COMPLEXES INVOLVED IN REGULATING APOBEC3G ANTIVIRAL ACTIVITY Molecular Assemblies Involved in Cellular APOBEC3G Complexes Several laboratories joined in efforts to characterize the protein and RNA components of the HMM hA3G RNP complexes (71, 116, 117). Tandem afﬁnity puriﬁcation and mass spectrometry have led to the identiﬁcation of at least 95 different proteins as participants in HMM hA3G complexes. Numerous cellular RNA binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in these HMM hA3G complexes, but most are present owing to their binding to resident RNAs rather than

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their direct assembly with hA3G (71, 116, 117). These complexes actually correspond to three well-characterized multisubunit RNPs present in human cells: (a) Staufencontaining, polysome-associated RNA granules, (b) Ro RNPs, and (c) prespliceosomes plus reservoirs for transcriptional regulators (116). Of note, the protein cofactors in the latter class are multifunctional, and many participate in Staufen-containing RNA granules or function as cytoplasmic regulators of translation. Staufen, a double-stranded RNA-binding protein, is best known for its role in the localization of speciﬁc mRNAs during Drosophila oogenesis. Mammalian homologs of Staufen (Staufen1 and Staufen2) have been implicated in the function of neuronal granules within dendrites of hippocampal neurons. After synaptic stimulation, these granules are transported to the synapse, where their mRNA cargoes are rapidly translated. The resultant protein products contribute to orchestrating changes in synaptic plasticity (118, 119). In general, RNA granules are >10 MDa macromolecular RNP complexes containing more than 100 proteins, including ribosomal subunits, scaffold proteins, translation machinery, RNA-binding proteins, helicases, and various decay enzymes (i.e., Staufen; Purα; Purβ; DbpA and DbpB; nucleolin; RNA binding motif protein 3; NFAR; FMR1; FXR1; FXR2; PABP; CBP80; CBP20; EF1α; Hsp70; Upf1; 60S and 40S ribosomal proteins; DDX1; DDX3; p68 RNA helicases; RNA helicase A; and hnRNP A/B, A0, A, D, and U) (118–121). Ro RNPs are the major RNP autoantigens recognized by sera from patients with various connective tissue diseases, although their normal function remains incompletely understood. In human cells, Ro RNPs contain one of the four human small Y (hY) RNAs (hY1, hY3, hY4, and hY5) and two core proteins (60-kDa Ro and 50-kDa La) (122). Almost all of the proteins mentioned above that participate in the formation of Staufen-containing RNA granules and Ro RNPs are readily de-

tectable in the puriﬁed HMM hA3G RNP complexes. An association of hA3G with stress granules (SGs) and processing bodies (PBs) instead of RNA granules has also been proposed (69, 71, 117). RNA granules, SGs, and PBs are in fact related, dynamic cytoplasmic RNA structures that control the localization, translation, and stability of their resident mRNA cargoes. However, RNA granules harbor highly speciﬁc mRNA sequences, whereas SG and PB are less discriminating. In addition to differing in their mRNA selectivity, RNA granules contain both the 60S and 40S ribosomal subunits, whereas SGs contain only small ribosomal subunits and PBs lack both subunits (118, 119). Staufen proteins are present in all three types of cytoplasmic RNA structures (118, 119). However, the RNA granules that associate with HMM hA3G contain both the 60S and 40S ribosomal subunits and harbor highly speciﬁc mRNA cargos including hA3G mRNA (116, 117); thus, they are more reminiscent of Staufen-containing, polysome-associated RNA granules rather than of SGs or PBs. Further, an association of endogenous proteins that are characteristic components of SGs (i.e., TIA-1) and PBs (i.e., Ago2 and decapping enzymes) has not been demonstrated in HMM hA3G complexes. Nevertheless, exogenously expressed components of SGs and PBs can be copuriﬁed with HMM hA3G complexes (69, 71). Under these conditions of forced expression, confocal microscopy further supports substantial colocalization of hA3G with cytoplasmic SGs and PBs (69, 71). Indeed, hA3G complexes are shifted reversibly between polysomes and dormant pools (likely, SGs) in response to translational inhibitors (117). Stressing the cells also induces the rapid redistribution of hA3G and a number of PB proteins (Ago1 and Ago2) to SGs (71). These ﬁndings imply that the dynamic spatial organizations between RNA granules and related cytoplasmic RNA complexes could affect the status of hA3G complexes, control A3G’s antiviral activity, and www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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Retroelement: any mobile genetic element that undergoes reverse transcription and then inserts the new DNA copy into the host cell genomes

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regulate translation/decay of mRNAs encoding this innate antiretroviral factor. Intriguingly, delineation of the RNA components in HMM hA3G complexes identiﬁed human Alu (Sx, Sp, Ya5, Ya8, and Yb8 subfamily) and small hY (hY1–5) endogenous retroelement RNA sequences (116). Activation of CD4 T cells with PHA and IL-2 induces high-level expression of these endogenous retroelement RNAs. Conversely, almost all of the protein cofactors that participate in the HMM A3G complexes are constitutively expressed in resting CD4 T cells but are not assembled into complexes (116). These ﬁndings raise the distinct possibility that the induced expression of Alu and hY RNAs forms the driving force for HMM hA3G complex assembly. The hA3G-dependent recruitment and speciﬁc enrichment of Alu and hY RNAs into Staufen-containing RNA granules further suggest a potential physiological function for these complexes (116). Speciﬁcally, endogenous nonautonomous retroelements (i.e., Alu and hY RNAs) (123, 124) likely form the natural cellular targets of hA3G (see below).

Intravirion APOBEC3G Complexes: Unexpected Interplay Between Host and Virus The fact that cellular hA3G principally resides in 5–15-MDa HMM RNP complexes in activated, virus-producing T cells prompted studies to determine which form of hA3G is actually incorporated into budding virions. Three models seemed possible: (a) Although unlikely, the entire HMM hA3G complex might be incorporated into budding virions; (b) hA3G residing in HMM complexes might be selectively extracted via interactions with HIV Gag and genomic RNA; and (c) newly synthesized hA3G not yet assembled into HMM complexes might be preferentially recruited into virions. When virions are examined for proteins that compose the HMM A3G complex, most are undetectable; those that are present do not appear to be recruited 332

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into virions in an hA3G-dependent manner (32, 117). These ﬁndings argue strongly against model a. In pulse-chase radiolabeling and size fractionation studies, hA3G is rapidly assembled (<30 min) into HMM complexes (32). Thus, only small quantities of newly synthesized LMM A3G are brieﬂy present in cells. However, pulse-chase studies revealed that virion hA3G is mainly recruited from this cellular pool of newly synthesized enzyme (32). Once hA3G becomes incorporated into the HMM complexes, it is no longer available for incorporation into virions. These unexpected ﬁndings also indicate that the small pool of newly synthesized LMM hA3G rather than the HMM A3G complex is the biologically relevant target of Vif. Although Vif promotes proteasome-mediated degradation of hA3G in the HMM complex (72), its mechanism of attack on LMM A3G remains undeﬁned. This attack could involve proteolytic degradation or effects that curtail virion encapsidation in the absence of degradation. What is the fate of the newly synthesized hA3G that is successfully incorporated into HIV virions? Because virion hA3G ultimately mediates high-level deamination of deoxycytidines in nascent minus-strand DNA formed during reverse transcription, it seemed likely that virion hA3G would be enzymatically active. However, interrogation of hA3G incorporated into virions revealed an absence of enzymatic activity (32). Apparently, the interaction of hA3G with HIV genomic RNA in the virion core leads to inhibition of its activity, analogous to the inactivation of cellular hA3G activity when it engages cellular RNAs in the HMM RNA-protein complexes (32, 72). Of note, when virions are produced under conditions of marked overexpression of hA3G, enzymatic activity of hA3G is readily apparent (36). However, under these nonphysiological conditions, excess packaging of the enzyme occurs outside of the core (32). Because this form of the enzyme is not bound to RNA, its enzymatic activity is not inhibited. The unexpected lack of enzymatic activity by virion-incorporated hA3G raised an

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important question: How is the enzyme ultimately activated? Intriguingly, the answer appears to involve the action of HIV-1 RNase H. Degradation of the viral genomic RNA by RNase H during reverse transcription not only generates the minus-strand DNA substrate of hA3G but also removes the inhibitory RNA bound to hA3G, thereby activating its deaminase activity (32). These ﬁndings highlight an unusual virus-host interaction where initiation of antiviral enzymatic activity of hA3G is contingent on the prior action of an essential viral enzyme. These studies also imply a dual strategy for restriction of HIV-1 by hA3G that could account for the deaminase-dependent and -independent antiviral activities described above (Figure 3). Initially, the enzymatically latent form of hA3G bound to HIV-1 RNA may impair the generation of minus-strand DNA by physically impeding reverse transcriptase on its viral RNA template. However, this physical block may be incomplete, and minus-strand viral DNA may be occasionally generated. Subsequently, hA3G deaminase activity is restored when RNase H degrades the viral RNA and leaves the singlestranded DNA template intact for plus-strand synthesis, allowing extensive deamination of the minus-strand DNA. The relative effectiveness of these two sequential antiviral actions of hA3G in different cellular environments could explain why HBV replication is inhibited by deaminase-dependent actions of hA3G in HepG2 cells but by deaminaseindependent action in Huh7 cells (64–67).

APOBEC3G AND OTHER MEMBERS OF THE EXTENDED APOBEC3 GENE FAMILY Evolutionary Expansion of APOBEC3 Deaminases The gene encoding hA3G is located on chromosome 22q13.2 within a cluster of highly related APOBEC3 genes (hA3A, hA3B, hA3C, hA3DE, hA3F, and hA3H) arrayed in a head-

to-tail manner (7, 10, 12, 125). In contrast, only a single APOBEC3 gene (mA3) is present in the syntenic chromosome 15 in rodents, suggesting that the APOBEC3 locus expanded after the genetic radiation of mice and humans, likely by tandem duplication and recombination with unequal crossover (Figure 2). Four of the human APOBEC3 proteins (hA3B, hA3DE, hA3F, and hA3G) and mA3 have two CDs, whereas hA3A, hA3C, and hA3H have only a single deaminase motif (7, 10, 12, 125). Phylogenetic analyses suggest that the primordial APOBEC3 likely contained only one CD, which might have evolved from AID or APOBEC2 during the onset of vertebrate speciation (10). The presence of two CDs may increase the antiviral potency of the APOBEC3 proteins by providing a means to separate the RNA binding and catalytic activities of the enzyme. In this regard, the single domain proteins, hA3A and hA3C, are notable for their inactivity or at best weak activity against HIV-1 (126, 127). In hA3G, both CDs are required for full antiviral activity, although they serve quite distinct functions. The N-terminal CD1 has been implicated in viral-RNA binding and virion encapsidation (51–53), whereas Cterminal CD2 confers deaminase activity (51– 54) and sequence speciﬁcity (42, 44, 45, 55– 57). This separation of functional domains is maintained in hA3B and hA3F, except the target-site preference for hypermutation conferred by CD2 differs from hA3G (5 -CC and 5 -TC are the favored dinucleotides for A3G and A3B/A3F deamination, respectively) (55– 57, 127). Conversely, in murine A3, the roles of these two domains are reversed; deamination is mediated by CD1, whereas encapsidation and dimerization are mediated by CD2 (128). Interestingly, the A3F enzymes from artiodactyls (cattle, pigs, and sheep) exhibit an enzymatically active CD1, which displays a broader dinucleotide deamination preference, and CD2, which confers deaminaseindependent antiviral activity (129). Because artiodactyls are positioned between rodents www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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and primates in most mammalian phylogenetic trees, artiodactyl A3F proteins likely represent evolutionary intermediates between the rodent and primate APOBEC3 enzymes (129). These ﬁndings highlight fascinating similarities and differences in this family of enzymes, including the emergence of double domain deaminases with specialized domain functions, the “ﬂip-ﬂopping” of these domain functions during evolution, and the development of different target site speciﬁcities characteristic of the various family members.

Tantalizing Insights into the Structure of APOBEC3 Deaminases Little is known about the structure of the APOBEC3 enzymes or how they engage their polynucleotide substrates. Two recent structures provide tantalizing insights. APOBEC2 corresponds to a single domain cytidine deaminase that is principally expressed in cardiac and skeletal muscle. Its function remains unknown. APOBEC2 is the closest known paralog of APOBEC3 for which a high-resolution structure is available (130). APOBEC2 crystallizes as a rod-shaped tetramer that can be viewed as two homodimers joined head to head and held together by extensive salt bridges, hydrogen bonding, and hydrophobic packing. Homodimers of APOBEC2 principally form as a result of hydrophobic beta-sheet interactions (130). The APOBEC2 dimer appears to be analogous to a monomer of APOBEC3, which contains two catalytic domains (131, 132). If a similar structural conﬁguration holds true for the APOBEC3 family members, this could explain how these family members can bind long polynucleotide substrates because their active sites are much more exposed than those of other known deaminases that act on free cytidine or cytosine. Small-angle X-ray scattering analysis of hA3G provides independent evidence for such an extended, symmetric structure (131), which could be a key to understanding the RNA-associated HMM hA3G complexes (72). Consistent with these mod334

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els of dimer interaction (130, 131), active APOBEC3 proteins, including hA3G, hA3F and hA3DE, have been reported to form homodimers and heterodimers in cells (57, 125). The crystal structure of APOBEC2 provides a template for constructing a model structure for hA3G (132). Mapping of functional and evolutionarily informative residues in hA3G CD1 demonstrates that critical residues cluster in well-deﬁned patches at the surface of the model. For example, amino acids 128–130 of hA3G are critical for its interaction with Vif, while R122 and W127 are required for efﬁcient encapsidation. Placing these ﬁndings in a detailed structural model suggests a surface hot-spot domain that includes R122, W127, and D128 and overlaps with a cluster of residues under positive selective pressure that includes T98, K99, R102, D128, and P129 (132). Strategies combining extensive site-directed mutagenesis and critical residue mapping with this structural model of full-length hA3G could speed rational antiviral drug design in the near future.

Diversified Antiviral Defense by APOBEC3 Family Members Several groups have worked to deﬁne the function and spectrum of antiviral activities of the various members of the human APOBEC3 family (Table 1). Apart from hA3G, several human APOBEC3 family members (e.g., hA3F, hA3B, and hA3DE) can inhibit HIV-1. Compared to hA3G, hA3F displays lethal, but less potent editing activity against vif HIV-1 (55–57) and is slightly less sensitive to the effects of Vif (55), possibly because a different binding interface is used (133, 134). Like hA3G, hA3F probably achieves its restrictive effect through a two-pronged sequential attack: initial impairment of the generation of reverse transcript products followed by mutational inactivation of successfully formed reverse transcripts (59, 135, 136). Human A3B also has moderate activity against HIV-1, but is quite resistant to Vif (56, 137). Although hA3B, hA3F,

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and hA3G all block HIV-1 replication when packaged into budding virions, only hA3F and hA3G are coordinately expressed in nonpermissive human T cell lines and tissues, including the primary cellular targets (CD4 T cells and macrophages) of HIV-1 (57); hA3B is barely detectable in human cell lines and tissues but is prevalent in various cancer cell lines (7, 137). Thus, A3G and A3F combine to form the dominant APOBEC3-mediate defense against HIV-1 in vivo. Like hA3G, hA3F also mediates postentry restriction of HIV-1. Although most recovered sequences are wild type, dG-to-dA hypermutations matching the recognized sequence preferences for both hA3G (CC) and hA3F (TC) are detectable in a minor subset of slowly formed reverse transcripts isolated from resting CD4 T cells infected with HIV1 (72). Furthermore, siRNA-mediated downregulation of hA3F expression in dendritic cells increases the permissiveness of these cells to HIV-1 infection (81). Human A3DE is the most recent APOBEC3 protein found to suppress HIV-1 infection (125). It is effectively encapsidated into budding virions and can mutate HIV-1 viral minus-strand DNA, albeit to a lesser extent than hA3G or hA3F. It preferentially deaminates AC, a dinucleotide motif that is distinct from that of other APOBEC3 members, but signatures of this type of mutation are evident in clinical HIV-1 isolates (125). In addition, hA3DE is more highly expressed than hA3F in biologically relevant cell targets such as CD4 T cells and macrophages (125). HIV is not the sole viral target of the APOBEC3 gene products. Indeed, each displays a different pattern of antiviral activity (Table 1). Human A3G actively suppresses the spread of HIV-1, SIV, and MLV; hA3F, hA3B, and hA3DE inhibit the replication of HIV-1 and SIV, but not MLV (56, 125, 137). Human A3C blocks SIV and HIV-1, although the anti-HIV effects are quite modest (127). Within the nucleus, hA3A blocks the replication of adeno-associated virus, which replicates as a single-stranded DNA in the nuclei of

infected cells (126). Strikingly, although failing to block HIV-1 replication when incorporated into vif HIV-1 virions, hA3A seems to be critical, likely acting in concert with hA3G, for restriction of HIV-1 infection in monocytes (82). In close parallel to the postentry restricting function of hA3G and hA3F against HIV-1, the deaminase-independent actions of hA3G, hA3F, hA3B, and hA3C sharply interfere with the HBV replication in cotransfected hepatoma cell lines. Mouse protein mA3 also restricts mouse mammary tumor virus, a betaretrovirus that replicates in lymphocytes, both in vitro and in vivo as highlighted by the increased vulnerability of mA3 knockout mice to MMTV infection (138).

Potential Roles of APOBEC3 Enzymes in HIV-1 Diversification and AIDS Progression Although current antiretroviral drugs are effective against AIDS, this may not be the case in the future. A major obstacle for controlling the spread of HIV/AIDS lies in the diversity and enormous evolutionary potential of the virus itself. Numerous HIV strains are contributing to the AIDS pandemic: two viral types (HIV-1 and HIV-2), 11 groups (M, N, and O for HIV-1 and A–H for HIV-2), and numerous subtypes, subsubtypes, and circulating recombinant forms have been described (99). The notorious genetic variation of HIV-1 has been attributed to a variety of factors, including (a) the high error rate of HIV-1 reverse transcriptase (approximately one nucleotide mistake per genome per replication cycle); (b) genomic recombination owing to the ability of reverse transcriptase to switch between the two virion-packaged viral RNA templates, coupled with the presence of multiple different proviruses in individually infected cells in vivo; and (c) the extensive and monotonous base substitutions of G to A (G-to-A hypermutations) that are evident in many viral genome sequences and account for 21% of the drug-resistance mutations against www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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Table 1

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Summary of restriction patterns by APOBEC proteinsa Other Viruses

Retroviruses

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HIV-1

SIVmac WT

SIVagm

WT

Δvif

APOBEC3A

−

−

APOBEC3B

+

+/−

+

+

+

+

+/−

+/−

APOBEC3C

−

+/−

−

+

−

+

+/−

−

APOBEC3DE

−

+/−

−

+

+

+

APOBEC3F

−

+

−

+

+

+

+/−

APOBEC3G Human

−

+

−

+

+

+

+

Chimpanzee

−

+

−

+

+

+

MAC

+

+

−

+

−

+

AGM

+

+

−

+

−

+

APOBEC3H

−

−

−

−

−

−

−

Mouse APOBEC3

+

+

+

+

+

+

−

Rat APOBEC1

+

+

Δvif

WT

PFV

Δvif

HTLV-1

EIAV

MLV

−

ΔBet

HBV

−

+ +

−

+

+/−

−

−

+

+

−

+

+

+

−

−

+

+

+

−

Subcellular Expression Profile

localization

LTR Retroelements IAP

MusD

Ty1

Non-LTR Retroelements L1

Alu

+

+

+

+

+

+

Keratinocytes, spleen, small intestine, monocytes, macrophages, colorectal adenocarcinoma, Burkitt’s lymphoma, chronic myelogenous leukemia

APOBEC3B

Keratinocytes, colon, small intestine, testis, ovary, stem cells, lung carcinoma, colorectal adenocarcinoma, Burkitt’s lymphoma, chronic myelogenous leukemia

N

+

+

APOBEC3C

Many tissues and a variety of cancer cell lines

N/C

+

+

+

+

APOBEC3DE

Many tissues

N/C

APOBEC3F

Many tissues and probably coexpressed with APOBEC3G

C

+

+

+

+/−

APOBEC3G Human Chimpanzee MAC AGM

Primarily expressed in the lymphoid and myeloid cell lineage, thymus, tonsil, bone marrow; also observed in testis, ovary, uterus, brain, heart, lung, liver, kidney, spleen, and pancreas, colorectal adenocarcinoma, and Burkitt’s lymphoma

C + + + +

+ − − +/−

+

− − − +/−

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−

APOBEC3A

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AAV

+

−

+

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APOBEC3H

Subcellular

LTR Retroelements

Expression Profile

localization

IAP

Peripheral blood lymphocytes, testis, ovary, fetal liver, skin, cerebellum, colon, small intestine

N/C

Mouse APOBEC3 Rat APOBEC1

C

MusD

Non-LTR Retroelements

Ty1

L1

+

+

−

+

N/C

a

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Alu

−

Abbreviations: +, restricted; −, not restricted; +/−, moderately restricted; blank, not determined. N, nuclear; C, cytoplasm; N/C, nucleo-cytoplasmic shuttling with primary signal retained in the nucleus; MAC/mac, Rhesus Macaque; AGM/agm, African green monkey; HIV, human immunodeﬁciency virus; SIV, simian immunodeﬁciency virus; HTLV-1, human T cell leukemia virus type-1; EIAV, equine infectious anemia virus; MLV, murine leukemia virus; PFV, primate foamy virus; HBV, hepatitis B virus; AAV, adeno-associated virus.

currently available protease and reverse transcriptase inhibitors (139). G-to-A hypermutation was ﬁrst noted in the HIV-1 env gene during propagation of HIV-1 virus in vitro (140). Sporadic G-to-A hypermutation sequences in clinical samples are also readily detected in multiple regions of the viral genome (141–145). These altered sequences seem to follow a speciﬁc pattern whereby hypermutation increases progressively from the 5 end of the genome to the central polypurine tract (PPT), then declines, and increases again in sequences preceding the 3 PPT (144). As shown by sequence analysis of these hypermutated HIV genomes, G-to-A transitions occur preferentially in a GA and GG dinucleotide context (140–145). Initially, this pattern was proposed to reﬂect dislocation mutagenesis in the case of GA mutations (140) and base misincorporation in the GG context, possibly stemming from suboptimal dCTP concentrations (142). The most prominent dinucleotide context (GG and GA) of mutations in the plus strand of the HIV-1 genome is consistent with the site preferences (CC and TC) for hA3G and hA3F, respectively, within the minus strand of proviral DNA. These ﬁndings suggest that G-to-A hypermutations might, in fact, result from the antiviral activity of different APOBEC3 family members (54). However, the signiﬁcance of such APOBEC3-mediated hypermutation in clinical HIV pathogenesis

remains uncertain. Nevertheless, it is striking that up to 60% of the guanines within speciﬁc segments of the HIV-1 genome are mutated. Massively edited genomes are frequently nonviable because they contain many missense and nonsense codon changes. Indeed, in a population-based study of HIV-1 proviral DNA sequences, G-to-A hypermutation conforming to expected hA3G and hA3F sequence preferences appears to be independently associated with at least a 0.7-log reduction in pretreatment viral load (146). Hypermutated viruses are also associated with slower disease progression and become predominant over time in long-term nonprogressive infection (147, 148), implying a protective role for such hypermutation. Genetic variation in APOBEC3 genes or critical cellular regulators (e.g., those involved in polyubiquitination or protein degradation) and natural variations in the expression levels of APOBEC3 proteins may impact HIV1 pathogenesis, as suggested by three recent reports. First, in an analysis of more than 3000 subjects, single nucleotide polymorphisms were identiﬁed in the hA3G gene. One of these codon-changing variants, H186R in exon 4, was polymorphic (∼37%) in African Americans, and the 186R/R genotype was strongly associated with more rapid decline in CD4 T cell numbers and accelerated progression to AIDS in HIV-infected individuals (149). www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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Retrotransposition: an intracellular process utilized by retroelements to replicate through reverse transcription of RNA intermediates and integration of the retroelement DNA into host cell genomes. Long interspersed nuclear element (LINE): an autonomous non-LTR retroelement with coding capacity for the protein machinery required for catalyzing its own retrotransposition Short interspersed nuclear element (SINE): a non-LTR retroelement that is dependent on LINE reverse transcription machinery for its retrotransposition

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Second, 12 single nucleotide polymorphisms of the Cullin5 gene were examined in ﬁve longitudinal cohorts of HIV-infected patients and grouped into two clusters of related haplotypes. Six major haplotypes in cluster II were linked with more rapid CD4 T cell loss and HIV-1 disease progression (150). Interestingly, hA3G-186R and Cullin5 haplotypes likely confer independent and additive effects on the rate of CD4 T cell loss (149, 150). Third, a recent study suggests a highly signiﬁcant inverse correlation between hA3G mRNA levels in peripheral blood mononuclear cells and levels of plasma HIV-1 RNA and CD4 T cell counts, both of which are predictors of HIV disease progression in treatment-naive patients. Furthermore, hA3G mRNA levels appear higher in longterm nonprogressors than in HIV-uninfected subjects and lowest in individuals with progressive HIV disease (151). Therefore, it is highly likely that human APOBEC3 proteins, particularly hA3G and hA3F, confer an innate antiviral resistance that is clinically and biologically relevant. These effects of the APOBEC3 proteins likely modify both the efﬁciency of HIV-1 transmission and the tempo of disease progression. Do G-to-A mutations induced by the innate APOBEC3 host-defense system actively contribute to genetic variation in HIV1? Because drug-resistant strains of HIV1 often exhibit G-to-A transitions in their genomic sequences (139), it is certainly conceivable that APOBEC3-mediated hypermutation importantly contributes to the evolution of such drug resistance mutations. The effects of hA3G are likely dose-dependent, because moderately elevated levels of hA3G are associated with occasional G-to-A transitions in the wild-type virus (23). These ﬁndings illustrate how the defensive actions of the APOBEC3, if incomplete, could undermine the effectiveness of current antiretroviral therapies. It is also possible that natural variations in Vif function may result in selective or parChiu

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tial neutralization of these APOBEC3 cytidine deaminases, thereby promoting viral sequence diversiﬁcation in HIV-1-infected persons. Indeed, defective vif alleles that cannot neutralize hA3G or hA3F are frequently detected in HIV-1 isolates and in infected patients (88). Concordantly, independently hypermutated proviruses with distinguishable patterns of G-to-A substitution attributable to deamination induced by hA3G, hA3F, or both are detectable in subjects carrying proviruses with completely or partly defective Vif variants (88). These studies suggest that sporadic inactivation of Vif likely occurs rather frequently in vivo. These apparent ﬂaws in the Vif counterstrike may be exploited by HIV-1 to accelerate viral sequence diversiﬁcation and evolution, enabling escape from both speciﬁc immune responses or antiviral drugs.

PARTICIPATION OF APOBEC3 ENZYMES IN THE CONTROL OF ENDOGENOUS RETROELEMENTS Mammalian Endogenous Retroelements The mammalian genome harbors many thousands of mobile genetic elements, including distinct families of DNA transposons and retrotransposons (123). Retrotransposons are mobile DNA sequences that integrate into the genome of host cells. New copies are generated by coupled transcription and reverse transcription followed by insertion of the new DNA into different sites in the genome. This process is termed retrotransposition as it does not involve the release of infectious particles. The major classes of endogenous retroelements in mammalian cells include elements that contain long terminal repeats (LTRs), such as endogenous retroviruses, and non-LTR retroelements, such as long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) (123). These retroelements are believed to have played an important role in evolution and

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speciation. However, mobilization of these elements can also be deleterious to the host cell, resulting in various genetic disorders and cancers. To limit the negative effects of retrotransposition, cells have adopted several strategies to restrict the mobility of transposable elements. Human endogenous retroviruses (HERVs) and many other non-LTR retroelements have been rendered replicationincompetent owing to the accumulation of inactivating mutations, promoter CpG methylation, or small RNA-mediated silencing (123, 152). However, some mammalian species exhibit more active retroelements. For example, although endogenous retroelements are four- to ﬁve-fold more abundant in the human genome than in the mouse genome (42% versus 8%–10%), retrotransposition activity is 100-fold higher in mouse cells (152). In mice, even endogenous retroviruses, including murine intracisternal A-particle (IAP) and MusD sequences, remained highly mobile. The singularly lower levels of retrotransposon activity found in primates intriguingly coincides with the expansion of the APOBEC3 gene cluster from a single gene in mice to seven genes in primates (7). The sequence variability of primate APOBEC3 indicates that this gene cluster has been subjected to strong positive selective pressure for more than 30 million years (153, 154). This selective pressure clearly antedates the appearance of the modern lentiviruses (∼1 Mya), further strengthening the notion that this gene family expanded in order to curtail the genomic instability caused by endogenous retroelements.

APOBEC3 Deaminases as Inhibitors of LTR Retrotransposons APOBEC3 proteins do in fact function as inhibitors of LTR retrotransposons. Human A3B, hA3C, hA3F, hA3G, and mA3 all effectively inhibit mouse IAP and MusD elements (126, 155, 156), whereas hA3C, hA3F, and hA3G inhibit the yeast Ty1 retrotrans-

poson (157, 158). APOBEC3 proteins exert dual inhibitory effects on these endogenous retroviruses, involving both a decrease in the number of transposed cDNA copies and extensive editing of the transposed copies (155, 159). These effects are reminiscent of the dual effects of hA3G in HIV-1 replication. In the mouse genome, many retrotransposon proviruses bear mutations consistent with APOBEC3-mediated deamination (155, 159). Interestingly, hA3A effectively inhibits IAP and MusD retrotransposition through a novel deamination-independent mechanism (156). Although none of the HERVs currently present in the human genome are replication competent, a pseudoancestral HERV-K DNA sequence was recently reconstructed based on the fossil record of ancient endogenous retroviruses. Of note, this reconstituted human retrovirus is effectively restricted by hA3F but not by hA3G (160).

HERV: human endogenous retrovirus

APOBEC3 Deaminases as Inhibitors of Non-LTR Retrotransposons What about the non-LTR retroelements that are continuing to retrotranspose in the human genome? These retroelements can be subdivided into autonomous LINEs (L1 being the most common), nonautonomous SINEs (Alu being the most prevalent), and processed pseudogenes (123). The average human diploid genome contains 80–100 active L1 retroelements, which encode the enzymes needed for their own retrotransposition. Alu elements constitute ∼10% of the human genome. These small retroelements lack protein-coding capacity but retrotranspose by “stealing” the reverse transcriptase/endonuclease enzymes encoded by the L1 open reading frame (ORF) 2 (123, 161). The replication cycle of L1 and Alu involves cytoplasmic RNA intermediates, reverse transcription in the nucleus, and integration of the newly formed DNA at novel chromosomal sites (Figure 6). Human APOBEC3 proteins, including hA3A, hA3B, hA3C, and hA3F, effectively inhibit L1 retroelements (70, 126, www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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b

LINE 1 (L1) (6 kb) 5' UTR ORF1

ORF2

SINE (Alu) (100–300 bp)

3' UTR AAAAAA

A B

AAAAAA

Human genome AAAAAA

Pol II-mediated transcription

AAAAAA

APOBEC3A/3B/3C

Integration

AAAAA

L1 RNA

AAAAAA

Pol II-mediated transcription L1 RNA

Pol III-mediated transcription Alu RNA

AAAA A

AAA AA

ORF2 AA AA A

AAAA

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Export

L1 ORF2-mediated reverse transcription

A

Nuclear pore

AAAA

ORF2 A AAA

A AAA

ORF2

Export

AA

A

Export

Import N U C L EU S

Translation

C YTOPLA YTO PLA SM SM AAAAA

L1 RNA 5'

ORF ORF1

L1 RNA 5'

ORF1

ORF2 L1 RNP A A AA A

assembly

Translation L1 RNA 5'

A A

AAAAA

APOBEC3B

Alu RNA AAA U UU

APOBEC3G

APOBEC3F ORF2 ORF1 ORF2

HMM APOBEC3G

Figure 6 Human APOBEC3 protects cells from the potentially detrimental effects of non-LTR retroelements. (a) Human APOBEC3 proteins, including hA3A, hA3B, hA3C, and hA3F, inhibit human LINE (long interspersed nuclear elements)-1 (L1) retroelements. Functional L1 elements are 6-kb long autonomous retroelements that contain an internal RNA polymerase II (Pol II) promoter within their 5 UTR, two open reading frames (ORF1 and ORF2), and a 3 UTR. L1 retroelements are characterized by ﬂanking target site duplications (short black arrows) that vary in length (usually 9–20 nucleotides) and by the presence of a poly(A)-rich tail. L1 retrotransposition involves the initial production of cytoplasmic RNA intermediates that in turn are reverse transcribed by ORF2 in the nucleus, and integration occurs at new genetic sites to complete the retrotransposition cycle. A number of human APOBEC3 proteins, including hA3A, hA3B, and hA3C, have been reported to inhibit retrotransposition of human L1 retroelements, reﬂecting their ability to enter the nucleus, where L1 reverse transcription and integration occur. Interactions of hA3B and hA3F with the L1 ORF2 protein have been suggested. (b) Human A3G proteins inhibit L1-dependent Alu retrotransposition through a novel nonenzymatic inhibitory mechanism directly targeting Alu RNA transcripts. Short interspersed nuclear elements (SINEs), including the most prominent and active member Alu, correspond to short RNA polymerase III (Pol III)-transcribed retroelements that contain an internal promoter but no protein coding capacity. Successful retrotransposition of Alu elements depends on their ability to “steal” the reverse transcriptase/endonuclease enzymes encoded by L1 ORF2. Human A3G impairs the retrotransposition of Alu by sequestering Alu RNA transcripts in the cytoplasmic HMM complexes, especially Staufen-containing RNA granules, away from the nuclear L1 machinery, thereby interdicting the retrotransposition cycle. Human A3A, hA3B, and, to a lesser extent, hA3C also inhibit Alu retrotransposition, although in this case the A3 proteins target the L1 machinery required for Alu retrotransposition (see panel a).

156, 162); hA3A, hA3B, and to a lesser extent hA3C also inhibit L1-mediated Alu retrotransposition (156). This ﬁnding could reﬂect the ability of these deaminases to en340

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ter the nucleus (126, 156), where L1 reverse transcription occurs. Human A3B and A3F may also directly interact with the L1 ORF2 protein through a highly homologous region

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present in these two deaminases. Of note, this domain is not present in hA3G (162) (Figure 6). Notably, hA3G does not affect the retrotransposition of L1 (70, 126, 155, 156, 162, 163), but it greatly inhibits L1-dependent retrotransposition of Alu retroelements (116, 164) (Figure 6). Catalytically inactive mutants of hA3G also effectively inhibit Alu retrotransposition, suggesting an enzymeindependent mechanism for hA3G action (116, 164). Experimentally, hA3G sequesters Alu RNAs in cytoplasmic HMM complexes, particularly Staufen-containing RNA granules, denying these retroelements access to the nuclear L1 machinery. These effects appear to explain how hA3G interdicts the Alu retrotransposition cycle (116). This inhibitory mechanism does not involve editing of the Alu RNA and also differs from hA3A- and hA3B-mediated inhibition of Alu retrotransposition, where the APOBEC3 proteins alter the activity of the L1 machinery in the nucleus. The lack of inhibitory effect of hA3G on autonomous L1 may reﬂect efﬁcient assembly of retrotransposing L1 RNA with its catalytic machinery encoded by the same RNA, a phenomenon termed cis preference, which minimizes hA3G’s opportunity to act. Although retrotransposition events occasionally occur in speciﬁc somatic cell types, L1 and Alu retrotransposition are most active in germ cells and during early embryogenesis, where extensive genome demethylation prevails and retroelements are thought to be transcribed and expressed (123, 152). Novel heritable insertions of L1 and Alu occur in ∼1 in 50 and ∼1 in 250 live human births, respectively (123, 152). Such retrotransposition events may also contribute to spontaneous abortions. The relatively high-level expression of APOBEC3 proteins in human testis and ovary (hA3G, hA3F, and hA3C) (7, 12) and embryonic stem cells (hA3B) (156) points to a physiologically relevant role for these DNA deaminases in the protection of these cells from the potentially deleterious effects of endogenous retroelement retrotransposi-

tion. The APOBEC3 family likely evolved in uniquely different ways to defend the integrity of the human genome against the attack leveled by various classes of endogenous retroelements.

THERAPEUTIC PERSPECTIVES AND FUTURE DIRECTIONS Vif Antagonists as Novel Therapeutics Advances in APOBEC3 biology provide a number of attractive targets for the development of novel anti-HIV-1 drugs. Enhancing hA3G/hA3F activities and inhibiting Vif function are attractive therapeutic strategies. Progress in deciphering how Vif counteracts hA3G action suggests various possibilities for developing a new class of anti-HIV drugs. Many HIV researchers regard the Vif-hA3G axis as the most attractive new drug target to emerge since the identiﬁcation of the HIV coreceptors. The ﬁrst and perhaps most attractive approach would be to identify smallmolecule inhibitors that selectively disrupt the binding of HIV-1 Vif to hA3G proteins. Such inhibitors would prevent Vif-mediated degradation of hA3G, promoting its effective encapsidation. Although these inhibitors would necessarily have to interfere with a proteinprotein interface, the fact that the D128K mutation completely blocks HIV-1 Vif binding is encouraging. Another point of attack might include blocking Vif recruitment of the E3 ligase complex by interfering with the binding of Cullin5 or Elongin C to Vif. However, a potential drawback to this approach is that Vif would still bind to hA3G and might be coencapsidated, with uncertain effects on A3G antiviral activity.

Potential Strategies to Boost the Expression of APOBEC3 Deaminases Identifying the signaling pathways that lead to APOBEC3 expression might be www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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therapeutically valuable if APOBEC3 levels could be safely boosted beyond the capacity of Vif to deplete the enzyme. For example, hA3G is present at low levels in resting T cells but is strongly induced when these cells are activated by phytohemagglutinin and IL2 (24). PMA stimulation of H9 T cells activates hA3G gene transcription via the MAP kinase signaling pathway (165). It might be possible to use cytokines (IL-2, IL-7, and IL15) to elevate the levels of hA3G/hA3F beyond the capacity of Vif to deplete the intracellular stores of these deaminases (77). Of note, interferon-α (IFN-α) treatment of macrophages markedly upregulates hA3G and A3A expression, and the induction of hA3G appears to be a major component of the IFN-α-induced anti-HIV response in these cells (82, 166). However, an appreciation of the respective contributions of these potential beneﬁcial effects must be weighed against the possible role of APOBEC3 enzymes in promoting viral diversity and drug resistance as well a mutation of the host genome. Additional information concerning the factors that regulate the levels of APOBEC3 expression might also provide a means to counteract Vif-mediated degradation. The speciﬁc enrichment of hA3G/hA3F mRNA in HMM hA3G complexes, particularly Staufen-containing RNA granules (116), along with translation initiation and elongation factors, provides an attractive mechanism to explain how destruction of the HMM complex by Vif (72) might impair de novo synthesis of hA3G (23, 24). Together with the proposed association of hA3G with SGs and PBs in response to the addition of translational inhibitors and agents that induce cellular stress (69, 71, 117), these ﬁndings suggest that the intracellular signals and molecular interactions governing the inducible formation, dynamic spatial localization, and controlled disassembly of RNA granules and related cytoplasmic RNA structures could serve as major regulatory switches controlling the expression and antiviral activity of hA3G/hA3F in human cells.

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The signiﬁcant upregulation of APOBEC3 (hA3G, hA3F, and hA3B) protein expression that occurs in primary hepatocytes in response to IFN-α provides a possible mechanism for interferon-mediated noncytolytic clearance of HBV (67). A better understanding of the potential participation of APOBEC3 proteins in this response could facilitate the identiﬁcation of new therapeutic strategies for HBV. Furthermore, population genetic analyses indicate that the hA3B gene is inﬂuenced by a 29.5-kb deletion polymorphism in the human genome and that the frequency varies signiﬁcantly among major ethnic groups (167). Because expression of hA3B in primary hepatocytes can be induced by IFN-α treatment, it will be interesting to determine whether the hA3B polymorphism correlates with the susceptibility of different human populations to infection with HBV or other hepadnaviruses.

Enforcing the Postentry Antiviral Activity of APOBEC3G The discovery of LMM hA3G as a postentry restriction factor in resting CD4 T cells and in cells within the monocyte lineage suggests it might be possible to recapitulate this activity or prevent its inactivation in cells normally permissive of HIV-1 infection (72, 80, 81, 166). A hA3G mutant constitutively residing in the LMM form, even after T cell activation or monocyte differentiation, could provide early intracellular resistance against incoming viruses. To avoid promiscuous editing of host genomic DNA, it will be important to determine whether catalytically inactive LMM hA3G can also mediate this postentry block. The conversion of LMM to HMM complexes after T cell activation merits careful assessment in terms of the signals and molecular pathways that induce this response. For example, cytokine stimulation appears to promote the assembly of hA3G into enzymatically inactive HMM complexes and thus creates a permissive state for HIV-1 in resting T cells in the absence of full-ﬂedged cellular

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activation and proliferation (76, 77). Similarly, it has been proposed that IFN-α locks hA3G in an LMM complex in activated CD4 T cells and confers anti-HIV-1 activity (168). Insights into the protein and RNA cofactors in HMM hA3G and the factors that govern complex assembly will undoubtedly be important for developing strategies to block HMM hA3G complex formation without compromising the ability of these HMM hA3G complexes to restrict endogenous retroelements retrotransposition.

Other Questions Meriting Further Investigation In summary, APOBEC3 proteins inhibit a range of exogenous viruses and endogenous mobile retroelements by multiple mechanisms. Many basic questions remain to be explored. The antiviral mechanisms of the APOBEC3 family appear to involve their intrinsic RNA binding and deaminase activities, yet uncertainty persists regarding the relative role and effectiveness of each. It is also unclear whether the mechanism employed by Vif

to counteract hA3G/hA3F activity solely involves protein degradation and virion exclusion. Neither is it known whether different APOBEC genotypes correlate with susceptibility to HIV infection and progression to AIDS or whether endogenous retroelements such as Alu and hY RNAs are the nucleating factors for HMM hA3G complex assembly and, if they are, whether downregulation of these endogenous retroelements would help to preserve hA3G in LMM form, thereby conferring potent postentry restricting activity even in activated CD4 T cells. Finally, the mobility of L1 and Alu elements often results in both insertional and postinsertional mutagenesis, which is commonly associated with cancer and leukemia development. It remains unclear whether, when, or how APOBEC3 proteins curtail such events in vivo and whether these proteins really function as inhibitors of tumorigenesis. Studies addressing these topics are urgently needed to derive clear insights into the exact relevance of the APOBEC3 family in retroviral pathogenesis in vivo and its potential role in maintaining host genomic stability.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We wish to acknowledge the outstanding contributions from various laboratories in the APOBEC ﬁeld that we were unable to cite owing to space limitations. Research in our laboratory is supported by funding from the National Institutes of Health Grants R01 AI065329–01 (to W.C.G.) and RR18928–01; the San Francisco Women’s HIV Interdisciplinary Network (National Institutes of Health Grant P01 HD40543 to W.C.G.); American Foundation for AIDS Research Grant 10652535-RFHF (to Y.-L.C.); and University of California San Francisco–Gladstone Institute of Virology and Immunology Center for AIDS Research Grant AI0277635P30GY. We thank John Carroll for assistance with graphics, Gary Howard and Stephen Ordway for editorial assistance, and Mr. Robin Givens for administrative support.

LITERATURE CITED 1. Gabuzda DH, Lawrence K, Langhoff E, Terwilliger E, Dorfman T, et al. 1992. Role of vif in replication of human immunodeﬁciency virus type 1 in CD4+ T lymphocytes. J. Virol. 66:6489–95 www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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2. Sova P, Volsky DJ. 1993. Efﬁciency of viral DNA synthesis during infection of permissive and nonpermissive cells with vif-negative human immunodeﬁciency virus type 1. J. Virol. 67:6322–26 3. von Schwedler U, Song J, Aiken C, Trono D. 1993. Vif is crucial for human immunodeﬁciency virus type 1 proviral DNA synthesis in infected cells. J. Virol. 67:4945–55 4. Madani N, Kabat D. 1998. An endogenous inhibitor of human immunodeﬁciency virus in human lymphocytes is overcome by the viral Vif protein. J. Virol. 72:10251–55 5. Simon JH, Gaddis NC, Fouchier RA, Malim MH. 1998. Evidence for a newly discovered cellular anti-HIV-1 phenotype. Nat. Med. 4:1397–400 6. Sheehy AM, Gaddis NC, Choi JD, Malim MH. 2002. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646–50 7. Jarmuz A, Chester A, Bayliss J, Gisbourne J, Dunham I, et al. 2002. An anthropoid-speciﬁc locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics 79:285–96 8. Teng B, Burant CF, Davidson NO. 1993. Molecular cloning of an apolipoprotein B messenger RNA editing protein. Science 260:1816–19 9. Harris RS, Petersen-Mahrt SK, Neuberger MS. 2002. RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators. Mol. Cell 10:1247–53 10. Conticello SG, Thomas CJ, Petersen-Mahrt SK, Neuberger MS. 2005. Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases. Mol. Biol. Evol. 22:367–77 11. Rogozin IB, Basu MK, Jordan IK, Pavlov YI, Koonin EV. 2005. APOBEC4, a new member of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases predicted by computational analysis. Cell Cycle 4:1281–85 12. OhAinle M, Kerns JA, Malik HS, Emerman M. 2006. Adaptive evolution and antiviral activity of the conserved mammalian cytidine deaminase APOBEC3H. J. Virol. 80:3853– 62 13. Espinosa R III, Funahashi T, Hadjiagapiou C, Le Beau MM, Davidson NO. 1994. Assignment of the gene encoding the human apolipoprotein B mRNA editing enzyme (APOBEC1) to chromosome 12p13.1. Genomics 24:414–15 14. Muto T, Muramatsu M, Taniwaki M, Kinoshita K, Honjo T. 2000. Isolation, tissue distribution, and chromosomal localization of the human activation-induced cytidine deaminase (AID) gene. Genomics 68:85–88 15. Mehta A, Kinter MT, Sherman NE, Driscoll DM. 2000. Molecular cloning of apobec1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA. Mol. Cell Biol. 20:1846–54 16. Yamanaka S, Balestra ME, Ferrell LD, Fan J, Arnold KS, et al. 1995. Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals. Proc. Natl. Acad. Sci. USA 92:8483–87 17. Muramatsu M, Sankaranand VS, Anant S, Sugai M, Kinoshita K, et al. 1999. Speciﬁc expression of activation-induced cytidine deaminase (AID), a novel member of the RNAediting deaminase family in germinal center B cells. J. Biol. Chem. 274:18470–76 18. Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553–63 19. Rada C, Williams GT, Nilsen H, Barnes DE, Lindahl T, Neuberger MS. 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deﬁcient mice. Curr. Biol. 12:1748–55

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129. Jonsson SR, Hache G, Stenglein MD, Fahrenkrug SC, Andresdottir V, Harris RS. 2006. Evolutionarily conserved and nonconserved retrovirus restriction activities of artiodactyl APOBEC3F proteins. Nucleic Acids Res. 34:5683–94 130. Prochnow C, Bransteitter R, Klein MG, Goodman MF, Chen XS. 2007. The APOBEC-2 crystal structure and functional implications for the deaminase AID. Nature 445:447–51 131. Wedekind JE, Gillilan R, Janda A, Krucinska J, Salter JD, et al. 2006. Nanostructures of APOBEC3G support a hierarchical assembly model of high molecular mass ribonucleoprotein particles from dimeric subunits. J. Biol. Chem. 281:38122–26 132. Zhang KL, Mangeat B, Ortiz M, Zoete V, Trono D, et al. 2007. Model structure of human APOBEC3G. PLoS ONE 2:e378 133. Tian C, Yu X, Zhang W, Wang T, Xu R, Yu XF. 2006. Differential requirement for conserved tryptophans in human immunodeﬁciency virus type 1 Vif for the selective suppression of APOBEC3G and APOBEC3F. J. Virol. 80:3112–15 134. Russell RA, Pathak VK. 2007. Identiﬁcation of two distinct HIV-1 Vif determinants critical for interactions with human APOBEC3G and APOBEC3F. J. Virol. 81:8201–10 135. Holmes RK, Koning FA, Bishop KN, Malim MH. 2007. APOBEC3F can inhibit the accumulation of HIV-1 reverse transcription products in the absence of hypermutation. Comparisons with APOBEC3G. J. Biol. Chem. 282:2587–95 136. Yang Y, Guo F, Cen S, Kleiman L. 2007. Inhibition of initiation of reverse transcription in HIV-1 by human APOBEC3F. Virology 365:92–100 137. Doehle BP, Schafer A, Cullen BR. 2005. Human APOBEC3B is a potent inhibitor of HIV-1 infectivity and is resistant to HIV-1 Vif. Virology 339:281–88 138. Okeoma CM, Lovsin N, Peterlin BM, Ross SR. 2007. APOBEC3 inhibits mouse mammary tumour virus replication in vivo. Nature 445:927–30 139. Berkhout B, De Ronde A. 2004. APOBEC3G vs reverse transcriptase in the generation of HIV-1 drug-resistance mutations. AIDS 18:1861–63 140. Vartanian JP, Meyerhans A, Asjo B, Wain-Hobson S. 1991. Selection, recombination, and G–>A hypermutation of human immunodeﬁciency virus type 1 genomes. J. Virol. 65:1779–88 141. Fitzgibbon JE, Mazar S, Dubin DT. 1993. A new type of G–>A hypermutation affecting human immunodeﬁciency virus. AIDS Res. Hum. Retroviruses 9:833–38 142. Vartanian JP, Meyerhans A, Sala M, Wain-Hobson S. 1994. G–>A hypermutation of the human immunodeﬁciency virus type 1 genome: evidence for dCTP pool imbalance during reverse transcription. Proc. Natl. Acad. Sci. USA 91:3092–96 143. Borman AM, Quillent C, Charneau P, Kean KM, Clavel F. 1995. A highly defective HIV-1 group O provirus: evidence for the role of local sequence determinants in G–>A hypermutation during negative-strand viral DNA synthesis. Virology 208:601–9 144. Janini M, Rogers M, Birx DR, McCutchan FE. 2001. Human immunodeﬁciency virus type 1 DNA sequences genetically damaged by hypermutation are often abundant in patient peripheral blood mononuclear cells and may be generated during near-simultaneous infection and activation of CD4+ T cells. J. Virol. 75:7973–86 145. Kieffer TL, Kwon P, Nettles RE, Han Y, Ray SC, Siliciano RF. 2005. G–>A hypermutation in protease and reverse transcriptase regions of human immunodeﬁciency virus type 1 residing in resting CD4+ T cells in vivo. J. Virol. 79:1975–80 146. Pace C, Keller J, Nolan D, James I, Gaudieri S, et al. 2006. Population level analysis of human immunodeﬁciency virus type 1 hypermutation and its relationship with APOBEC3G and vif genetic variation. J. Virol. 80:9259–69 www.annualreviews.org • The APOBEC3 Cytidine Deaminases

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147. Wang B, Mikhail M, Dyer WB, Zaunders JJ, Kelleher AD, Saksena NK. 2003. First demonstration of a lack of viral sequence evolution in a nonprogressor, deﬁning replication-incompetent HIV-1 infection. Virology 312:135–50 148. Wei M, Xing H, Hong K, Huang H, Tang H, et al. 2004. Biased G-to-A hypermutation in HIV-1 proviral DNA from a long-term nonprogressor. AIDS 18:1863–65 149. An P, Bleiber G, Duggal P, Nelson G, May M, et al. 2004. APOBEC3G genetic variants and their inﬂuence on the progression to AIDS. J. Virol. 78:11070–76 150. An P, Duggal P, Wang LH, O’Brien SJ, Donﬁeld S, et al. 2007. Polymorphisms of CUL5 are associated with CD4+ T cell loss in HIV-1 infected individuals. PLoS Genet. 3:e19 151. Jin X, Wu H, Smith H. 2007. APOBEC3G levels predict rates of progression to AIDS. Retrovirology 4:20 152. Maksakova IA, Romanish MT, Gagnier L, Dunn CA, van de Lagemaat LN, Mager DL. 2006. Retroviral elements and their hosts: insertional mutagenesis in the mouse germ line. PLoS Genet. 2:e2 153. Sawyer SL, Emerman M, Malik HS. 2004. Ancient adaptive evolution of the primate antiviral DNA-editing enzyme APOBEC3G. PLoS Biol. 2:e275 154. Zhang J, Webb DM. 2004. Rapid evolution of primate antiviral enzyme APOBEC3G. Hum. Mol. Genet. 13:1785–91 155. Esnault C, Heidmann O, Delebecque F, Dewannieux M, Ribet D, et al. 2005. APOBEC3G cytidine deaminase inhibits retrotransposition of endogenous retroviruses. Nature 433:430–33 156. Bogerd HP, Wiegand HL, Doehle BP, Lueders KK, Cullen BR. 2006. APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells. Nucleic Acids Res. 34:89–95 157. Dutko JA, Schafer A, Kenny AE, Cullen BR, Curcio MJ. 2005. Inhibition of a yeast LTR retrotransposon by human APOBEC3 cytidine deaminases. Curr. Biol. 15:661–66 158. Schumacher AJ, Nissley DV, Harris RS. 2005. APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast. Proc. Natl. Acad. Sci. USA 102:9854–59 159. Esnault C, Millet J, Schwartz O, Heidmann T. 2006. Dual inhibitory effects of APOBEC family proteins on retrotransposition of mammalian endogenous retroviruses. Nucleic Acids Res. 34:1522–31 160. Lee YN, Bieniasz PD. 2007. Reconstitution of an infectious human endogenous retrovirus. PLoS Pathog. 3:e10 161. Dewannieux M, Esnault C, Heidmann T. 2003. LINE-mediated retrotransposition of marked Alu sequences. Nat. Genet. 35:41–48 162. Stenglein MD, Harris RS. 2006. APOBEC3B and APOBEC3F inhibit L1 retrotransposition by a DNA deamination-independent mechanism. J. Biol. Chem. 281:16837–41 163. Turelli P, Vianin S, Trono D. 2004. The innate antiretroviral factor APOBEC3G does not affect human LINE-1 retrotransposition in a cell culture assay. J. Biol. Chem. 279:43371– 73 164. Hulme AE, Bogerd HP, Cullen BR, Moran JV. 2007. Selective inhibition of Alu retrotransposition by APOBEC3G. Gene 390:199–205 165. Rose KM, Marin M, Kozak SL, Kabat D. 2004. Transcriptional regulation of APOBEC3G, a cytidine deaminase that hypermutates human immunodeﬁciency virus. J. Biol. Chem. 279:41744–79 166. Peng G, Lei KJ, Jin W, Greenwell-Wild T, Wahl SM. 2006. Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti-HIV-1 activity. J. Exp. Med. 203:41–46

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167. Kidd JM, Newman TL, Tuzun E, Kaul R, Eichler EE. 2007. Population stratiﬁcation of a common APOBEC gene deletion polymorphism. PLoS Genet. 3:e63 168. Chen K, Huang J, Zhang C, Huang S, Nunnari G, et al. 2006. Alpha interferon potently enhances the antihuman immunodeﬁciency virus type 1 activity of APOBEC3G in resting primary CD4 T cells. J. Virol. 80:7645–57

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:317-353. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Thymus Organogenesis Annu. Rev. Immunol. 2008.26:355-388. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Hans-Reimer Rodewald Institute for Immunology, University of Ulm, D-89070 Ulm, Germany; email: [email protected]

Annu. Rev. Immunol. 2008. 26:355–88

Key Words

First published online as a Review in Advance on November 30, 2007

thymus medulla, thymus cortex, germ layers, thymus epithelial stem and progenitor cells, transcription factors, cervical thymus, reaggregate organ cultures, Cre recombinase, fate mapping, nude blastocyst complementation

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090408 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0355$20.00

Abstract The epithelial architecture of the thymus fosters growth, differentiation, and T cell receptor repertoire selection of large numbers of immature T cells that continuously feed the mature peripheral T cell pool. Failure to build or to maintain a proper thymus structure can lead to defects ranging from immunodeﬁciency to autoimmunity. There has been long-standing interest in unraveling the cellular and molecular basis of thymus organogenesis. Earlier studies gave important morphological clues on thymus development. More recent cell biological and genetic approaches yielded new and conclusive insights regarding the germ layer origin of the epithelium and the composition of the medulla as a mosaic of clonally derived islets. The existence of epithelial progenitors common for cortex and medulla with the capacity for forming functional thymus after birth has been uncovered. In addition to the thymus in the chest, mice can have a cervical thymus that is small, but functional, and produces T cells only after birth. It will be important to elucidate the pathways from putative thymus stem cells to mature thymus epithelial cells, and the properties and regulation of these pathways from ontogeny to thymus involution.

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INTRODUCTION

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The classical embryological view of thymus development that had been perpetuated for decades has undergone major revisions within the past few years. Advancing the understanding of thymus organ development has been slow by comparison to the progress in research addressing cellular pathways and underlying molecular mechanisms of developing T cells. Why are experiments addressing the development of the thymus interesting? The unique function of the thymus in establishment and maintenance of the T cell arm of the immune system is intimately linked to specialized functions of thymus stromal cells and the thymus architecture (1, 2). These cells are of major immunological relevance for the intrathymic selection of a selftolerant and self-MHC-restricted T cell antigen receptor (TCR) repertoire (3–6). It is broadly recognized that thymus structure is key to these speciﬁc immunological properties of the thymus. This, combined with the realization that thymus structure and its development are, in large part, still uncharted territory, has spurred increased interest in thymus organogenesis. Novel experimental access has been facilitated by the development of experimental tools such as, to name a few, stromal cell isolation by phenotype-based cell sorting (e.g., 7– 9), dissociation and reaggregation of stromal cell subsets to probe their functional capacity in vitro (10, 11) and in vivo (e.g., 12–15), or global gene expression analyses to determine the pattern of self-antigen expression in thymus epithelial cell (TEC) subsets (5). Key questions in thymus organogenesis surround the fundamental cellular and molecular mechanisms that lead to the formation of a normal thymus. Areas of interest include the transient or permanent germ layer contribution to thymus structure, that is, origin of TEC, and other stromal elements; the quest for thymusbuilding and/or maintaining stem or progenitor cells; an understanding of the turnover of thymus stromal elements in the steady-state

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thymus; and the role of thymus stromal cells in thymus involution or pathology-associated transient thymus malfunctions (16). In particular, quantitative answers to many of these questions would be useful. For instance, the number of cells with TEC-forming potential (TEC progenitors) needs to be determined, as well as their clonogenic potential, steps of commitment, life span, etc. Thymus stroma can be viewed as all nonhematopoietic components of the thymus that are functionally deﬁned as those elements, regardless of their origin and lineage, that constitute the thymus structure, and hence provide the matrix on which thymocytes develop (Figure 1). A simple, but useful classiﬁcation of stroma lacking the pan-hematopoietic marker CD45 is based on keratin expression, in that keratin+ cells represent thymus epithelium, and keratin− cells are a mixture of mesenchymal cells. Keratin+ cells are composed of two major subsets referred to as cortical TEC (cTEC) and medullary TEC (mTEC). Keratin− cells—by default collectively considered as mesenchymal cells—include ﬁbroblasts (17), nonﬁbroblastic mesenchymal cells (9), capsule- and septae-forming connective tissue cells, and endothelial cells forming the typical thymus vasculature (9, 18, 19). Finally, dendritic cells and macrophages that are CD45+ hematopoietic cells are also important elements of thymus stroma. Stroma is not only heterogeneous at a given time point, but its composition also varies considerably over time (20). Such restructuring can reach extreme forms under thymus-ablating conditions (steroid treatment, irradiation, cachectic conditions) or with age-associated thymus involution, events that seriously impair thymus function (16). The cellular heterogeneity of thymus structure, as is true for most organs, poses intrinsic difﬁculties to analyze the development or function of given cell types in the physiological context and their transient or permanent contribution to the thymus structure and function. Moreover, direct from indirect

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Phenotypes

Cell types

Medullary TEC +

Keratin

Cortical TEC

Origins

Third pharyngeal pouch endoderm TEC progenitors; Foxn1-dependent

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CD45–

Capsule Septae Keratin–

Endothelium Fibrobasts Nonfibroblastmesenchyme

Neural crest mesenchyme early in ontogeny with minor contribution to adult thymus?; replenished from “local” mesenchyme? Foxn1-independent

Thymus

Thymocytes Dendritic cells CD45+

Macrophages

Hematopoietic stem cells; continuous colonization

B cells Figure 1 Major cell types in the thymus and their developmental origin. The thymus can be divided into hematopoietic cells (CD45+ ) that are transient passengers and resident stromal cells (CD45− ). CD45− cells include two lineages: Thymus epithelial cells (TEC, Keratin+ ) that originate from pharyngeal pouch endoderm (third pouch in the mouse) and mesenchymal cells (Keratin− ), which are a mixture of cell types that contribute to various structures of the thymus such as capsule or vasculature. The origin of the mesenchyme appears heterogeneous. The ratio of CD45+ to CD45− cells is about 50 to 1, but most of the depicted cell types can be isolated based on phenotype from thymus cell suspensions following enzymatic digestion of the thymus.

phenotypes are not easily distinguishable because defects in one cell type may cause alterations in other cell types as well. This limitation also holds true for conclusions drawn about the relationship between overall organ architecture and T cell development, and vice versa, or, in short, the concept of crosstalk (21).

Until about 2001, the prevailing view of thymus organogenesis was, at least in part, based on early morphological studies that had to rely on extrapolation from standing pictures to moving cells (22). Anatomical but also functional data were obtained by grafting experiments in developing birds (23, 24). Collectively, these studies led to the view that www.annualreviews.org • Thymus Organogenesis

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epithelial cells of endodermal and ectodermal origin (22) and mesenchymal cells of neuroectodermal (neural crest) origin (23, 24) all contributed directly or indirectly to the thymus. Directly means that cells and their progeny constitute thymus structure, as is the case for pharyngeal pouch–derived epithelium, and indirectly means that cells provide inductive signals in trans. Neural crest (NC)derived mesenchyme is thought to fall in this latter category. To what extent it also contributes to thymus structure has not been fully resolved. The mechanism that establishes the separation into the inner, morphologically lighter zone, the medulla, and the outer, morphologically darker zone, the cortex, had apparently been settled many years ago. A look at the evolution of the immune system strongly suggests that medulla-cortex organization is functionally important because, as soon as there was a thymus, this architectural hallmark of the thymus was present (25). According to an earlier model of cortex and medulla development (22), a layer of pharyngeal pouch epithelium (endoderm) was the source of medullary epithelium, whereas its surrounding cortex was derived from a layer of ectodermal epithelium. The latter was donated by the cervical vesicle, ectodermal epithelium that comes closely into the proximity of the endodermal pouch at one point in ontogeny (embryonic day 10.5 in the mouse). Hence, medulla-cortex organization was supposed to be established from two cell layers by a mechanism involving invagination and circumferential growth. The model implying cell layer movements was revised by the ﬁnding that the medullary epithelium is composed of single epithelial cell–derived islets that coalesce to form larger medullary areas in the adult thymus (13, 26). Hence, the medulla develops from few progenitors, and the extent of progenitor proliferation establishes the boundaries between medulla and cortex. The remarkable capacity of cell suspensions of puriﬁed fetal thymus epithelial cells to reaggregate in vitro (11) and to form a functional thymus when grafted under the kid-

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ney capsule (12) paved the way to examine the potential of fetal epithelial cells, or subsets thereof. Based on this approach, a TEC stem/progenitor phenotype was proposed a few years ago (14, 15), but the exclusivity of this phenotype was questioned recently (27). Hence, the search to identify, enumerate, and functionally characterize true thymic stem or progenitor cells is only beginning. In fact, it is not clear whether self-renewing thymic epithelial stem cells exist, and if so, to what extent they are involved in the generation, or regeneration, of the thymus. Grafting techniques were further reﬁned by mixing in single, genetically marked cells into a donor thymus, followed by grafting of this tagged tissue, and subsequent visualization of single cell–derived medullary and cortical TEC progeny (26, 28, 29). Moreover, genetic activation of single thymic epithelial cells in athymic nude mice showed that one TEC progenitor could form small yet functional thymus units, again composed of medulla and cortex. This can happen, at least experimentally, early after birth in the thorax and hence later than normal and away from its normal physiological place, the third pouch (26; discussed in 29). On the basis of dyemarking and embryo culture techniques and in line with earlier studies on thymus development in birds (23), researchers abandoned the dual germ layer origin model of mouse thymus epithelium in favor of a single endodermal origin model (30, 31). By deﬁnition, the identiﬁcation of clonal common medulla and cortex progenitors also calls for a common germ layer origin (26, 28). The structural components of the thymus have been only poorly accessible for a long time, mostly owing to difﬁculties or inefﬁciency in retrieving these cells from the solid organ. This, however, is a prerequisite, for instance, to probe the stromal cell subsets functionally (10) or phenotypically (8). Monoclonal antibodies and other reagents that speciﬁcally recognize subtypes of stromal cells have been instrumental in dissecting the thymus structure (8, 32–36). Mutations

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can now be introduced into thymus epithelium by tissue-speciﬁc gene targeting (37) that has been applied to thymus stromal cells (38, 39), or by nude mouse [ forkhead box N1 gene (Foxn1nu/nu )] blastocyst complementation (9). Mice expressing Cre recombinase speciﬁcally in ancestors and/or their progeny of thymus epithelial cells (Foxn1Cre ) (39, 40) will prove useful not only for studying gene function in thymus organogenesis but also for clarifying the origin and lineage relationship of stromal cell subsets (fate mapping). Strategies for gene targeting in, and fate mapping of, thymus epithelial cells are depicted in Figure 2. Visualization of thymus epithelium by expression of markers such as enhanced green ﬂuorescent protein (Egfp) (Foxn1Egfp ) (41) or enzymes (Foxn1LacZ ) (39, 40) under the control of TEC-speciﬁc genes should also shed new light onto the development and maintenance of TEC and reveal temporal and special changes in expression patterns of TEC genes. Finally, genetic screens in vertebrates other than mice, e.g., zebraﬁsh, are ongoing and aim at the identiﬁcation of new genes that control thymus organogenesis (42, 43). Rather than attempting a comprehensive coverage of all current knowledge of thymus organogenesis, this review focuses on signiﬁcant recent advances in the ﬁeld, starting with a brief primer on the embryological origin of the thymus in phylogeny and concluding with the recently identiﬁed functional second thymus in mice, located in the neck.

PHYLOGENY OF THYMUS ORGANOGENESIS The appearance of the thymus in evolution is linked to the appearance of lymphocytes expressing highly diverse antigen-recognition receptors based on DNA recombination of variable (V), diversity (D), and joining ( J) gene elements. This diversity, combined with stringent selection processes forced onto developing lymphocytes, allowed for self-nonself discrimination that is a condition of cellmediated adaptive immunity (44–47). The

thymus evolved as the primary lymphoid organ to fulﬁll these functions, that is, generation of a large and selected T cell repertoire. In light of recent ideas on anatomical microcompartments that serve as specialized hematopoietic and stem cell niches, it is noteworthy that T cells required an entire organ, and not merely a niche. The thymus is an autonomous organ, physically separated from the general primary hematopoietic sites such as the bone marrow. The destructive potential of T cells, once released into the body following incomplete or faulty selection in an only poorly separated niche, could have necessitated the emergence of an entirely separate organ. No thymus is known in species more primitive than vertebrates. Among vertebrates, only jawed, and not jawless (agnatha such as lamprey), species have a thymus (44–46). The precise embryological origin of the thymus, the number of thymus organs per animal, and the ﬁnal anatomical positions of thymus lobes all differ markedly in different species (Figure 3) (reviewed in 48). The common theme is that the thymus always originates from pharyngeal pouches that arise as specialized pockets of the foregut endodermal tube. Pharyngeal pouches harbor primordia for organs and tissues later found in chest, neck, or head regions, including the thymus, and the parathyroid gland (49). The origin of the thymus in the inner layer of an embryonic gut ancestor is reminiscent of GALT (gut-associated lymphoid tissue), which is a key lymphoid structure in species prior to the appearance of a thymus. Thus, the thymus may have evolved as a GALT derivative (45). The most primitive thymus-bearing species are cartilaginous ﬁsh (e.g., sharks and rays). In sharks, thymus anlagen are located in the second to sixth pouch, whereas they are found in the second pouch in frogs, in the second and third in reptiles, and in the third and/or fourth in bony ﬁsh, birds, and mammals (Figure 3). Thus, species are ﬂexible in positioning of the thymus anlage somewhere along the pharyngeal foregut endoderm. Numbers and positions of the www.annualreviews.org • Thymus Organogenesis

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ﬁnal thymus, or thymi, can also be variable. Chickens have seven, sharks ﬁve, and urodele (e.g., salamander) amphibians three thymus pairs, while many teleost ﬁsh species, anuran amphibians (e.g., frogs), and many mammals have only one thymus composed of two bilat-

eral lobes. The position of thymus in the neck and/or in the chest in different mammals is discussed in the context of the cervical thymus in the mouse. In some species, each thymus has a private anlage. For instance, in sharks, ﬁve thymus

Strategies for gene targeting in and fate mapping of thymus epithelium

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Conditional knockout or marker switch driven by Foxn1Cre, KeratinCre , or other loci Mouse with floxed genes of interest or floxed stopper preventing marker gene expression in the absence of Cre

Foxn1Cre mouse KeratinCre mouse Others

X Foxn1nu/nu blastocyst Cre-dependent deletion of floxed genes in TEC or Cre-dependent activation of marker genes in TEC

Principle The Foxn1 gene acts in cis in TEC. Hence Foxn1nu/nu cells cannot be rescued by Foxn1+/+ cells in trans. In Foxn1+/+ ES into Foxn1nu/nu blastocyst chimeric mice, TEC originate from Foxn1+/+ ES cells. Hence, TEC in such mice bear the homozygous mutations from the ES cells. Advantage

Nude blastocyst complementation is arguably a technique forcing most, if not all, TEC to carry the desired mutation.

Disadvantage Nude blastocyst complementation is laborious because it requires generating chimeras by repeated blastocyst injections. In addition, homozygous null ES cells are required, and the method is constitutive and not conditional. 360

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Foxn1Cre drives Cre expression in all or the vast majority of Foxn1-expressing cells at any time in ontogeny, leading to homozygous deletion in floxed alleles in mTEC and cTEC. This also works using KeratinCre mice, or Cre mice that drive expression of Cre in all or subsets of TEC.

Tissue-specific gene targeting of TEC is a versatile approach that could be applied to all floxed genes of interest. Incomplete or late deletion via Foxn1Cre (or KeratinCre) may lead to a mosaic in TEC. The spatial distribution of deleted versus nondeleted TEC is a challenge to analyze.

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primordia each give rise to one thymus lobe positioned along each side of the body. Thus, multiple thymi arise in a one-anlage-to-onethymus ratio (Figure 3). In contrast, in the chicken, the anlagen in the third and fourth pouches give rise to one immature thymus that subdivides secondarily into multiple individual thymus lobes positioned along the neck (Figure 3) (50). As we begin to think in terms of organ progenitor cells, it is likely that the original pharyngeal anlage in the chicken harbors a certain number of progenitor TEC that are partitioned into separate cell clusters, each of which gives rise to one ﬁnal thymus lobe.

CELLULAR BASIS OF THYMUS ORGANOGENESIS: ENDODERMAL EPITHELIUM AND THE GERM LAYER ORIGIN OF THE THYMUS In analogy to general organ development, thymus organogenesis has been divided into several consecutive steps: (a) positioning; (b) budding and outgrowth of the thymus anlage from the third pouch; (c) detachment of the primitive thymus from its endodermal basis; and (d ) patterning, differentiation, and migration of the thymus toward its ﬁnal anatomical position (51, 52). Positioning refers to pouch formation at the prospective site where epithelial cells will later undergo commitment toward

thymus and parathyroid fates. Morphological three-dimensional reconstructions (22) indicate that, on embryonic day 9 (developmental timing is from analyses of the mouse), the pharyngeal pouch constitutes a double-layered membrane composed of an ectodermal and an endodermal cell sheet. On day 9.5, these layers blend together, and it is likely that the precise germ layer origin of these epithelial cells in the third pouch can no longer be assigned with certainty solely based on morphology. Later, on day 10.5, parts of the ectodermal cervical vesicle come into close contact with the endoderm of the third pouch. It was thought that these ectodermal cells rapidly proliferate and ﬁnally surround the endodermal tissue. These and earlier (53) observations formed the basis for the long-held textbook view (54– 56) of a double germ layer origin of the thymus with endodermal and ectodermal origins of medullary and cortical epithelium, respectively. In contrast, on the basis of grafting experiments in birds, researchers concluded that thymus epithelium is derived from a single germ layer, the endoderm (23, 24), that required the presence of NC mesenchyme (see below). More recently, the question of the germ layer origin of thymus epithelium was readdressed, this time in the mouse (30). Examination of the histiogenesis of the thymus during the critical period (E10.5 to E12) conﬁrmed that third pouch endoderm and third cleft

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 2 Strategies for gene targeting and fate mapping of thymus epithelium. (a) Mutations can be targeted to the thymus epithelium, and gene function can be analyzed in TEC by the generation of chimeric mice made by injection of Foxn1+/+ embryonic stem (ES) cells into nude (Foxn1nu/nu ) blastocysts, termed nude blastocyst complementation (9). This strategy takes advantage of the fact that all thymic epithelial cells in such chimeras (9), as well as in aggregation chimeras of Foxn1+/+ and Foxn1nu/nu embryos (149, 150), are derived from the Foxn1+ origin because the nude gene acts in cis and cannot be rescued in trans (149). The use of Foxn1+/+ ES cells bearing homozygous null alleles in a gene of interest will result in a thymus in which TEC lack the gene that is deleted in the ES cell. Nude complementation has been used to study the role of vascular endothelial growth factor (Vegf )-A in thymus epithelium, a gene that is heterozygous lethal if mutated in all cells of an embryo. (b) Thymus epithelium can be genetically modiﬁed using TEC-speciﬁc Cre recombinase deleter mice such as Foxn1Cre (40), or various KeratinCre (e.g., 26) mice. These lines can be used to delete ﬂoxed genes or to activate Cre-dependent reporter loci (fate mapping). The ﬁdelity and inclusiveness of the Cre activity determines how speciﬁc and how quantitative the experiment will be. Advantages and disadvantages are listed. www.annualreviews.org • Thymus Organogenesis

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Figure 3 Origin of the thymus in phylogeny: a variation of the theme. A phylogenetic comparison of the origins of the thymus in shark (a), chicken (b), and mouse (c) demonstrates that thymus anlagen can be found in different pharyngeal pouches (pp) and that the ratio of mature thymus lobes per pharyngeal pouches can differ. In the shark, each thymus lobe has its own anlage (a), whereas the chicken splits one thymus, from two pouches, into seven lobes (b). In the mouse, the thoracic thymus originates from the third pouch. It remains to be determined whether the cervical thymus in mice develops akin to the chicken or the shark thymus. 362

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ectoderm indeed make contact. Evidently, this contact does not result in a compound structure incorporating cells of both germ layer origins. Instead, there are signs of apoptosis at the contact site, suggesting a cell loss, presumably on the ectodermal side (30). Moreover, when the outer pharyngeal surface of E10.5 embryos was dye-labeled in vitro and these ectodermally tagged embryos were cultured for 30 h, there were no labeled cells found in the thymus. This indicates, again, that ectodermal epithelium does not contribute to the thymus proper. In addition to these experiments involving in vitro dye labeling and embryo culture techniques (57), endoderm-only third pouch tissue was dissected from E9 embryos and grafted into nude recipients. These grafts developed into functional thymi with medulla-cortex architecture (30). There is also genetic evidence for a common germ layer origin of cortical and medullary epithelium from tetraparental chimeric mice generated by injection of ES cells into MHC-mismatched blastocysts (13). There was no clear relationship between the origin of mTEC islets, discussed later in the context of TEC progenitors, and the origin of the cortex immediately surrounding a medullary islet (13). In other words, a medullary islet of ES cell origin could be localized in cortical epithelium of its own (ES), but also of the opposite (blastocyst) origin. Hence, there was no sign that a common origin translates into local units composed of adjacent mTEC and cTEC. However, a common germ layer origin of mTEC and cTEC, and a late developmental split into mTEC and cTEC, might imply that, overall, the origins of mTEC and cTEC are proportional. The relative contribution of the ES cell and the blastocyst to the tested organs (thymus, skin, liver, heart) was random, suggesting that these organs developed independently (Figure 4). In contrast, comparison of the overall contribution of ES cell or blastocyst to medullary and cortical epithelium revealed a straight line pointing at a common origin and close

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CONTRIBUTION OF NEURAL CREST (NC) MESENCHYME TO THYMUS ORGANOGENESIS: TRANSIENT OR LONG LASTING? NC-derived mesenchyme is crucial for thymus organogenesis and thymus function (reviewed and further references in 1, 51, 58, 65, 66). In the original description of this phenomenon in bird embryos, it was demonstrated that NC-derived mesenchyme colonizes the branchial arches, surrounds the

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relationship of cells forming medulla and cortex in ontogeny (58). These data also imply that the common origin holds true for the entire thymus epithelium. Collectively, independent experimental approaches have now provided compelling evidence that thymus epithelium is derived from the endodermal layer of the third pharyngeal pouch only (reviewed in 52, 59). Budding and outgrowth of the thymus anlage from the third pouch coincides with onset of expression of the Foxn1 gene (60). Foxn1 expression is ﬁrst detected in a subset of epithelial cells in the third pouch on embryonic day 11.5 in mouse development (61–63). Doublestaining for expression of Foxn1 versus glial cells missing 2 [Gcm2], a transcription factor gene required for parathyroid organogenesis (64), reveals adjacently located epithelial cell clusters whereby Foxn1+ cells are located in the ventral part of the third pouch while Gcm2 marks the dorsal aspects of the same pouch (62). Because Foxn1 and Gcm2 are essential for thymus (60, 61) and parathyroid (64) development, respectively, these cell clusters likely represent the anlagen for each or the two third pouch–derived organs. However, a direct and exclusive precursor-product relationship between Foxn1- or Gcm2-expressing cells in the pouch and the mature organs has not been established. This would require the puriﬁcation of cells based on their expression of these transcription factors and a prospective test of their potential.

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Figure 4 Common origin of medullary and cortical TEC in tetraparental (chimeric) mice. Mice generated as described by injecting embryonic stem (ES) cells into MHC-mismatched blastocysts (13) were analyzed for ES cell versus blastocyst contributions to skin, heart, liver, and thymus utilizing microsatellite differences in ES cell and blastocyst genomic DNA. The contributions of ES cell and blastocyst to thymus medulla and cortex were determined by histological analysis of the MHC class II haplotypes indicative of ES cell or blastocyst origin. None of the organs showed any linkage of the origins except for thymus cortex and medulla.

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thymic epithelium, and forms perivascular mesenchyme (23). Ablation of cephalic NC in birds prevented any contribution to or induction of thymus epithelium by NC-derived mesenchyme, and this resulted in small thymi with delayed development and poor function (24). These early reports were conﬁrmed in principle and extended by many subsequent experiments that showed an important role for NC in thymus development in vivo (reviewed in 51, 52) and in organ culture systems (10, 67, 68; reviewed in 1). Examples of genetic pathways involved in mesenchymethymus epithelium interactions are provided below. Given that mesenchymal-epithelial contact takes place early in development and that mesenchymal cells are abundant in the adult thymus, as speciﬁed in the introduction, it has been debated whether or not embryonic NCderived mesenchyme and adult thymus mesenchyme share a precursor-product relationship (reviewed in 66). Several reports address this point in mice generated with the aim to express Cre recombinase speciﬁcally in the NC lineage. In combination with appropriate Cre activity–dependent reporter mice, NCderived cells and their progeny should be permanently labeled in this system. Mesenchyme marked by Wnt1Cre surrounds the E13.5 thymus as a massively stained, thick layer. However, labeled cells become rare as soon as the thymus grows, owing to the rapid proliferation of thymocytes. In the adult thymus, the overall contribution of Wnt1Cre -marked cells appears too low to account for major mesenchymal components such as the capsule, the septae, or intrathymic ﬁbroblast (66, 69). Some cells of unknown character are, however, present in the adult thymus (66). A similar result of transient but not permanent participation was obtained using a different NC-speciﬁc marker gene, myelin protein zero (P0). By ﬂow cytometry on thymus cells from E13.5, as many as 30% of all stromal cells are labeled by P0Cre (70), which is the ﬁrst quantitative estimate of the contribution of NC to the developing thymus. Again, few marked

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cells persist at later stages. In vitro, P0Cre marked cells from the fetal thymus could be developed into melanocyte and glial cell lineages, further supporting their NC origin. The idea that the thymus contains cells related to neuronal/glial lineages is not new (71), but there is currently no evidence to deﬁne functions for such cells in the thymus. Finally, a cautionary note may be warranted here because, as in other fate mapping experiments, the result will ultimately depend on the strategy used. Random transgenic integration may yield more mouse-to-mouse variability than targeted knockin approaches, and the result, here low or no contribution of NC cells to the adult thymus, depends on the properties of any particular Cre driver mouse and the sensitivity of the reporter detection. A possible molecular link between NCderived mesenchyme and thymus epithelium is provided via ﬁbroblast growth factors (Fgfs, also termed keratinocyte growth factor [KGF]) and their receptors (FgfR). Fgf7 and Fgf10 are expressed by the mesenchyme surrounding the embryonic thymus epithelium, and the latter expresses FgfR2IIIb. Defects in this signaling pathway perturb thymus development (72), demonstrating a growth-promoting role for mesenchyme toward thymic epithelium. Signals via Fgfs also induce TEC proliferation (73, 74) and protect thymus epithelium from injury by irradiation (75) or by conditions of graftversus-host disease (76). Another role of thymus mesenchyme could be the presentation of growth factors such as IL-7 or c-kit ligand to thymocytes, but currently no data support this idea.

GENES AFFECTING THYMUS DEVELOPMENT PRIOR TO THYMUS SPECIFICATION Mutations in a number of genes, including Hoxa3 (77), Eya1 (78, 79), Six1 (79, 80), Pax1 (81–84), Pax3 (85), Pax9 (86, 87), and Tbx1 (88), lead to thymus aplasia, or hypoplasia, or failure of the thymus lobes to migrate

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toward the chest (reviewed and further references in 51, 52, 89). These genes are expressed in multiple cell lineages during development, and hence their loss of function causes pleiotropic defects in embryonic development. It is, therefore, often difﬁcult to distinguish the primary function of these genes in thymus organogenesis, e.g., in TEC progenitors, from an upstream function, e.g., in formation or patterning of pharyngeal structures or in NC migration. It is obvious that the thymus cannot develop normally if the third pouch is absent as a scaffold in which TEC progenitors arise. The effects of such mutations are therefore upstream of thymus organogenesis itself. Moreover, it is also possible that some of these genes are required both upstream of organogenesis and later in the thymus epithelium itself. Along this line, Hoxa3 (90), Pax1 (82), and Pax9 (91) are expressed in thymus epithelial cells. Because this was mostly measured by RT-PCR in cell populations, frequencies and phenotypes of TEC expressing these genes are unknown. TECspeciﬁc deletion of such ubiquitous pathway genes will be required to resolve their function in thymus development beyond the general defects that may be nonspeciﬁc for the thymus. An example of TEC targeting was blocking of Bmp signaling in TEC by expression of Noggin under the control of the Foxn1 promoter. Whereas blocking of Bmp signaling in premigratory NC by transgenic expression of Noggin in NC affected thymus development indirectly (92), Foxn1-driven inhibition of the Bmp pathway demonstrated, in addition to the NC defect, a role for Bmp signaling intrinsic in TEC (93). This is compatible with expression of Bmp family members in the thymus epithelial anlage (94). Another interesting gene that belongs to this gene category, and that is related to DiGeorge syndrome (95–98), is the T box gene Tbx1. DiGeorge syndrome is caused genetically by heterozygous deletions within chromosome 22q11 and clinically by malformations of pharyngeal arch arteries (cardiac

outﬂow tract) and heart, parathyroid hypoplasia, and absence or ectopic location of the thymus (96, 99). Hallmarks of this phenotype are recapitulated in mice lacking Tbx1 (88, 100), a gene that is located in the deleted region in humans (88, 100). Tbx1−/− mice display agenesis of pharyngeal pouches 2–4 and concomitant loss, or malformation, of pharyngeal pouch–derived organs and tissues (thymus, parathyroid gland, cardiac outﬂow tract) (88, 100). Tbx1 is expressed in the pharyngeal pouch endoderm but also in the core mesoderm of the pharyngeal apparatus and the pharyngeal ectoderm but not in NC-derived mesenchyme (101). Thus, Tbx1 expression may play distinct roles in different anatomical sites (e.g., endoderm versus mesoderm) during development. Pharyngeal pouches fail to develop in mice in which the Tbx1 deﬁciency is restricted to the endoderm by means of preferential deletion in pharyngeal endoderm using another Fox family gene locus expressing Cre, Foxg1Cre . Hence, this mutant recapitulates the defects known from the constitutive null mice, including absence of the thymus (102). This demonstrates that Tbx1 expression in the pharyngeal endoderm is required for thymus development. However, mice that lack Tbx1 expression selectively in the pharyngeal mesoderm, but not the endoderm, also have a hypoplastic pharynx with impaired pharyngeal endoderm and lack a thymus (103). Conditional reversion from a defective to a functional Tbx1 allele in pharyngeal mesoderm, but not endoderm, is sufﬁcient to rescue major defects known from the Tbx1 null phenotype (pharyngeal patterning, cardiovascular defects) but does not restore thymus development (103). In conclusion, expression of Tbx1 both in the pharyngeal core mesoderm and in the pharyngeal endoderm is a prerequisite for thymus development. Within the third pouch, Tbx1 expression coincides rather with the parathyroid than the thymus anlage (104), and it remains to be determined if Tbx1 is expressed in TEC and whether it plays a role in their development.

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EPITHELIAL PATTERNING AND DIFFERENTIATION, AND CROSSTALK BETWEEN THYMOCYTES AND EPITHELIUM

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Coinciding with hematopoietic colonization that initially occurs around E12 and prior to vascularization, the immature thymus undergoes further patterning and differentiation (reviewed in 51, 105, 106). Morphologically, this stage leads to the ﬁrst signs of medullacortex separation. This compartmentalization is associated with changes in keratin expression patterns in the epithelium. During ontogeny, and in the adult thymus, TEC subsets express different members of the keratin family. Major populations of adult mTEC and cTEC have been distinguished by their keratin (K) 5+ K8− and K5− K8+ phenotypes, respectively (107, 108 and references therein). This dichotomy is, however, not absolute because K5 is also expressed in some cTEC, and K8 is also found in some mTEC (107). Regarding the onset and pattern of K5 and K8 expression in ontogeny, third pouch epithelium at E11.5 was reported as K5− K8+ (15, 108), whereas others found coexpression of K5 and K8 already at this stage, even with high expression of K5 (14). A prominent TEC population coexpresses K5 and K8 on days 12 and 13 (14, 15, 108). Using these and other markers, so-called double-positive TEC are thought of as progenitors of mature singlepositive K5+ K8− medullary and K5− K8+ cortical TEC phenotypes (108–110). This idea was based on the fact that K5+ K8+ cells precede the appearance of mature K5+ K8− and K5− K8+ TEC in ontogeny. Moreover, large clusters of K5+ K8+ TEC are maintained in mutants with massive early blockade in T cell development such as compound KitW /W and common γ chain (γ c− ) (111), or RAG2−/− γ c− mice (thymus stromal phenotypes reviewed in 105). Thirdly, and further discussed in the context of TEC progenitors, MTS24+ TEC, a controversial TEC progenitor phenotype (27), also coexpresses K5 and K8 (14, 15). In

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any case, the initial patterning of the embryonic thymus is dependent on expression of Foxn1 in the epithelium but is independent of hematopoietic colonization. The transition from immature TEC phenotypes, such as abundant K5+ K8+ cells, to the full medulla-cortex organization is perturbed in mice in which T cell development is blocked at immature stages. That the TEC architecture is somehow inﬂuenced by the presence or absence of particular stages of thymocytes development has been viewed as an interdependence between thymocytes and stroma (36, 107, 112) and has been referred to as crosstalk (21). In the original case, it was suggested that the thymus stroma was permanently damaged unless it had proper contact with developing pro-T cells (CD44+ CD25− ) and that this contact needed to take place at fetal stages of TEC development (112). The transgenic mouse (hCD3ε26tg) on which these conclusions were based expressed 40– 60 copies of a human CD3ε transgene (113, 114). T cell development in this mouse was blocked at an early CD4− CD8− (doublenegative) stage prior to expression of CD25, and numbers of residual thymocytes were very low. This paucity of thymocytes and the severe block in thymocyte development were suspected as the cause of aberrant adult TEC structure characterized by poorly discernable cortex and an abundance of cells coexpressing K5 and K8 (107, 112). Subsequent studies found, however, that the hCD3ε26tg thymus was not simply devoid of thymocytes, but harbored B cells (114). Hence, it cannot be excluded that it is not the absence of developing thymocytes that perturbs thymus organogenesis but the aberrant B cells or their development that contributes to the adult stromal cell phenotype. This interpretation would question the speciﬁcity of crosstalk between pro-T cells and TEC. Another concern regarding models of crosstalk in thymus organogenesis has been the inability to distinguish alterations in the epithelial compartment owing to lack of

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signals from developing thymocytes to TEC from epithelial reactions to a state of inactivity. The latter scenario could lead indirectly to defects in the maintenance of the epithelial cells. Along this line, a recent reevaluation of the developing thymus in hCD3ε26tg mice found normal proportions of immature K5+ K8+ , as well as single-positive K5+ K8− and K5− K8+ TEC when compared to wildtype thymus (115). These data imply that thymus organogenesis may be quite normal in hCD3ε26tg mice and that the architectural alterations seen in this mutant are secondary to regular TEC development, a conclusion that would support the argument against a role of pro-T cell–mediated signaling for the development of thymic epithelial cells (115). The suggestion that the thymus stromal cell architecture is damaged at long-term unless proT cells contact the epithelium at the proper time in ontogeny is also contradicted by experiments made in KitW /W γ c− mice, a mutant in which thymocyte development was completely abrogated (116). The severely dysmorphic thymus structure of this mutant could be reverted to a structurally normal and functional thymus when grafted postnatally into a recipient that provided wild-type hematopoietic stem cells (111). Finally, the fact that the human thymus of severe combined immunodeﬁciency (SCID) patients can be reconstituted by transplantation of normal bone marrow stem cells (117) further supports the argument against continuous stroma defects caused by a block in T cell development. Collectively, much speculation has surrounded the interesting crosstalk concept. A crosstalk mechanism should involve cell surface molecules such as receptor-ligand pairs that can transmit signals in both directions, and the absence of such signals might cause a speciﬁc phenotype on the nonreceiving end. Some molecularly deﬁned cases for bidirectional crosstalk between thymocytes and thymus epithelium exist. One documented example of a crosstalk mechanism from thymocytes into epithelial cells is the defect

in the mTEC compartment in mice lacking components of the TNF-TNF receptor family. Expression of the lymphotoxin-β receptor (LTβR) on thymocytes and of a LTβR ligand on mTEC are required for normal cellularity and architecture of mTEC. Mutations that interrupt this signaling pathway lead to structural defects associated with faulty selection and autoimmunity (118). Moreover, the recent ﬁnding that expression of RANK ligand on a CD4+ CD3− inducer cell population promotes the maturation of RANK-expressing CD80− Aire− mTEC progenitors into CD80+ Aire+ mTECs (119) can also be viewed as a form of crosstalk, albeit between a highly specialized and rare lymphoid cell and a speciﬁc stage of TEC. There are other candidate molecules that could mediate signaling from stroma to thymocytes and back. For instance, the receptor tyrosine kinase Kit is expressed on proand pre-T cells, and the membrane-bound ligand [Kit ligand (KL), or stem cell factor (Scf )] is expressed on stromal cells (120). Kit signaling into thymocytes via expression of KL in TEC is crucial for T cell development (121), and KL can indeed signal into epithelial cells (122). However, T cell development was permissive in a KL mutant (Sl17H ) (H.-R. Rodewald, unpublished data) in which the cytoplasmic tail of KL is nonfunctional (123). Hence, there is currently no evidence for KL-mediated crosstalk in the thymus. Further potential examples are Notch and Notch ligand pairs. Notch-1 is expressed on proand pre-T cells, and Notch ligands, including Delta-like (Dll)1 and Dll4, are expressed in thymus epithelium (reviewed in 124, 125). Dll1 or Dll4 could signal into the epithelium and induce changes in the microenvironment that could ultimately be involved in the thymus phenotype of Notch-1 mutants, including an abundance of B cells instead of T cells in the thymus (124, 125). However, it is not known whether Notch-Notch ligand–driven mechanisms play a role in crosstalk or in TEC development, as has been speculated (28).

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POTENTIAL, FREQUENCIES, AND PHENOTYPES OF THYMUS EPITHELIAL STEM/PROGENITOR CELLS Progenitors for mTEC

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Measurements of stem or progenitor cell activity, and the prospective isolation by phenotype and subsequent analysis of progenitor potential, ultimately require clonal assays. Although single cell–based assays to study TEC development in vitro are still lacking, cellular and genetic strategies have been developed to follow the fate of single TEC progenitors or their activity in vivo (13, 26, 28). Clonal progenitor activity for TEC was initially identiﬁed for the medullary lineage in chimeric mice and in grafts of reaggregate fetal thymus organ cultures (RFTOC) (13) and has recently been conﬁrmed independently by genetic means (26). The former approach was based on techniques that allow disassembly of embryonic or fetal thymus by enzymatic digestion, puriﬁcation of stromal cells or subsets thereof, and reassembly into thymus reaggregates in vitro (10). These RFTOC are functional in that they support T cell development in vitro, but the TEC architecture of RFTOC in vitro is quite different from that of a normal thymus in vivo. In RFTOC assembled from puriﬁed TEC in the absence of thymocytes, distinct mTEC or cTEC phenotypes were present, but these cells did not form medulla-cortex structures. In contrast, RFTOC grafted into recipient mice not only supported steady-state T cell development, but also achieved proper thymus architecture, including regular medullacortex organization (12, 126). This remarkable capacity of TEC to build functional thymus architecture from reaggregated cell suspensions suggested that mTEC and cTEC, randomly arranged in the reaggregates in vitro, could migrate within the graft to segregate into cortex and medulla. Such sorting out would have required active migration, or at least directed movement, and recognition

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of the neighboring cells as mTEC or cTEC. However, experiments using RFTOC assembled from two distinct donor strains, followed by transplantation of these mixed RFTOC, revealed an alternative mechanism and provided evidence for clonal events during epithelial organogenesis of the thymus (13; reviewed in 106). Cellular products of mTEC progenitor activity were visible in the form of epithelial medullary islets, the majority of which were either of one or the other, but not of mixed, origin (13). Medullary epithelial islet formation also occurred during normal thymus organogenesis in vivo, as shown in chimeric mice made by injection of ES cells into MHC-mismatched blastocysts. Here, individual epithelial islets stemmed from either ES cell or blastocyst origin. Hence, medullary islets arise from single progenitors. With age, the islet-like character of mTEC is harder to recognize, as many mTEC islets coalesce to form conﬂuent regions of medullary epithelium. The existence of clonal medullary islets was recently conﬁrmed genetically (26) (see below), and the principle of islet formation in chimeric thymus grafts has been used as an assay to assess the potential of phenotypically deﬁned embryonic mTEC subsets (127) or the role of MHC class II expression for the selection of Foxp3+ regulatory T cells on medullary epithelium (128). Serial sectioning showed that one thymus lobe of a mouse at two weeks postnatal contained ∼300 medullary areas (according to this deﬁnition, a medullary area is larger than a medullary islet). These medullary areas were composed of one to three islets. Hence, up to ∼900 mTEC progenitors are sufﬁcient to generate the entire medulla in one thymus lobe of a mouse (13). This estimate is in a strikingly similar order of magnitude as the estimated 1500 TEC in the E13.5 thymus that express the tight junction proteins claudin (Cld)-3 and 4 (Cld3,4) (127). Already on E10.5 in development, Cld3,4+ cells were found in the apical epithelial layer in the third pouch thymus anlage. Subsequently, Cld3,4+

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cells showed a clustered arrangement that partially overlapped with general mTEC markers such as MTS10 or UEA-1. In the adult thymus, Cld3,4 expression marked a subset of mTEC expressing the autoimmune regulator gene Aire (127). Cld3,4+ Aire+ cells are presumably fully mature mTEC that are busy in promiscuous expression of tissue-restricted self-antigens (TRA) (reviewed in 5). By ﬂow cytometry, several TEC subpopulations were identiﬁed and separated based on Cld3,4+ and UEA-1 expression. Interestingly, Cld3,4+ cells, irrespective of UEA-1 expression, gave rise to only mTEC and not cTEC when mixed into RFTOC and grafted into nude mice, whereas Cld3,4low UEA-1− cells gave rise to both mTEC and cTEC. These data suggest that commitment to the mTEC lineage can take place already by E13.5, at least in cells deﬁned by Cld3,4 expression. Collectively, the very early and restricted expression of the tight junction proteins of Cld3,4 marks an mTEC pathway. Because the medullary clusters that arise from Cld3,4+ progenitors are very similar in size and frequency to the medullary islets described earlier, it is possible that Cld3,4 expression marks the entire mTEC pathway at some point in the differentiation tree of TEC. It is not clear whether mTEC progenitors are only active during ontogeny, or whether they continue to generate mTEC de novo in the adult thymus. Clonogenic, mTEC-isletsforming mTEC progenitors, or their activity, have not been detected in the adult thymus. For a long time, TEC were considered postmitotic cells that constitute an epithelial network that is constructed in ontogeny and later maintained. However, several reports recently found TEC proliferation indicating rapid epithelial turnover in the steady-state thymus (5, 20, 129). There are considerable differences in the reported proliferation rates, ranging from as many as 23% of MHC class II+ CD45− TEC (the majority of which are mTEC) incorporating BrdU after three days of continuous labeling (20) to as few as 8% of mTEC incorporating BrdU after one week of label-

ing (129). This would translate into half-lives of about six days in one case versus four weeks in the other case; the latter is similar to a third estimate of six weeks (5). It is obvious from the above considerations that there are large gaps in our understanding of the sequence of events during mTEC differentiation from mTEC progenitors to mature mTEC. Speciﬁcally, the relationship of mTEC differentiation stages to mTEC function (TRA expression and presentation; full maturation as antigen-presenting cells) and mTEC turnover (proliferation versus cell death) are only poorly understood. On the one hand, islets of mTEC vary in diameter from a minimum of 60 × 40 to a maximum of 170 × 170 μm and harbor between 5 and 45 epithelial cells in a two-dimensional lattice (13). On the other hand, numbers of cells expressing a particular TRA in the medulla appear even lower than the cells per islet (129, 130; reviewed in 5). Because an mTEC islet is originally made by a single progenitor, cells within an islet might be further diversiﬁed into subclones (131). It is not known whether this diversiﬁcation is stable or whether individual mTEC change their expressed TRA pattern over time. It will be necessary to better deﬁne stages of mTEC differentiation from mTEC progenitors via immature to mature mTEC and to shed light on the signals that regulate this developmental progression as well as on the capacity of TEC at each stage to present antigens and ultimately guide TCR repertoire selection on thymocyte populations (5, 119, 129, 131, 132).

Common mTEC and cTEC Progenitors It was speculated early on that mTEC and cTEC share a common progenitor (109, 110). The idea was based on coexpression of markers such as keratins on TEC early in thymus organogenesis, as discussed above in the context of the germ layer origin. More recently, the generation of a complete and functional thymus environment in RFTOC grafts was www.annualreviews.org • Thymus Organogenesis

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also taken as evidence of a common progenitor for both mTEC and cTEC. Transplantation of RFTOC assembled from embryonic day 12.5 (14) or fetal day 15.5 (15) TEC led to the formation of a thymus composed of both medulla and cortex (discussed in 133). Gill et al. (15) interpreted their data as direct evidence of thymic progenitor cells giving rise to both cortical and medullary epithelial lineages, and Bennett et al. (14) argued strongly in the same direction. These polyclonal (bulk) experiments in fact demonstrate that the populations of cells used to assemble the grafts contained all the cells required for a functionally and structurally normal thymus. What they could not answer was whether or not a common progenitor for mTEC and cTEC existed and, if so, whether it was responsible for the generation of both mTEC and cTEC in the grafts. This key property of a common progenitor, clonogenicity, has only recently been addressed (26, 28; reviewed in 29). Single epithelial cells isolated from yellow ﬂuorescent protein (YFP) expressing embryonic E12.5 thymus were injected into a nonﬂuorescent host thymus, and such single cell–tagged thymi were transplanted into recipient mice to allow for full thymus development. Immunohistological analysis showed that, in each case of positive reconstitution, the single embryonic epithelial cell had produced both mTEC and cTEC. Because the donor cells were puriﬁed via expression of a pan-TEC marker [EpCAM1; antibody G8.8 (34)], no particular TEC subset was selected, implying that a large proportion of epithelial cells in the day 12 thymus has dual potential for mTEC and cTEC and that their progeny persists in the adult thymus (28). A different approach addressing progenitor activity in thymus organogenesis was based on epithelial cell tracing using genetic in situ labeling (26). Cre recombinase under the control of the human Keratin 14 promoter (K14Cre ) effectively acted as a random and very rare switch that turned on YFP expression in TEC. Although no labeled cells were

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found in the thymus at birth, numbers of mice with labeled cells and numbers of YFP+ TEC per thymus increased with age after birth. At all times analyzed, labeled progeny remained very rare, consistent with Cre-mediated YFP expression only in single or in very few TEC progenitors. Three patterns of progeny were noted: (a) mTEC clusters only, reminiscent of medullary islets; (b) cTEC clusters only; or (c) mTEC plus cTEC progeny (26). These results strongly suggest that K14Cre randomly marked TEC progenitors and their progeny and that this labeling event could occur over developmental stages that covered common to committed TEC progenitors. If the genetic hit occurred at an early postnatal age, TEC progenitors, endowed with the listed potentialities, persist at least until that age in a normal thymus. After the putative mTEC versus cTEC branch in TEC differentiation, cells probably migrate considerable distances. This is evident from the space that was observed between mTEC and cTEC progeny of common origin in the adult medullary and cortical zones, respectively (26). The notion of TEC migration, or perhaps passive movement, during development would also ﬁt the topological dissociation of mTEC versus cTEC of the same origin in chimeric mice (13). Collectively, based on cellular (13, 28) and genetic (26) evidence, the thymus harbors TEC progenitors that, at the single cell level, contribute to the formation of thymus epithelial structure. Except for an estimate on total numbers of mTEC progenitors required (see above), there is currently no information on the total number of common mTEC and cTEC progenitors that normally engage in the building of a thymus. The marking of common TEC progenitors left unanswered whether or not single TEC progenitors are capable of forming functional thymus units. This was addressed again in the K14Cre mouse, but in this case the Cre recombinase was used to revert a nonfunctional Foxn1 allele to a functional Foxn1 allele akin to correcting the nude mutation in the thymus in vivo. The Foxn1nu/nu thymus is

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composed of epithelial cysts that do not support T cell development. When crossed to a Cre-dependent LacZ reporter, K14Cre marked rare TEC that, in this case, were located in the wall of the nude thymus cysts. When Cre reverted the loss-of-function Foxn1 allele back to a functional Foxn1 allele, an event that occurred again randomly in single postnatal epithelial cell precursors, small units of thymus tissue developed (26). These neo-thymi showed all tested hallmarks of a normal thymus, including medullary and cortical organization and expression of Aire. Mice bearing such thymi had elevated numbers of immunocompetent T cells that, unlike the few T cells found in nude mice, expressed a diverse TCR repertoire. These experiments demonstrate that the block that occurs in TEC development in the nude mouse does not lead to a complete loss of TEC progenitors. Rather, TEC progenitors may enter a stage of dormancy or may be continuously generated de novo in nude mice. Once genetically reverted to wild type, they can recapitulate normal ontogeny and complete their differentiation into functional TEC. It is noteworthy that this development can take place outside of the physiological location, the third pouch, because the cystic thymus rudiments in the nude mouse are at this age in the chest. For anatomical and kinetic reasons, it is unlikely that the inductive signals that are provided to the thymus during normal ontogeny, e.g., by NC-derived cells, are available to those thymi that use their second chance (29). This lack of proper context could at least in part be responsible for their small size (26).

TEC Progenitor Phenotypes In the aforementioned single cell experiments, TEC progenitor cells have not been physically puriﬁed except by the panepithelial marker EpCAM1 (28). Are there cell surface markers known that identify TEC progenitors? This would be an important prerequisite for analyses of the prospective potential of these cells. Previous experiments

have suggested that MTS24 is a marker for embryonic TEC progenitors, a proposal that met with some interest (133, 134). As noted before, RFTOC assembled from embryonic (14) or fetal (13, 15) TEC developed into functional thymi in vivo. Although earlier studies showed thymus development in RFTOC grafts made from several hundred thousand fetal TEC deﬁned as MHC class II+ CD45− cells (12, 13), others reported thymus formation using much lower numbers of cells using the MTS24 marker for positive puriﬁcation. Twelve thousand ﬁve hundred TEC from E12.5 (14), or 2,500 from day 15.5 fetal (15), contained all the cells required for a thymus. Even when only 500 cells were transplanted, nude recipients showed signs of transiently functional thymus (14). In analogy to other progenitor systems, it can be assumed that the frequency of progenitors is low among all TEC. Are MTS24+ TEC rare cells, as has been suggested (133)? On E10.5, MTS24 is broadly expressed in the endodermal pharyngeal pouches (15) (including those that do not give rise to a thymus). By histology, most (14, 15), if not all (27), epithelial cells on day 11.5 and 12.5 express MTS24. By ﬂow cytometry, essentially all thymus epithelial cells, gated as pan-cytokeratin+ or EpCAM1+ cells on E12.5 and 13.5, are MTS24+ (27), suggesting that at this stage of development, MTS24− cells are nonepithelial cells. Consistent with these data, and not surprisingly, only MTS24+ (that is, epithelial) and not MTS24− (that is, nonepithelial) cells from E12.5 have thymus-forming capacity (14). Is MTS24 a progenitor marker at later stages of thymus development? On day 15.5, about half of all TEC still express MTS24 before the frequency of MTS24+ cells declines to a few percent at later fetal and adult stages (14, 15, 27, 135). One report found thymus potential exclusively in MTS24+ and not MTS24− epithelial from day 15.5 thymus (15). However, a recent reassessment of TEC progenitor activity using MTS24 expressionbased puriﬁcation did not reproduce these ﬁndings using larger cell numbers from www.annualreviews.org • Thymus Organogenesis

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14- or 16-day-old thymi and concluded that both MTS24+ and MTS24− epithelial cells were similarly potent in forming a functional thymus (27). The available evidence suggests the following order of events: MTS24 is initially a nonthymus-speciﬁc marker of pharyngeal endoderm cells; it then marks all TEC around day 12.5 and is expressed on day 15.5 on a major subset of TEC. At this stage, thymus potential is included in both MTS24+ and MTS24− epithelial cells. From the adult thymus, MTS24+ cells can be retrieved, but their function remains to be determined (135). If MTS24 is perhaps not “the” TEC progenitor marker, is there any evidence for stemness among TEC? Interestingly, TEC express genes such as Nanog, Oct4, and Sox2, which are commonly found in stem cells, and the expression of these hallmark genes is reduced in TEC from Aire-deﬁcient mice (91). Because gene expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in populations of TEC, it will be important to determine the frequencies of TEC and their maturation stages that express Nanog, Oct4, and Sox2. Perhaps TEC stem or progenitors will be found within these cells or among cells expressing p63 (136) (see below).

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GENES AFFECTING THYMUS DEVELOPMENT AFTER THYMUS SPECIFICATION Foxn1, the Gene Mutated in the Nude Mouse There is no deﬁnitive marker that indicates epithelial cell commitment toward thymus fate prior to expression of Foxn1. Functionally speaking, Foxn1 is the single most important gene known to be essential speciﬁcally for thymus epithelial development (60, 61). Therefore, it is worthwhile to take a closer look at properties of Foxn1 such as expression, regulation, and function. Foxn1 is a protein belonging to the family of forkhead box transcription factors (137–139). In addition to Foxn1, other members of this gene family also 372

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play important roles in the immune system, such as Foxp3, which determines the development and function of regulatory T cells, and Foxo factors, which are involved in apoptosis and proliferation, and hence leukocyte homeostasis (138, 139). Foxn1 is characterized by a winged-helix/forkhead DNA-binding domain and a transcriptional activation domain (42, 140). In the original nude mouse allele (Foxn1nu ), a single base pair deletion in exon 3 causes a frame shift leading to a truncated Foxn1 protein lacking both the DNA-binding and the activation domain (60). Homozygous Foxn1nu/nu mice had the same thymus and skin phenotypes as compound heterozygous mice bearing one engineered null allele of Foxn1 (Foxn1− ) and one natural mutant allele of Foxn1 (Foxn1nu ), formally showing that Foxn1 is allelic to the nude gene (61). In mice homozygous for a Foxn1 allele lacking exon 3 (Foxn1/ ), thymus epithelium developed beyond the block in Foxn1nu/nu mice. Foxn1 encodes a Foxn1 protein with a large deletion in the N-terminal part of Foxn1 (141). The phenotype of Foxn1/ mice overall resembled that of a hypomorphic mutant in which TEC phenotype and organization suggest an arrest at a K5+ K8+ stage. In contrast to the cystic Foxn1nu/nu thymus, the Foxn1/ thymus had a more continuous structure permissive to support T cell development, albeit only poorly and with a delay in fetal thymocyte development. It is not clear whether this TEC phenotype results from a speciﬁc requirement for the N-terminally deleted amino acids at later stages of TEC development, perhaps beyond the K5+ K8+ stage, or whether the Foxn1 allele encodes a Foxn1 protein with an overall reduced functional activity. The stability of the Foxn1 protein encoded by this particular allele has not been determined. Little is known about upstream factors that regulate Foxn1 expression. Through transgenic work, DNA fragments and promoter elements that permit gene expression under the control of Foxn1 have been described (41, 93, 142). It has been proposed that Foxn1 expression is regulated through members of the Wnt

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family (143). Given that no single or compound Wnt mutant mouse has been reported that resembles a nude thymus phenotype, further clariﬁcation of the role of Wnt family members directly upstream of Foxn1 would be helpful. In brief, there is very little information on how Foxn1 transcription is initiated in development or maintained in the adult thymus. On the basis of a Foxn1lacZ knockin allele, in situ hybridization, and antibody staining, investigators have found that Foxn1 expression in thymic epithelium is ﬁrst detectable on E11.5 (61–63), a stage preceding colonization of the thymus by hematopoietic progenitors. Analysis of a Foxn1lacZ allele demonstrates that Foxn1 is expressed in most, if not all, thymic epithelial cells both at embryonic stages and in the adult (61). However, a more recent study that used an anti-Foxn1 antibody suggests that both Foxn1+ keratin+ and Foxn1− keratin+ thymic epithelial cells exist in the embryonic thymus (E13) and that the percentage of Foxn1− keratin+ TEC is even as high as 80% in the adult thymus (63). If subsets of adult TEC differ, in fact, in expression of Foxn1, it would be interesting to deﬁne how those Foxn1+ and Foxn1− subsets differ with regard to the relative stages of maturation, cellular age, turnover, or their functional capacities to promote T cell development. Although the extent to which adult TEC actively express Foxn1 is controversial, fate mapping of TEC using a Foxn1Cre allele convincingly showed that most, if not all, TEC arise from Foxn1+ progenitors, or at least transit through a stage of ubiquitous Foxn1 expression (39, 40). As mentioned earlier, genetic activation of Foxn1 in single TEC led to the appearance of units of productive thymi in an otherwise nude thymus. This underscores the idea that Foxn1 is expressed in thymus-forming progenitors (26). Foxn1-dependent cells in the thymus can be visualized in nude blastocyst complementation (Figure 2) (9) by injection of Gfp+ Foxn1+ ES cells into Gfp− Foxn1nu/nu blastocysts (Figure 5). On thymus tissue sec-

c

a ES cells Gfp + Foxn1 +/+

Estimation of number and location of Foxn1-dependent TEC by visualization of Gfp+ nuclei DAPI = all nuclei Gfp= Foxn1-dependent TEC

b

Blastocysts Gfp – Foxn1nu/nu

Figure 5 Visualization of Foxn1-dependent thymus epithelium. The rare representation of TEC among all thymus cells is demonstrated in chimeric mice generated by injection of Gfp+ Foxn1+/+ embryonic stem (ES) cells (a) into Gfp− Foxn1nu/nu blastocysts (b). Percentages, phenotypes and tissue distribution of Gfp+ cells can be determined by ﬂow cytometry (not shown) and by histology (c). The thymus section is from a chimera with very low (<5%) ES cell contribution to thymocytes or thymic mesenchyme, and hence the Gfp+ cells represent mostly Foxn1-dependent thymus epithelium. Note the abundance of mTEC (inner area) and, by comparison, the paucity of cTEC (outer area), which implies that the density of the two TEC types is different.

tions, Gfp expression is conﬁned to the cell’s nucleus by nuclear localization and indicates the position of cells from ES cell origin. In the example shown in Figure 5, there was minimal contribution (<5%) of the ES cell to hematopoietic cells (thymocytes) and to nonepithelial stromal cells, and hence the vast majority of Gfp+ cells in the thymus represent cells that are dependent in development or maintenance on Foxn1 expression, that is, they are TEC. Overall, the density of these Foxn1-dependent TEC is much higher in the medulla than in the cortex. This is perhaps surprising given that the cortex space is completely ﬁlled with keratin+ cells, but the distribution of Gfp+ cells indicates that mTEC www.annualreviews.org • Thymus Organogenesis

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and cTEC appear to cover different thymus volumes per cell. The conclusion that mTEC outnumber cTEC in situ is certainly compatible with the observation made by many laboratories that proportionally more mTEC than cTEC are routinely retrieved from stromal cell preparations. Lack of Foxn1 expression in nude mice becomes phenotypically evident as early as E12.5 or 13.5 when the Foxn1-deﬁcient thymus anlage fails to grow adequately when compared to wild-type thymus (22, 42, 144, 145). The nude thymus rudiment is also characterized by a near absence of hematopoietic cells (22, 42, 146), possibly related to loss of expression of the chemokines CCL25 (ligand of CCR9) and CXCL12 (ligand of CXCR4) in the embryonic nude thymus (147). Interestingly, embryonic nude thymic epithelial cells are also deﬁcient in expression of the Notch ligands Dll1 and Dll4 (145). Although the question whether Notch-Notch ligand interactions and concomitant T cell commitment take place before or after entry into the thymus is still debated (see 148 for a recent discussion), lack of Notch ligands in the nude thymus may be prohibitive for T cell development and, as such, may contribute to the alymphoid nude thymus phenotype. Virtually all medullary and cortical TEC in chimeras constructed from Foxn1+/+ plus Foxn1nu/nu embryos (149, 150) or from Foxn1+/+ ES cells plus Foxn1nu/nu blastocysts (9) were from the Foxn1+ but not Foxn1nu origin. This demonstrates that Foxn1 acts in a cell-autonomous manner in thymic epithelial cells. The action of the Foxn1 gene in cis, with no rescue in trans, implies that the essential target genes of the Foxn1 transcription factor do not include a gene encoding a soluble factor such as a growth factor or a cytokine, or at least none that plays a substantial role in the nude thymus phenotype. Although genes have been identiﬁed that are differentially expressed between Foxn1+ and Foxn1nu TECs (144), functionally crucial target genes are unknown. This may appear surprising given that Foxn1 has been known for some time. It

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should be noted, however, that it has been impossible to study the true function of Foxn1 either in the development or in the maintenance of thymus epithelium in cell lines in vitro. This necessitates analyses of primary thymic epithelial cells that are rare cells (on the order of a few percent of total thymus cells) in a normal thymus and are even rarer in a nude thymus. Hence, although cell numbers of primary wild-type and mutant TEC are permissive for genome-wide expression analyses, low cell numbers have precluded biochemical studies, including the identiﬁcation of relevant DNA binding sites or protein cofactors that regulate the transcription factor activity of Foxn1 in primary TEC. As a consequence, the molecular function of Foxn1 in thymic epithelium remains enigmatic. Likewise, it will be of interest to determine the role of Foxn1 beyond the developmental block in nude mice, that is, in differentiated thymus epithelium and perhaps during thymus involution.

Traf6 and RelB TEC differentiation is completely blocked in Foxn1nu/nu mice at a stage precluding any support of T cell development. In contrast, thymus development is permissive in mice deﬁcient in the NF-κB component RelB or the TNF receptor-associated factor Traf6. Nevertheless, both of these mutants show major defects in TEC structure and function that may indicate faulty TEC differentiation or maintenance. Traf6 is a signal transducer in the NFκB pathway that activates IκB kinase (IKK) in response to proinﬂammatory cytokines. IKK converts the RelB/p100 dimer to RelB/p52, which activates target genes in the nucleus (reviewed in 151). Based on an NF-κB activity reporter mouse, the NF-κB pathway is used in the developing thymus in cells with a medullary location (152). In the absence of Traf6 (153), the thymus architecture is disorganized and the medulla-cortex separation is blurry. Medullary TEC populations are either missing or lack expression of certain cell surface markers (UEA-1− but K5+ phenotype).

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These mutants suffer from autoimmunity that correlates strongly with lack of Aire+ TEC, concomitantly reduced expression of tissuerestricted self-antigens on the TEC side, and an absence of Foxp3+ regulatory T cells in the thymus. These defects are indeed stromal cell–intrinsic, as shown by transfer of autoimmune symptoms by thymus grafting. RelB expression was markedly reduced in thymus stroma ex vivo and in TEC cell lines and could be restored by reintroduction of Traf6. The diminished expression of RelB is a plausible molecular mechanism because earlier work has shown that RelB-deﬁcient mice also have severe medullary defects that render mice autoimmune (154–156). The phenotype of the Traf6-deﬁcient thymus has been proposed to be independent of LTβR ligand signaling on mTEC (118) because LTβR-induced activation of NF-κB was unaffected by lack of Traf6 (153). Overall, the importance of different NF-κB pathways in thymus epithelium is not entirely clear because Traf6-independent NFκB activity was noted in the aforementioned reporter mouse (152). The molecular defects in mice lacking RelB or Traf6 give novel insights into the genetic requirements for a key thymus function, tolerance induction. However, further interpretations of these and other studies into TEC defects are complicated by the ambiguity of whether the gene defects cause ablation of a cell type (e.g., Aire-expressing mTEC) or alter the gene expression (e.g., Aire in otherwise normal TEC).

p63 Expression of p63, a homolog of the tumor suppressor p53, is required for the normal development and maintenance of many epithelial tissues. The widespread epithelial defects in mice lacking p63 are consistent with a crucial function of this gene in epithelial stem cells. p63−/− mice possess very small thymi that were recently analyzed in detail (136, 157). p63 was expressed in all K8+ thymic epithelial cells on E12 and continues to be

expressed later in ontogeny in some but not all medullary and cortical TEC. Numbers of thymocytes were very low, but T cell development was normal in p63−/− thymi. Hence, mutant TEC are, in principle, functional (136, 157). This also holds true for hematopoietic progenitors, as shown by T cell development from p63−/− fetal liver stem cells transferred into Rag-2-deﬁcient mice. Further experiments show that TEC from p63−/− thymi have reduced expansion potential in a general epithelial cell colony assay (136). Using single cell suspensions from rat thymus, Senoo et al. (136) also developed an interesting assay for continuous culture of thymus epithelium that was considered a thymus epithelial stem cell assay. Cells that expanded in this assay expressed p63, and knock-down of p63 strongly impaired the colony size, directly supporting the argument for an important role of p63 in TEC or TEC stem cell maintenance. Because support of T cell development is the ultimate functional test for TEC, it would be interesting to know whether TEC that arise in this stem cell assay are functional. An additional mechanism for the abnormally small p63−/− thymus may be the reduced expression of p63 target genes, some of which are familiar as they play a role in thymus organogenesis. Notably, FgfR2-IIIb, which was mentioned earlier as a crucial receptor involved in mesenchymal-epithelial interactions (72), as well as Jag2 were downregulated (157). Collectively, these recent ﬁndings on the role of p63 in TEC development, and possibly TEC stem cell maintenance, may open new experimental access to the identity of thymus stem cells and their molecular regulation.

FUNCTIONAL CERVICAL THYMUS IN MICE Incidence Recently, evidence has been provided that mice have a cervical thymus that is indeed www.annualreviews.org • Thymus Organogenesis

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functional in its ability to be colonized by bone marrow–derived progenitors, to generate thymocytes that are TCR repertoire selected according to the laws of positive and negative selection, and to export mature immunocompetent T cells. Terszowski et al. (41) found cervical thymus in chimeras generated as controls for mutant chimeras initially made to test the function of Tbx1 in thymus organogenesis. Dooley and colleagues (158) searched for ectopic thymus in the neck based on the similarity of thymus epithelium with nonthymic epithelium such as respiratory epithelium (90) and on the observation that samples of human parathyroid showed inclusions of ectopic thymus (158). Both reports provided evidence for a strain-dependent incidence of cervical thymi ranging from ∼50% (158) to ∼90% (41) in BALB/c mice and the lower but still signiﬁcant frequency of ∼30%

a

b Foxn1:: Egfp

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Functional cervical thymi in mice. (a) Visualization of bilateral cervical (neck) thymi in a Foxn1::Egfp reporter mouse, and (b) histological ¨ comparison of May-Grunwald-Giemsa-stained tissue sections from the thoracic thymus and one cervical thymus lobe in a C57BL/6 mouse. The functional properties of neck thymus have recently been described (41, 158). 376

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(158) to ∼50% (41) in C57BL/6 mice. These differences might be explained by the different counting of either all detectable (41) or only medially located thymi (158). As other tools of visualization of thymus become available (39, 40, 41), frequencies of cervical thymus in mice can be measured more precisely. Examples of cervical thymi are shown in a mouse line (FVB × C56BL/6 backcross) transgenic for a Foxn1Egfp reporter gene in which Foxn1-expressing cells are visualized speciﬁcally by green ﬂuorescence (41) (Figure 6a), and in a normal C57BL/6 mouse in which two thoracic and one cervical lobe are ¨ displayed as May-Grunwald-Giemsa-stained tissue sections (Figure 6b). Hence, although cervical thymi are not detectable in every single mouse in the strains analyzed so far, two of the most commonly used laboratory strains (BALB/c and C57BL/6) frequently have cervical thymi. Although the neck region of nude mice has, to my knowledge, not formally been examined for cervical thymi, the known lack of functional T cells in this mutant and the expression of Foxn1 in TEC in the neck thymus (41, 158) support the argument for a common defect in the generation of thoracic and cervical thymi in the nude mouse.

The cervical thymus in mice is not without precedent, because neck thymus is known from other mammals. Primitive mammals such as marsupials (pouched mammals) provide interesting examples. Most marsupials have only thoracic thymus, whereas others, interestingly, have both thoracic and cervical thymi (kangaroo or possum), and yet others have only a cervical thymus (koala) (159). In some of these species, cervical thymus tissue can be mingled with parathyroid tissue. In sheep, cattle, and pigs, the thymus has cervical and thoracic parts belonging to one thymus and therefore they are connected to each other (160). In humans, case reports of diseaseassociated cervical thymi (161–163) suggest

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that cervical thymus is rare (see references in 41, 158) and is caused by failure of the thymus to properly descend to its ﬁnal mediastinal location. However, others have estimated that the cervical thymus in humans may be quite frequent, with an incidence of 50% in adults (further references in 158) and 60% in children (164). Collectively, precise ﬁgures on the normal incidence of neck thymus in human are difﬁcult to extract from the literature (for further references and considerations, see also 162), and a deﬁnitive answer to this question may require further and more systematic imaging data.

Origin of the Cervical Thymus in Mice and the Relationship of Thymus and Parathyroid The following possibilities can be considered for the origin of the second thymus. 1. The cervical thymus originates from the third pouch as the thoracic thymus does, but it takes the parathyroid route of migration. This could occur as a result of imprecise separation of the parathyroid and thymus domains (62) within the third pouch epithelium, leading to the partition of thymus progenitors into the parathyroid anlage and vice versa (see below). This would be reminiscent of the model of secondary splitting of the thymus anlagen in chicken (Figure 3). In this regard, it is noteworthy that thymus and parathyroid organs do not only develop from the same pharyngeal pouch but also share, at least in mice, some functional similarities. Some 70% of Gcm2-deﬁcient mice survive despite a complete absence of parathyroids (64). In mice, but not in humans (165), the thymus was identiﬁed as the auxiliary source of parathyroid hormone (PTH) (64). However, though expression of Gcm2 is restricted to the parathyroid and is required for the development of precursors for this organ

(104), the PTH-producing cells in the thymus colocalize with Gcm1, a gene homologous to Gcm2, that is expressed in clusters of cells in the thymus (166). The lineage of these Gcm1-expressing cells in the thymus is unknown. 2. The anlage for the cervical thymus arises independently in a different pouch, and, up to now, these few TEC progenitors may have escaped detection. An origin in a separate pharyngeal pouch would be akin to the multiple thymus anlagen in the shark (Figure 3) and could be considered an atavism. 3. There could be a delayed epithelial speciﬁcation toward the cervical thymus fate at an unknown location in the neck. In this case, a search for cells expressing Foxn1 between days 14 and 18 outside the thoracic thymus may provide a clue. This latter idea is supported by the notion that the development of the cervical thymus is delayed by about one week compared to the thoracic thymus (41) (see below).

Developmental Kinetics of the Cervical Thymus The identiﬁcation of the primitive anlage for the cervical thymus in the neck of newborn mice also led to the surprising result that thoracic and cervical thymi do not develop in parallel but asynchronously (41). Cells in the cervical thymus anlage in the neck of newborn mice express Foxn1 and MHC class II, indicative of their thymus identity, but, at this stage, the overall TEC structure is still primitive, with many cells coexpressing K5 and K18 and poor epithelial cell compartmentalization. In addition, there are only few and only immature thymocytes present in the neck thymus at this stage (41). TCR DJβ and V(D)Jβ rearrangements are undetectable in the neck on fetal day 18.5, further supporting the lack of thymic function in the neck before birth. Based on the kinetics and a maturational comparison of cervical and thoracic thymus, the www.annualreviews.org • Thymus Organogenesis

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cervical thymus appears at birth as immature as the major thymus one week earlier, that is, on days 12 or 13 in embryonic development. The developmental delay between the thoracic and the cervical thymus is also evident by a burst of CD4+ CD8+ cells as late as one week after birth in the neck thymus, as opposed to the same burst, on a larger scale, in the thorax thymus on fetal day 18. The presumed onset of thymopoiesis in the neck only after birth could make the cervical thymus miss the wave of production of Vγ5-expressing thymocytes (G. Terszowski & H.-R. Rodewald, unpublished) that are the precursors of the canonical TCR-expressing dendritic epidermal T cells (167). Along these lines, asynchronous functioning of thoracic and cervical thymus within individual mice may give clues as to which properties of the thymus are organautonomous and which are regulated at the level of the organism or at the level of the bone marrow that is producing thymus-bound progenitors.

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Anatomy Cervical thymi in the mouse are often single unilateral lobes, but two bilateral (Figure 6) or even two unilateral lobes can also be found. Cervical thymi are usually located medially, that is, on the inside of the large cervical vessels, and along the ventral region between the thyroid or even more cranially toward the submandibular glands and caudally toward the lower part of the neck. However, in no instance could we ﬁnd open connections to the chest or a continuous chest-neck-thymus in mice. The position of the cervical thymus is usually superﬁcial, but cervical thymus lobes are also present between or under muscle strings (for anatomical positions, see 41, 158). Overall, the distribution of cervical thymus is much wider than previously suggested on the basis of previous anatomical reports of thymus tissue located within or next to the thyroid (168, 169). Likewise, the view that mice, similar to sheep or cattle, have a cervical part of

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their thymus (170) is not supported by recent studies (41, 158). Many of these discrepancies, and the fact that cervical thymus in mice has by and large been overlooked in immunology, could be explained by the fact that cervical thymus lobes may have been considered cervical lymph nodes. Cervical thymi can easily be distinguished from lymph nodes by histology or ﬂow cytometry using thymus-speciﬁc markers (41, 158).

Function of the Cervical Thymus and Implications Several differences have been noted comparing cervical and thoracic thymus. Cervical thymi are mostly single small lobes composed of medulla and cortex, whereas thoracic thymi are, of course, composed of multiple medulla and cortex structures (41, 158) (Figure 6). Thymic ﬁbroblasts are found more locally restricted in the neck than the chest thymus (158). The cortex in the cervical thymus contains clusters of CD4+ CD8− cells that are unusual among normal cortical CD4+ CD8+ cells (41). The cervical thymus has between 105 and 2 × 105 cells but these cell numbers can also be smaller or be as large as 106 . This cellularity is low when compared to the thoracic thymus (∼108 cells) but large when compared to, say, single gut cryptopatches, intestinal areas harboring ∼103 lymphoid progenitors (171, 172). When tested in model systems for positive and negative TCR repertoire selection, thoracic and cervical thymus obey the same wellknown rules (41, 158). Moreover, the cervical thymus can export functional T cells when grafted into nude mice (41, 158), and these T cells mount T cell help required for an antibody response, including class switching (41). The contribution of the cervical thymi to the overall T cell pool in a mouse that has both thoracic and cervical thymi is unknown, and it may be proportional to the size differences. Likewise, the contribution of cervical thymi to peripheral T cells in a

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mouse that underwent thoracic thymectomy remains to be determined. The cervical thymus contains Foxp3+ thymocytes in their normal medullary location, suggesting that the neck thymus provides both effector and regulatory T cells to the periphery (158). This raises some interesting questions regarding the possible role of the cervical thymus in models of autoimmunity that are driven by thoracic thymectomy shortly after birth (173). Because cervical thymi have not been removed in these experiments, it remains to be determined whether T cells produced by the cervical thymus postnatally contribute to this phenomenon or not, and if so, why they cannot be controlled by regulatory cells that can also be generated in the cervical thymus. Hints that mice have a thymus in their neck date back to 1964. A histological section from the parathyroid region of a BALB/c mouse neck included thymus tissue (168; discussed in 174, 175). Moreover, cervical thymus embedded in the thyroid gland in 80% of female diabetes-prone NOD but not in normal control mice had been reported (169). Cervical thymi in lieu of normal thoracic thymi were identiﬁed in Pax9-deﬁcient mice, providing a case for ectopic thymus as a result of failed migration toward the thorax (87). Anatomical drawings, on the other hand, suggested that

mice, similar to sheep (160), have a cervical part of the thymus (170), a view not supported by the recent analyses (41, 158). In any case, the function of the cervical thymus in mice was clearly unknown. Moreover, as far as we know, neither the proper anatomical location (as opposed to cervical lymph nodes) nor the incidence had been reported. It is clear now that the cervical thymus, though not detectable in every individual mouse, is not restricted to the BALB/c strain, and it is also not restricted anatomically to the thyroid/parathyroid area, nor is it part of the thoracic thymus. Arguments surrounding early hints of a cervical thymus, and the fact that it had been ignored in subsequent experimentation, have recently been exchanged (for a discussion and further references, see 174– 176). Because the cervical thymus is capable of generating and releasing functional T cells that render athymic mice immunocompetent, at least toward antigens tested so far, it may have confounded thoracic thymectomy experiments. Determining to what degree this is the case requires further work. Finally, comparative analyses of thoracic and cervical thymi may yield insights into the regulation of thymus organogenesis, the location dependency of thymus functions, and possibly thymus aging.

DISCLOSURE STATEMENT The author is not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS I thank past and present members of my laboratory, in particular Carmen Blum, Verena ¨ ¨ Buhrmann, Susanna Muller, Sabine Paul, Greg Terszowski (Ulm), and Corinne Haller (Basel), for their contributions to our studies addressing thymus development. I am grateful to Graham ¨ Fehling, Reinhard Pabst, and Nick Anderson, Thomas Boehm, Louis Du Pasquier, Hans Jorg Trede for advice and discussions, Louis Du Pasquier for drawings incorporated into Figure 3, Virginia Papaioannou for providing Gfp-marked ES cells (Figure 5), and Thomas Boehm for collaborating on the Foxn1::Egfp reporter mouse (Figure 6). Thymus research in my laboratory is supported by grants from the Deutsche Forschungsgemeinschaft (SFB 497-B5 and Klinische Forschergruppe 142-P8).

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66. Petrie HT. 2002. Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production. Immunol. Rev. 189:8–19 67. Shinohara T, Honjo T. 1996. Epidermal growth factor can replace thymic mesenchyme in induction of embryonic thymus morphogenesis in vitro. Eur. J. Immunol. 26:747–52 68. Suniara RK, Jenkinson EJ, Owen JJ. 2000. An essential role for thymic mesenchyme in early T cell development. J. Exp. Med. 191:1051–56 69. Jiang X, Rowitch DH, Soriano P, McMahon AP, Sucov HM. 2000. Fate of the mammalian cardiac neural crest. Development 127:1607–16 70. Yamazaki H, Sakata E, Yamane T, Yanagisawa A, Abe K, et al. 2005. Presence and distribution of neural crest-derived cells in the murine developing thymus and their potential for differentiation. Int. Immunol. 17:549–58 71. Kranz A, Kendall MD, von Gaudecker B. 1997. Studies on rat and human thymus to demonstrate immunoreactivity of calcitonin gene-related peptide, tyrosine hydroxylase and neuropeptide Y. J. Anat. 191:441–50 72. Revest JM, Suniara RK, Kerr K, Owen JJ, Dickson C. 2001. Development of the thymus requires signaling through the ﬁbroblast growth factor receptor R2-IIIb. J. Immunol. 167:1954–61 73. Erickson M, Morkowski S, Lehar S, Gillard G, Beers C, et al. 2002. Regulation of thymic epithelium by keratinocyte growth factor. Blood 100:3269–78 74. Rossi SW, Jeker LT, Ueno T, Kuse S, Keller MP, et al. 2007. Keratinocyte growth factor (KGF) enhances postnatal T-cell development via enhancements in proliferation and function of thymic epithelial cells. Blood 109:3803–11 75. Min D, Taylor PA, Panoskaltsis-Mortari A, Chung B, Danilenko DM, et al. 2002. Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation. Blood 99:4592–600 76. Rossi S, Blazar BR, Farrell CL, Danilenko DM, Lacey DL, et al. 2002. Keratinocyte growth factor preserves normal thymopoiesis and thymic microenvironment during experimental graft-vs-host disease. Blood 100:682–91 77. Manley NR, Capecchi MR. 1998. Hox group 3 paralogs regulate the development and migration of the thymus, thyroid, and parathyroid glands. Dev. Biol. 195:1–15 78. Xu PX, Zheng W, Laclef C, Maire P, Maas RL, et al. 2002. Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid. Development 129:3033– 44 79. Zou D, Silvius D, Davenport J, Grifone R, Maire P, Xu PX. 2006. Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1. Dev. Biol. 293:499–512 80. Laclef C, Souil E, Demignon J, Maire P. 2003. Thymus, kidney and craniofacial abnormalities in Six1 deﬁcient mice. Mech. Dev. 120:669–79 81. Dietrich S, Gruss P. 1995. Undulated phenotypes suggest a role of Pax-1 for the development of vertebral and extravertebral structures. Dev. Biol. 167:529–48 82. Wallin J, Eibel H, Neubuser A, Wilting J, Koseki H, Balling R. 1996. Pax1 is expressed during development of the thymus epithelium and is required for normal T-cell maturation. Development 122:23–30 83. Su DM, Manley NR. 2000. Hoxa3 and Pax1 transcription factors regulate the ability of fetal thymic epithelial cells to promote thymocyte development. J. Immunol. 164:5753–60 84. Su D, Ellis S, Napier A, Lee K, Manley NR. 2001. Hoxa3 and pax1 regulate epithelial cell death and proliferation during thymus and parathyroid organogenesis. Dev. Biol. 236:316–29 www.annualreviews.org • Thymus Organogenesis

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85. Conway SJ, Henderson DJ, Copp AJ. 1997. Pax3 is required for cardiac neural crest migration in the mouse: evidence from the splotch (Sp2H) mutant. Development 124:505– 14 86. Peters H, Neubuser A, Kratochwil K, Balling R. 1998. Pax9-deﬁcient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities. Genes Dev. 12:2735–47 87. Hetzer-Egger C, Schorpp M, Haas-Assenbaum A, Balling R, Peters H, Boehm T. 2002. Thymopoiesis requires Pax9 function in thymic epithelial cells. Eur. J. Immunol. 32:1175– 81 88. Jerome LA, Papaioannou VE. 2001. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat. Genet. 27:286–91 89. Hollander G, Gill J, Zuklys S, Iwanami N, Liu C, Takahama Y. 2006. Cellular and molecular events during early thymus development. Immunol. Rev. 209:28–46 90. Dooley J, Erickson M, Farr AG. 2005. An organized medullary epithelial structure in the normal thymus expresses molecules of respiratory epithelium and resembles the epithelial thymic rudiment of nude mice. J. Immunol. 175:4331–37 91. Gillard GO, Dooley J, Erickson M, Peltonen L, Farr AG. 2007. Aire-dependent alterations in medullary thymic epithelium indicate a role for Aire in thymic epithelial differentiation. J. Immunol. 178:3007–15 92. Ohnemus S, Kanzler B, Jerome-Majewska LA, Papaioannou VE, Boehm T, Mallo M. 2002. Aortic arch and pharyngeal phenotype in the absence of BMP-dependent neural crest in the mouse. Mech. Dev. 119:127–35 93. Bleul CC, Boehm T. 2005. BMP signaling is required for normal thymus development. J. Immunol. 175:5213–21 94. Patel SR, Gordon J, Mahbub F, Blackburn CC, Manley NR. 2006. Bmp4 and Noggin expression during early thymus and parathyroid organogenesis. Gene. Expr. Patterns 6:794–99 95. Greenberg F. 1993. DiGeorge syndrome: an historical review of clinical and cytogenetic features. J. Med. Genet. 30:803–6 96. Naiche LA, Harrelson Z, Kelly RG, Papaioannou VE. 2005. T-box genes in vertebrate development. Annu. Rev. Genet. 39:219–39 97. Perez E, Sullivan KE. 2002. Chromosome 22q11.2 deletion syndrome (DiGeorge and velocardiofacial syndromes). Curr. Opin. Pediatr. 14:678–83 98. Packham EA, Brook JD. 2003. T-box genes in human disorders. Hum. Mol. Genet. 12:37– 44 99. Scambler PJ. 2000. The 22q11 deletion syndromes. Hum. Mol. Genet. 9:2421–26 100. Lindsay EA, Vitelli F, Su H, Morishima M, Huynh T, et al. 2001. Tbx1 haploinsufﬁciency in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410:97–101 101. Chapman DL, Garvey N, Hancock S, Alexiou M, Agulnik SI, et al. 1996. Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206:379–90 102. Arnold JS, Werling U, Braunstein EM, Liao J, Nowotschin S, et al. 2006. Inactivation of Tbx1 in the pharyngeal endoderm results in 22q11DS malformations. Development 133:977–87 103. Zhang Z, Huynh T, Baldini A. 2006. Mesodermal expression of Tbx1 is necessary and sufﬁcient for pharyngeal arch and cardiac outﬂow tract development. Development 133:3587– 95 104. Liu Z, Yu S, Manley NR. 2007. Gcm2 is required for the differentiation and survival of parathyroid precursor cells in the parathyroid/thymus primordia. Dev. Biol. 305:333–46

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143. Balciunaite G, Keller MP, Balciunaite E, Piali L, Zuklys S, et al. 2002. Wnt glycoproteins regulate the expression of FoxN1, the gene defective in nude mice. Nat. Immunol. 3:1102– 8 144. Bleul CC, Boehm T. 2001. Laser capture microdissection-based expression proﬁling identiﬁes PD1-ligand as a target of the nude locus gene product. Eur. J. Immunol. 31:2497– 503 145. Tsukamoto N, Itoi M, Nishikawa M, Amagai T. 2005. Lack of Delta like 1 and 4 expressions in nude thymus anlages. Cell. Immunol. 234:77–80 146. Itoi M, Kawamoto H, Katsura Y, Amagai T. 2001. Two distinct steps of immigration of hematopoietic progenitors into the early thymus anlage. Int. Immunol. 13:1203–11 147. Bleul CC, Boehm T. 2000. Chemokines deﬁne distinct microenvironments in the developing thymus. Eur. J. Immunol. 30:3371–79 148. Jenkinson EJ, Jenkinson WE, Rossi SW, Anderson G. 2006. The thymus and T-cell commitment: the right niche for Notch? Nat. Rev. Immunol. 6:551–55 149. Blackburn CC, Augustine CL, Li R, Harvey RP, Malin MA, et al. 1996. The nu gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors. Proc. Natl. Acad. Sci. USA 93:5742–46 150. Martinic MM, Rulicke T, Althage A, Odermatt B, Hochli M, et al. 2003. Efﬁcient T cell repertoire selection in tetraparental chimeric mice independent of thymic epithelial MHC. Proc. Natl. Acad. Sci. USA 100:1861–66 151. Karin M, Lin A. 2002. NF-κB at the crossroads of life and death. Nat. Immunol. 3:221–27 152. Dickson KM, Bhakar AL, Barker PA. 2004. TRAF6-dependent NF-κB transcriptional activity during mouse development. Dev. Dyn. 231:122–27 153. Akiyama T, Maeda S, Yamane S, Ogino K, Kasai M, et al. 2005. Dependence of selftolerance on TRAF6-directed development of thymic stroma. Science 308:248–51 154. Burkly L, Hession C, Ogata L, Reilly C, Marconi LA, et al. 1995. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature 373:531–36 155. Weih F, Carrasco D, Durham SK, Barton DS, Rizzo CA, et al. 1995. Multiorgan inﬂammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-κB/Rel family. Cell 80:331–40 156. Zuklys S, Balciunaite G, Agarwal A, Fasler-Kan E, Palmer E, Hollander GA. 2000. Normal thymic architecture and negative selection are associated with Aire expression, the gene defective in the autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). J. Immunol. 165:1976–83 157. Candi E, Ruﬁni A, Terrinoni A, Giamboi-Miraglia A, Lena AM, et al. 2007. Np63 regulates thymic development through enhanced expression of FgfR2 and Jag2. Proc. Natl. Acad. Sci. USA 104:11999–2004 158. Dooley J, Erickson M, Gillard GO, Farr AG. 2006. Cervical thymus in the mouse. J. Immunol. 176:6484–90 159. Haynes JI. 2003. The marsupial and monotreme thymus, revisited. J. Zool. London 253:167–73 160. Yamashita A, Miyasaka M, Trnka Z. 1985. Early post-thymic T cells: studies on lymphocytes in the lymph from the thymus of sheep. In Immunology of the Sheep, ed. B Morris, M Miyasaka, pp. 165–86. Basel: Editiones Roche 161. Tovi F, Mares AJ. 1978. The aberrant cervical thymus. Embryology, pathology, and clinical implications. Am. J. Surg. 136:631–37 162. Millman B, Pransky S, Castillo J III, Zipfel TE, Wood WE. 1999. Cervical thymic anomalies. Int. J. Pediatr. Otorhinolaryngol. 47:29–39 www.annualreviews.org • Thymus Organogenesis

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163. Shah SS, Lai SY, Ruchelli E, Kazahaya K, Mahboubi S. 2001. Retropharyngeal aberrant thymus. Pediatrics 108:E94 164. Chu WC, Metreweli C. 2002. Ectopic thymic tissue in the paediatric age group. Acta Radiol. 43:144–46 165. Maret A, Bourdeau I, Ding C, Kadkol SS, Westra WH, Levine MA. 2004. Expression of GCMB by intrathymic parathyroid hormone-secreting adenomas indicates their parathyroid cell origin. J. Clin. Endocrinol. Metab. 89:8–12 166. Hashemolhosseini S, Hadjihannas M, Stolt CC, Haas CS, Amann K, Wegner M. 2002. Restricted expression of mouse GCMa/Gcm1 in kidney and thymus. Mech. Dev. 118:175– 78 167. Hayday AC, Pennington DJ. 2007. Key factors in the organized chaos of early T cell development. Nat. Immunol. 8:137–44 168. Law LW, Dunn TB, Trainin N, Levy RH, eds. 1964. Studies of Thymic Function, Vol. 2, pp. 105–20. Philadelphia: Wistar Inst. Press 169. Many MC, Drexhage HA, Denef JF. 1993. High frequency of thymic ectopy in thyroids from autoimmune prone nonobese diabetic female mice. Lab. Invest. 69:364–67 170. Popesko P, Rajtova V, Horak J. 1992. A Colour Atlas of the Anatomy of Small Laboratory Animals, Vol. 2. Bratislava: Wolfe 171. Kanamori Y, Ishimaru K, Nanno M, Maki K, Ikuta K, et al. 1996. Identiﬁcation of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. J. Exp. Med. 184:1449–59 172. Rocha B. 2007. The extrathymic T-cell differentiation in the murine gut. Immunol. Rev. 215:166–77 173. Asano M, Toda M, Sakaguchi N, Sakaguchi S. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387–96 174. Miller JF. 2006. Investigating a second thymus in mice. Science 312:1597–98 175. Rodewald HR. 2006. Investigating a second thymus in mice (response). Science 312:1598 176. von Boehmer H. 2006. Immunology. Thoracic thymus, exclusive no longer. Science 312:206–7

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:355-388. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury1 and Judy Lieberman2 1

Dana Farber Cancer Institute and Department of Radiation Oncology, 2 Immune Disease Institute and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115; email: Dipanjan [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:389–420

Key Words

First published online as a Review in Advance on November 28, 2007

cytotoxic T lymphocyte, cytotoxic granule, natural killer cell, serine protease, granule exocytosis, serpin

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090404 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0389$20.00

Abstract The granzymes are cell death–inducing enzymes, stored in the cytotoxic granules of cytotoxic T lymphocytes and natural killer cells, that are released during granule exocytosis when a speciﬁc virus-infected or transformed target cell is marked for elimination. Recent work suggests that this homologous family of serine esterases can activate at least three distinct pathways of cell death. This redundancy likely evolved to provide protection against pathogens and tumors with diverse strategies for evading cell death. This review discusses what is known about granzyme-mediated pathways of cell death as well as recent studies that implicate granzymes in immune regulation and extracellular proteolytic functions in inﬂammation.

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INTRODUCTION

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The granzymes (granule enzymes) are a family of highly homologous serine proteases contained in cytotoxic granules of innate and adaptive immune killer cells. Their major job is to induce cell death to eliminate viruses and tumor cells. The granzymes may also play a role in immune regulation by controlling the survival of activated lymphocytes and may also regulate inﬂammation by acting on extracellular substrates. There are ﬁve human granzymes and ten mouse granzymes, expressed from three gene clusters. Granzyme A and granzyme B (GzmA, GzmB) are the most abundant granzymes. GzmB, which cleaves after aspartic acid residues in many of the same substrates as the caspases, has been the most extensively studied, but recent studies have begun to elucidate the roles of and cell death pathways activated by GzmA and the other (so-called orphan) granzymes. Killer cells, including natural killer (NK) cells, cytotoxic CD4 and CD8 T cells, and even some regulatory T cells (Tregs), express highly variable and tightly regulated patterns of granzymes that depend on both cell type and mode of activation, but investigators are only beginning to study what controls the expression of each of the granzymes. The cytotoxic granules also contain perforin, needed to deliver the granzymes into the target cell. When cytotoxic T lymphocytes (CTLs) and NK cells form an immune synapse with a speciﬁcally recognized target cell destined for elimination, cytotoxic granules move to the immune synapse where the cytotoxic granule membrane fuses with the killer cell membrane, releasing the granule contents into the synaptic cleft. The granzymes are then delivered into the target cell (but not the killer cell), where they initiate at least three distinct pathways of programmed cell death. The killer cell is a serial killer that escapes this encounter unharmed and can then seek and destroy another target cell. This review focuses on what is known about the biochemistry, gene regulation, cell

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biology, functions, and inhibitors of the granzyme family, with special attention to what has been learned in the past ﬁve years since the last comprehensive review in this series by Russell & Ley (1).

GENE EXPRESSION The granzyme serine proteases are encoded in three distinct clusters in humans and mice: GzmA and GzmK, both tryptases, on chromosome 5 (human) and 13 (mouse); GzmB and GzmH on chromosome 14 (human) and their mouse counterparts (GzmB and GzmC) also on chromosome 14; and GzmM, which cleaves after Met or Leu, on chromosome 19 (human) and chromosome 10 (mouse) (Figure 1). GzmB cleaves after aspartic acid, whereas GzmC and GzmH have the speciﬁcity of chymotrypsin, cleaving after aromatic amino acids. The GzmB cluster also encodes for cathepsin G and mast cell chymase. The mouse GzmB cluster is uniquely expanded by multiple gene duplications to encode, in addition, GzmD, E, F, G, L, and N. Nothing is known about these mouse-speciﬁc enzymes, but investigators have hypothesized that they may have evolved to defend against speciﬁc common mouse pathogens (1).

Expression in Noncytolytic Lymphoid Cells Granzymes in the past were thought to be expressed uniquely only by NK cells and CTLs, which could be either CD8+ or cytolytic CD4+ cells, usually of the Th1 lineage (1). However, granzyme transcripts can be ampliﬁed by RT-PCR from mouse progenitor T cells (2). GzmB transcripts are found in prothymocytes in fetal liver, whereas mRNA for GzmA, B, and C are found in immature double-negative thymocytes. Although GzmA transcripts are found in thymocytes with the potential to develop into CD8+ cells, GzmA activity is detected only in the most mature CD4− CD8+ thymocytes. These results suggest post-transcriptional regulation

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GzmA

1 29

65

70

114

262 aa

125

206

26

63

68

116

20

60

65 108

127

119

208

219

254

262 aa 197

20

60

65 108

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208

25

62

67 111

122

Catalytic activity (GzmB): Asp-I-Xaa- >> -Asn-I-Xaa- > -Met-I-Xaa-Ser-I-Xaa-

262 aa 196

Catalytic activity (GzmH): Phe-I-Xaa

239

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GzmM 1

Locus: Chymase

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GzmH

1

Catalytic activity: -Arg-I-Xaa-, -Lys-I-Xaa- >> -Phe-I-Xaa-

262 aa

GzmB

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Locus: Tryptase

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GzmK

1

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Locus: Metase

257 aa 201

212

Catalytic activity (GzmH): Met-I-Xaa, Leu-I-Xaa

249

Trypsin-like serine protease domain

Active site 2, conserved Asp

Active site 1, conserved His

Active site 3, conserved Ser

gzmM gzmA gzmK gzmB gzmH

Active site 1

Active site 2

Active site 3

SHLCGGVLVHPKWVLTAAHCL KTICAGALIAKDWVLTAAHCN HHVCGGVLIDPQWVLTAAHCQ LKRCGGFLIQDDFVLTAAHCW RKRCGGILVRKDFVLTAAHCQ

DLALLQLDGKVK DLKLLQLTEKAK DIMLVKLQTAAK DIMLLQLERKAK DIMLLQLERKAK

APCKGDSGGPLVCG DSCNGDSGSPLLCE DSCKGDSGGPLICK TSFKGDSGGPLVCN TGFKGDSGGPLVCK

Figure 1 The family of human granzymes are encoded in three clusters.

of granzyme protein (see below for additional examples). Whether there might be any function for granzyme transcripts in early progenitor T cells is unknown. In the past few years, it has become clear that granzymes are expressed in a broader array of cells and have additional noncytolytic functions in regulating lymphocyte survival and immune tolerance, as well as promoting inﬂam-

mation and potentially enhancing lymphocyte migration by proteolyzing extracellular proteins or cell surface receptors. Some of these additional functions do not require perforin and may be activated by cells that express granzymes, but not perforin. GzmB, but not GzmA, is expressed in Tregs and plays an important perforin-dependent role in Treg function in mice (3, 4). B-chronic www.annualreviews.org • Granzyme-Mediated Cell Death

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lymphocytic leukemia (B-CLL) cells treated with interleukin-21 (IL-21) produce low levels of GzmB, which can be enhanced by adding either CpG oligodeoxynucleotide or anti–B cell receptor (anti-BCR) antibody (5). In this setting, GzmB induces apoptosis of both the GzmB-expressing cells and untreated bystander B-CLL cells. Similarly, the combination of IL-21 and anti-BCR induces GzmB in benign human B cells, Epstein-Barr virus–transformed lymphoblasts, and many lymphoma cell lines. These results suggest that inducing GzmB expression could open new approaches to the therapy of B-CLL and other B cell malignancies.

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Expression of Granzymes in Nonlymphoid Cells GzmB is expressed in many different types of myeloid cells, generally without perforin. Within the immune system, GzmB is expressed in human plasmacytoid dendritic cells (pDCs) (6). There are comparable levels of GzmB transcripts in resting and activated pDCs, but signiﬁcantly higher levels of GzmB protein in activated cells, suggesting post-transcriptional regulation of expression. GzmB is also expressed in both normal and neoplastic human mast cells in vitro and in vivo (7). GzmB localizes to speciﬁc granules in human mast cells and is secreted upon activation as it is in CTL. Low levels of perforin mRNA and protein can be detected in one human mast cell line, HMC-1, but not in another (LAD-2) or in primary mast cells derived from cord blood and skin. In mice, only skin-associated mast cells and bone marrow– derived in vitro–differentiated mast cells make GzmB protein, but lung mast cells do not (8). Neither GzmA nor perforin are detected in mouse mast cells. Because the GzmB gene is encoded within a few hundred kilobases of mast cell proteases, the GzmB genomic region may be open and active in mast cells, allowing coordinated GzmB expression with mast cell chymase and tryptase. In human basophils, which are developmentally related to 392

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mast cells, IL-3-mediated GzmB induction in the absence of GzmA and perforin expression has also been reported (9). Expression of GzmB in mast cells and basophils suggests a role of GzmB in mediating allergic disease. In fact, GzmB has been found in bronchoalveolar lavage ﬂuid after allergen exposure. Several studies have also provided evidence for the expression of GzmB and perforin in human neutrophils, but this is controversial (10–13). GzmB is also expressed in the absence of perforin in the human reproductive system. It is detected by immunohistochemistry in developing spermatocytes and in placental trophoblasts (14). It is also produced in response to follicle-stimulating hormone by granulosa cells of the human ovary (15). In addition, GzmB protein has been detected in a subset of primary human breast carcinomas and in chondrocytes of articular cartilage (16). The speciﬁc expression of GzmB, but not perforin, in these different cell types suggests a noncytotoxic role for GzmB in these cells. The other granzymes expressed in nonlymphoid cells are mouse GzmK and GzmM. The GzmM transcript is expressed at low levels in the photoreceptor cells of the retina in the mouse (17). Interestingly, an alternatively spliced form (aGM) is exclusively expressed in these cells at much higher levels. Like GzmM, GzmK has an alternatively spliced form exclusively expressed in the brain (18). The physiological signiﬁcance of the alternative transcripts of GzmM and GzmK is yet to be determined.

Extracellular Signals Regulating Granzyme Expression Early studies with T cell populations showed that naive T cells do not express granzymes, and most activated CD8 T cells coexpress granzymes and perforin. However, the kinetics and expression level of the individual granzymes and perforin vary in different clonal populations in vitro and in vivo and depend on how they are activated (19–21). Most circulating CD8+ T lymphocytes that

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express any granzyme express both GzmA and GzmB, but some cells are positive for only one granzyme. Single-cell expression proﬁles of granzymes, perforin, and IFN-γ have been investigated in in vitro– and in vivo–activated CD8+ T cells using RT-PCR in mice (22) and intracellular staining and ﬂow cytometry in humans (4). Individual T cells show unexpected diversity in the expression of these genes. Although some pairs of genes (perforin and IFN-γ) are expressed more frequently than others, no speciﬁc combination of genes is consistently coexpressed. During in vitro activation of mouse naive lymphocytes with antibodies to CD3, CD8, CD11a, and IL-2, the expression of GzmA and GzmC is consistently delayed compared with cytolytic activity and expression of perforin and GzmB (22). When mouse CTLs are activated in vivo by inﬂuenza virus infection, cytotoxicity is evident in the lung but not in the mesenteric lymph nodes. Most antigen-speciﬁc tetramer+ CD8 T cells in the lung one week after infection express both GzmA and GzmB, and about one-third of them also express perforin. Moreover, there is no difference in the kinetics of induction in vivo of GzmA, GzmB, or perforin. In addition, GzmC is not induced by inﬂuenza infection in vivo. The diversity of expression of individual granzyme and perforin genes suggests that each gene is regulated independently, although these genes will likely share some common transcription factor recognition sites and epigenetic changes. It is likely that differences in TCR avidity, costimulatory and inhibitory receptor engagement, cytokine milieu, type and state of activation of the antigen-presenting cell, and presence of helper or regulatory CD4 T cells will inﬂuence the induction of the granzyme and perforin genes. Moreover, the cell’s prior history of activation will affect cytolytic gene expression during subsequent encounters with antigen. Surprisingly little is known about this subject. The perforin and granzyme genes are induced during T cell activation. However, the only signal shown to upregulate GzmA and B

and perforin consistently is IL-2 (23). Previously, the pleiotropic properties of IL-2 made dissociating its effects on T cell survival and proliferation from its effects on gene expression difﬁcult (24). However, a recent study in mice showed that IL-2 regulates perforin and granzyme expression directly and independently of its effect on CD8+ T cell survival and proliferation (25). Mice genetically deﬁcient in IL-2 retain the ability to elicit a CTL response against many viruses, tumors, and allografts (26, 27), although there are deﬁciencies in cytotoxicity under certain conditions (28). The other γc -dependent cytokines (IL-4, IL-7, IL-9, IL-15, and IL-21) are the most likely candidates for substituting for IL2 in its absence. IL-15 is particularly important because along with the γ-chain it also shares the β-chain with the IL-2 receptor. IL15 induces the expression of perforin, GzmA, GzmB, IFN-γ, and Fas ligand in primary mouse lymphocytes (29). IL-21 works synergistically with IL-15 to upregulate GzmA and GzmB expression in mouse CD8 T cells (30). In vivo in mice, IL-21 exhibits potent antitumor function by enhancing NK and CD8 T cell cytotoxicity (31). Similarly, in human peripheral blood CD8 T cells, IL-15 and IL-21 both activate GzmB and perforin expression, but interestingly IL-21 does so without inducing CD8 T cell proliferation (32). Other cytokines implicated in regulating granzymes and perforin are the IL-6/IL-12 family. IL-12 induces the cytotoxic activity of NK cells and enhances their expression of perforin (33). IL-27 treatment of activated CD8+ T cells signiﬁcantly augments GzmB and, to a lesser extent, perforin expression (34).

Transcriptional Regulation of Granzymes Induction of the expression of granzyme transcripts requires at least two independent stimuli—activation of the TCR and costimulation via cytokines of the γc family. The signals from several distinct signal transduction pathways are integrated in the nucleus in www.annualreviews.org • Granzyme-Mediated Cell Death

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the form of transcription factors that bind to granzyme gene regulatory elements and activate transcription. Early studies identiﬁed a 243-bp fragment upstream of the mouse GzmB transcription start site that potentially regulates GzmB transcription (35). This region contains binding sites for two ubiquitous transcription factors, activating transcription factor/cyclic AMP-responsive element binding protein (ATF/CREB) and activator protein-1 (AP-1), and two lymphoidspeciﬁc factors, Ikaros and core-binding factor (CBF/PEBP2) (36). Several of these transcription factor–binding sites are evolutionarily conserved between the human and mouse GzmB promoters (37, 38). Analysis of reporter assays using promoters that had been systematically mutated at these sites in primary cells and cell lines revealed subtle differences in the importance of some transcription factors in primary cells versus cell lines. For example, AP-1, CREB, and CBF were not as important for transcription in primary cells as they appeared to be in cell lines (37, 39). These studies suggested that combinations of transcription factors (particularly AP-1 and CBF) are required to activate GzmB expression in primary cells. The most compelling difference between the mouse and human GzmB gene promoter is the importance of the Ikaros site in human GzmB expression. Mutations introduced into the Ikaros-binding site of the human, but not mouse, GzmB promoter abrogate expression in primary T cells (37, 39). Two recent studies have shown a role for signal transducer and activator of transcription 1 (STAT1) in the transcriptional regulation of GzmB. Mouse splenocytes stimulated with IFN-α typically express GzmB. This IFN-α effect is abrogated in STAT1-deﬁcient mice (40). IL-27-mediated enhancement of GzmB and perforin expression is also impeded in STAT1-deﬁcient mice (34). IL-27-induced augmentation of GzmB and perforin expression is also dependent on the T-box family transcription factor T-bet (34). Another Tbox family member, Eomesodermin, was recently identiﬁed in activated CD8+ T cells in

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mice and shown to drive perforin, GzmB, and IFN-γ expression when ectopically expressed in mouse Th2 CD4+ cells (41). In the converse experiment, expression of a dominantnegative Eomesodermin impaired GzmB expression and cytolytic activity in CD8+ T cells. Thus, these two T-box family members, T-bet and Eomesodermin, appear to regulate GzmB expression cooperatively. Little is known regarding the transcriptional regulation of GzmA. Two groups looking to identify transcriptional targets of the glucocorticoid receptor found that GzmA expression is signiﬁcantly upregulated in a glucocorticoid-sensitive human pre-B acute lymphocytic leukemia line (42–44). Interestingly, GzmA expression in these cells induces apoptosis, which can be prevented by treatment with a synthetic GzmA inhibitor (43). Further study of the GzmA promoter did not ﬁnd a glucocorticoid responsive element (GRE) upstream of the transcriptional start site, but did ﬁnd a GRE in the ﬁrst intron (42). In fact, a novel 5 variant GzmA transcript starts 290 bp downstream of this intronic GRE (45). These alternative promoters generate two GzmA transcripts with different ﬁrst exons (exon1a and 1b). GzmA transcripts isolated from CTL contain exon1a, which encodes an N-terminal leader peptide to direct the nascent protein into the lumen of the endoplasmic reticulum (ER). Glucocorticoid treatment induces the exon1bcontaining transcript and also suppresses the exon1a transcript (45). The exon1b encodes a shorter leader peptide, which may cause aberrant subcellular localization of mature GzmA. Glucocorticoid-induced apoptosis of leukemia cells might then be due to the GzmA promoter switch and production of GzmA that is atypically released into the cytosol.

Post-Transcriptional Regulation of Granzymes An intriguing recent study suggests that GzmB and perforin gene expression may be

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post-transcriptionally regulated (46). Mouse NK cells in a resting state from pathogen-free animals have abundant GzmB and perforin mRNA but no corresponding protein and no substantial cytotoxicity. Upon activation, these cells show a dramatic increase in GzmB and perforin protein, but a minimal change in mRNA levels. Both resting and activated mouse NK cells have high levels of GzmA. This report appears to contradict previous studies showing that resting mouse and human NK cells do not need to be activated to develop cytotoxicity (47–49). The reason for the discrepancy is unclear but could result from low levels of asymptomatic infection in mice used in earlier studies, leading to prior activation of NK cell precursors. Unlike sterile laboratory mice, human subjects are chronically exposed to commensal organisms that activate the innate immune system as well as to a variety of pathogens. Therefore, human NK cells constitutively express perforin and GzmA and GzmM (but not GzmB). However, to our knowledge, no one has looked at granzyme expression in human NK cells in the immediate postnatal setting. In addition to the difference in granzyme mRNA and protein in T cell progenitors mentioned above, there are other examples that suggest post-transcriptional gene regulation. There are comparable levels of GzmB transcripts in resting and activated human pDC, but signiﬁcantly higher levels of GzmB protein in activated cells (6). Human mast cells upon activation express higher levels of GzmB mRNA than are found in CTL, but the corresponding protein levels are considerably lower in mast cells (7). Mouse memory CTLs express abundant GzmB mRNA but no protein (50). All these results point toward a general mechanism of prearming cytotoxic lymphocytes with effector mRNAs, allowing these cells to respond rapidly to external stimuli. This type of gene regulation is well known to regulate cytokine expression, presumably for the same purpose.

GRANZYME STRUCTURE The granzymes are homologous to trypsin, and their overall structure is similar to trypsin and other related serine proteases. Human GzmA, human and rat GzmB, and human pro-GzmK have all now been crystalized to yield high resolution structures (51– 54a) (Figure 2). The active granzymes are produced by cleavage of a dipeptide from the N terminus of the proenzyme. Activation is likely accompanied by a radical conformational change because a monoclonal antibody to GzmA does not recognize pro-GzmA. Pro-GzmK has a more rigid structure lacking an open active site, compared with the solved structures for the active granzymes. Detailed information about the conformation surrounding the active site of GzmA and GzmB has provided the structural basis for understanding how subtle differences in the active site conformation lead to substantial differences in substrate speciﬁcity. As a consequence, mouse GzmB is preferentially able to cleave mouse procaspase-3, whereas human GzmB is better able to cleave the human homolog. GzmA differs from the other granzymes in forming a covalent homodimer; the other granzymes are monomeric. Dimerization creates an extended site for substrate binding that is believed to confer a high degree of speciﬁcity to GzmA for its substrates. In particular, because of the extended exosite for substrate binding, GzmA substrates do not share a common short peptide sequence around the cleavage site. The crystal structures for the granzymes should provide useful tools for identifying small molecule inhibitors.

GRANZYME BIOSYNTHESIS AND STORAGE IN TARGET CELLS The granzymes are expressed with a signal sequence that directs them to the ER. Cleavage of the signal peptide produces

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a

b

β5 β6

180°

α2 R99

β3

β4 β1

His57 Ser195

β2

10 β10

Asp102

N T

Q

β12 12 β11 11

β9

E218

β7

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A'

T40

S

N218A

β8 α1

H57 S195 K

K219

Figure 2 Crystal structure of the GzmA homodimer. (a) GzmA is a disulﬁde-linked dimer in which the two active sites, indicated on the right (His57-Asp102-Ser195), face in opposite directions. The surface of the molecule contains concentrations of basic amino acids, which may explain the preference for acidic protein substrates through binding outside the active site through an extended exosite. (b) The SET protein is an important target of GzmA, whose cleavage triggers its unique pathway of DNA damage. Model of how the SET peptide surrounding the GzmA cleavage site ﬁts into the GzmA active site. [Figures based on the structure obtained by Hink-Shauer and colleagues (54a), reprinted with permission.]

an inactive proenzyme that contains an Nterminal dipeptide that needs to be cleaved to produce an active protease. In the Golgi, a mannose-6-phosphate tag is added, a sorting signal for transporting the proenzyme to lysosomes. Cytotoxic granules are specialized secretory lysosomes, maintained at an acidic pH with a distinctive electron dense core on electron micrographs. Within the cytotoxic granule, the N-terminal dipeptide is removed by cathepsin C (dipeptidyl peptidase I) (55). However, both mice and humans genetically deﬁcient in cathepsin C have only partially reduced granzyme activity and cytolytic function and modestly reduced immune defense against viral infection (56, 57). This suggests that there are alternate ways to activate the proenzyme. In fact, IL-2 treatment stimulates cathepsin C–independent dipeptide cleavage in NK cells from patients with PapillonLefevre syndrome who have loss of function of cathepsin C (58). Granzymes, which are highly basic, are bound along with perforin to the acidic serglycin proteoglycan within the granule, which probably keeps them inactive. Serglycin is responsible for the elec396

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tron dense core and may help to enhance the granzyme storage capability of the granules (59). Granzyme proteolytic activity is also negligible at acidic pH. Therefore, during protein synthesis and processing and when stored within the granule, several mechanisms are at work to make sure the granzymes are not proteolytically active.

GRANZYME RELEASE, UPTAKE, AND TRAFFICKING IN TARGET CELLS When a CD8 T cell or NK cell is activated by its antigen receptor, the lytic granules move to cluster around the microtubule organizing center and then align along the immunological synapse (reviewed in 60). The granule membrane fuses with the killer cell plasma membrane, releasing its contents, including perforin and granzymes, into the synapse. In CTL, granule fusion appears to localize to a distinct (secretory) region of the central cluster (c-SMAC) that is separate from the signaling domain containing the T cell receptor and associated kinases (61).

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However, the organization of granule fusion at the synapse may differ among types of killer cells, and cytotoxicity and granule fusion may occur even in the absence of a stable synapse (62). Granzymes likely dissociate from serglycin before they enter target cells (63). Granzymes bind to the target cell membrane by electrostatic interactions (granzymes are very positively charged with pIs ∼9–11, and the cell surface is negatively charged) (64–66) and also by speciﬁc receptors, such as the cation-independent mannose-6-phosphate receptor (67). However, speciﬁc receptors are not required for binding and cytotoxicity (64, 68, 69). Entry of granzymes into the target cell cytosol is generally mediated by perforin, but how perforin accomplishes this is still unclear (reviewed in 70). The original model of granzyme entry through plasma membrane pores formed by perforin (perforin is a poreforming protein with homology to complement) is generally no longer considered valid. A revised model posits that perforin makes microscopic holes in the plasma membrane that cause a calcium inﬂux, which triggers a cellular plasma membrane response and rapid endocytosis of granzymes and anything else bound to the cell surface (71). In fact, entry is dynamin-dependent (72) and results in the formation of giant endosomes containing both granzymes and perforin (71; D. Keefe, J. Thiery, and J. Lieberman, manuscript in preparation). Within minutes, the granzymes escape (through perforin pores in the endosome?) and ﬁnd their way into the cytosol. Although some key granzyme targets are cytosolic [i.e., BH3 interacting domain death agonist (bid) and inhibitor of caspase activated DNase (ICAD) for GzmB], other important targets are in other membrane-bound cellular compartments, including the nucleus and mitochondrion. GzmA and GzmB rapidly translocate to the nucleus (73, 74), where proteolytic cleavage of key substrates is important to induce programmed cell death by both GzmA (SET, Ape1, lamins, histones, Ku70, PARP1) and GzmB (lamin B, PARP1). Nu-

clear translocation of the granzymes may be mediated by importin-α (75). We have recently found that GzmA also trafﬁcs into the mitochondrial matrix, which is necessary for it to initiate mitochondrial damage (D. Martinvalet, D.M. Dykxhoorn, R. Ferrini, J. Lieberman, manuscript submitted). Genetic mutations that affect perforin function or granule exocytosis are associated with profound immunodeﬁciency and the familial hemophagocytic lymphohistocytosis syndrome (77–81). The killer cell on the other side of the synapse is not injured by its own granules. One important protective mechanism against autodestruction is provided by the expression in the killer cell cytoplasm of irreversible granzyme inhibitors called serpins (see next section). However, although serpins that inactivate GzmB have been shown in killer cells, no serpins are known that can inactivate GzmA. Another protective mechanism occurs via externalization of a cytotoxic granule membrane protein (cathepsin B), capable of proteolytically inactivating perforin, to the killer cell plasma membrane during granule fusion (82). Cathepsin B is thought to protect the killer cell membrane from any perforin that is redirected to the CTL side of the synapse. However, killer cells from cathepsin B–knockout mice survive encounters with target cells unharmed (83). This suggests that other mechanisms likely exist to protect killer cells from their own agents of destruction. Although perforin is the major molecule responsible for granzyme delivery, under some circumstances other molecules may serve that function. For example, bacterial and viral endosomolysins can substitute for perforin in vitro [and are widely used as laboratory reagents for intracellular delivery (84)] and may potentially play a similar role in vivo. The heat shock protein hsp70, which is known to chaperone some peptides across cell membranes, can also carry GzmB (and presumably other granyzmes) into cells (85). Hsp70 is found on the surface of some stressed cells www.annualreviews.org • Granzyme-Mediated Cell Death

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or tumor cells and may help to remove these cells from the body.

GRANZYME INHIBITORS—ENDOGENOUS, VIRAL, AND SYNTHETIC

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The regulation of proteolytic enzymes in tissues by endogenous inhibitors is critical to maintaining homeostasis and preventing undesirable damage. Although the trafﬁcking of granzymes within CTLs is designed to minimize leakage of active enzyme out of granules, any stray molecules in the cytoplasm could cause cell death (86). During granule exocytosis, some granzymes might inadvertently reenter the effector cells. Because CTLs typically kill several targets in succession without harming themselves, an important question is how CTLs protect themselves from their own cytotoxic molecules. One way is by expressing granzyme-speciﬁc inhibitors, members of the serpin (serine proteinase inhibitor) superfamily (87). Serpins are the largest and most broadly distributed superfamily of protease inhibitors, with more than 1500 family members (88, 89). Serpins inactivate their targets either by covalently and irreversibly binding to the active site of the enzyme or by forming noncovalent complexes that are tight enough to resist the denaturing conditions of SDS-PAGE gel electrophoresis (89, 90).

GzmB Endogenous Inhibitors The only intracellular inhibitor of human GzmB is the nucleocytoplasmic serpin, proteinase inhibitor-9 (PI-9). PI-9 is expressed by lymphocytes (87, 91), dendritic cells (DCs) (92), cells at immune privileged sites (testis and placenta) (14, 93, 94), endothelial and mesothelial cells (95), and ﬁnally mast cells (96). This in vivo distribution pattern supports the idea that PI-9 protects effector, accessory, and bystander cells from ectopic GzmB during an immune response. PI-9 gene expression is induced by modulators of inﬂam398

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mation like lipopolysaccharide, IFN-γ, and IL-1β (97, 98). PI-9 expression is enhanced by estrogen and hypoxia because of estrogen responsive elements and hypoxia inducible factor 2 (HIF-2)-binding sites, respectively, in the PI-9 promoter (99, 100). In particular, PI-9 is induced by hypoxia in neuroblastomas (101). The mouse counterpart of PI-9 is serine proteinase inhibitor-6 (SPI-6) (102). SPI6 is expressed in CTL and NK cells (103) and is upregulated during DC maturation (104). Overexpression of SPI-6 in target cells protects them from CTL killing (105). In SPI-6 transgenic mice, increased numbers of CTLs persist long after viral clearance, suggesting that SPI-6 protects CTLs from selfdestruction (103). However, somewhat paradoxically, GzmB-deﬁcient mice do not have increased numbers of CTL after viral infection, raising questions about the interpretation of the SPI-6 transgenic study (103). However, CTL from mice genetically deﬁcient in SPI-6 have increased cytosolic GzmB and reduced viability (106). One surprising ﬁnding is a breakdown of the integrity of the cytotoxic granules in SPI-6-deﬁcient CTLs (106). The exact mechanism behind this collapse of cytotoxic granules is unclear. Early studies with solid tumors and lymphomas in humans and mice suggested that overexpression of PI-9 or SPI-6 may be a mechanism by which tumors evade the GzmB/perforin pathway (105, 107). In these studies there is no comparison of serpin expression in tumor cells relative to corresponding normal tissues, thus making the results difﬁcult to interpret. Also, only a small subset of human lymphoma cell lines express PI9 (107). These issues have been addressed in recent studies. In cultured human hepatoma cells, induction of endogenous PI-9 by IFN-γ or estrogen partially blocks CTLand NK-induced apoptosis (98, 99). Similarly, induction of increasing amounts of endogenous PI-9 by estrogen in a human breast cancer line (MCF-7) progressively increases its resistance to NK-mediated cytolysis (108).

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PI-9 expression in pediatric acute lymphoblastic leukemias also correlates with resistance to cytolysis in vitro (109). Importantly, PI-9 expression is an important determinant of disease-free survival time of melanoma patients following immunotherapy (110). However, endogenous PI-9 and Bcl2 expression by some human lymphomas do not confer any resistance to cytolysis by in vitro–activated CTLs or NK cells (111). This study assumes that the cytotoxicity of an in vitro–activated cytotoxic lymphocyte is comparable to the in vivo scenario. Measuring cytotoxicity using highly activated cytotoxic lymphocytes in vitro may exaggerate the effectiveness of these cells (112) and thus underestimate the protective capacity of antiapoptotic molecules. The ability of serpins to make tumors resistant to immune cell destruction will likely depend on the level of expression of the serpin and of other antiapoptotic molecules, such as bcl-2 family members and survivin.

GzmA Endogenous Inhibitors No intracellular inhibitors of GzmA have yet been identiﬁed. However, some trypsin inhibitors also inhibit GzmA. GzmA is bound and irreversibly inhibited in the circulation by two trypsin inhibitors, α-2 macroglobulin and antithrombin III (113). Extracellular GzmA complexed to proteoglycans is resistant to these two protease inhibitors (114). A recent study identiﬁed another GzmA inhibitor, pancreatic secretory trypsin inhibitor (PSTI), from pancreatic secretions (115). PSTI is found in the blood, particularly in patients with severe inﬂammation and tissue destruction (116, 117). Extrapancreatic or blood PSTI might regulate extracellular activity of GzmA. Unlike the other two GzmA inhibitors, PSTI inhibits GzmA complexed to proteoglycans (115). It is still not clear whether any of these GzmA inhibitors are expressed in cytotoxic lymphocytes.

Viral Granzyme Inhibitors The pox virus–encoded cytokine response modiﬁer A gene (CrmA) is the ﬁrst viral inhibitor that was found to inhibit GzmB (118). CrmA directly binds and inhibits GzmB both in vitro and in vivo. Overexpression of CrmA in target cells inhibits CTL-mediated cell death. However, CrmA also strongly binds and inhibits caspases-1 and -8 and weakly inhibits other caspases like caspase-3; therefore, it is difﬁcult to pinpoint the importance of GzmB inhibition in these studies (119). Parainﬂuenza virus type 3 speciﬁcally inhibits GzmB by degrading GzmB mRNA in infected T cells (120). Importantly, GzmA transcripts are not affected by this virus. The mechanism of virus-mediated GzmB mRNA decay is not known. Human GzmB is inhibited by the adenoviral assembly protein (Ad5-100K) by a unique unserpin-like mechanism (121). In adenovirus-infected cells, Ad5-100K rapidly complexes with GzmB and gets cleaved very slowly at speciﬁc sites. GzmB that enters the infected target cell upon CTL attack is saturated by the abundant Ad5-100K protein. Importantly, the slow kinetics of the cleavage reaction ensures that there is always a molar excess of Ad5-100K protein relative to GzmB. Unlike CrmA, which is just an antiapoptotic factor, Ad5-100K is also necessary for virus assembly (121). It impedes human GzmB but does not inhibit caspases or other apoptotic pathways (122). Interestingly, the inhibitory activity of Ad5-100K is speciﬁcally directed against human GzmB and not its mouse or rat homolog. A recent study shows how CTLs have gotten around adenovirus resistance. In adenovirus-infected cells, the Ad5-100Kmediated GzmB inhibition is released by the action of an orphan granzyme, GzmH. GzmH cleaves Ad5-100K to rescue GzmB activity (123). Both GzmB and GzmH target the same adenoviral proteins, DNA-binding protein (DBP) and Ad5-100K. The direct cleavage of essential viral proteins by granzymes is

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a novel mechanism by which cytotoxic cells rapidly and directly block viral replication (123). Additionally, the different speciﬁcities of the granzymes allow distinct substrate processing, leading to synergistic antiviral activity. Viruses have evolved pathways to evade or inhibit granzymes and block apoptosis. This is the ﬁrst example of how the unique catalytic speciﬁcities of granzymes combine to counter a viral challenge.

Synthetic Inhibitors Synthetic inhibitors of granzymes are powerful tools both for research (identiﬁcation of peptide substrate speciﬁcity and determination of granzyme function) and potentially for therapeutic applications (immune suppression during autoimmune diseases and organ transplantation). There are several classes of granzyme inhibitors, including isocoumarin derivatives, peptide chloromethyl ketones,

PFN Gzms

SET Ape1 HMGB2 Histone H1, core histones

Perforin

Gzm A

ICAD Bid

Lamins

DNA-PKCS

Ku70

Gzm B Gzm A Gzm B

NuMa Lamin B

Caspases 3, 7

PARP1 Tubulin

NUCLEUS

Figure 3 Granule exocytosis-mediated cell death. When a CTL or NK cell recognizes a target cell, cytolytic granules containing perforin (PFN) and granzymes move to the immune synapse, and the granule membranes fuse with the killer cell plasma membrane, releasing PFN and granzymes into the synapse. PFN facilitates the entry of granzymes into the cytosol of the target cell. The most abundant granzymes are GzmA and GzmB. GzmA activates cell death independently of the caspases, whereas GzmB activates the caspase pathway both directly by cleaving the caspases and indirectly by cleaving key caspase substrates. Some of the key substrates of human GzmA and GzmB are shown. Both GzmA and GzmB trafﬁc to the nucleus by an unknown pathway, where many of the nuclear substrates are cleaved. 400

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and peptide phosphonates, but the major limitation has been a lack of speciﬁcity (113). Modiﬁcations that increase speciﬁcity generally diminish efﬁciency. Thornberry and colleagues (124) recently reported the identiﬁcation of a novel class of human GzmB inhibitors. The key feature of these compounds is a 1,2,3-triazole moiety that is crucial for their selectivity and cellular efﬁcacy. Future work with these inhibitors will determine their importance in studying the biology of granzymes.

PROGRAMMED CELL DEATH PATHWAYS INITIATED BY THE GRANZYME ALPHABET SOUP We are just beginning to understand how granzymes, other than GzmB, activate cell death, as laboratories have begun to express active recombinant forms of many of these enzymes. Now all ﬁve of the human enzymes have been expressed. Granzymes likely activate at least three (and probably more) distinct pathways of cell death (Figure 3, Table 1). We know most about cell death by GzmB, which activates the caspase apoptotic pathway by cleaving caspase-3 and also proteolyzes many of the important caspase substrates directly. However, there is good evidence that GzmB can also activate other pathways of cell death (particularly in the mitochondrion) that remain to be worked out. GzmA activates cell death that has all the morphological features of apoptosis but is completely caspase-independent and involves novel mitochondrial and DNA damage pathways. GzmC (in mouse) and GzmH (in humans) also activate caspase-independent cell death with a pronounced mitochondrial phenotype. There is some evidence that GzmM may activate autophagy. Understanding in detail the workings of these multiple roads to cell death will help us understand the many strategies for protection against intracellular pathogens and cellular transformation and how viruses and tumors evade immune destruction. Inevitably, research in this area will also help us

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Table 1

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Features of the distinct cell death pathways induced by the granzymes

Granzyme

A

B

C/H

K

M

Cytolytic CD8 T cells

++

++

+

+

Cytolytic CD4 T cells

+

+

CD4 Tregs

−

+

NK cells

+

+/−

Myeloid cells

−

+

Rapid loss of membrane integrity

+

+

+

+

?

Annexin V staining

+

+

+

+

?

Chromatin condensation

+

+

+

+

?

DNA damage

+

+

+

+

?

Mitochondrial depolarization

+

+

+

+

?

Caspase activation

−

+

−

−

?

Oligonucleosomal DNA fragmentation

−

+

−

−

?

Single-stranded DNA nicks

+

−

+

+

−

TdT labeling

+

+

+

+

?

Klenow labeling

+

+

+

+

−?

Inhibition by bcl-2 overexpression

−

+

?

?

?

Cytochrome c release

−

+

+?

?

?

Mitochondrial swelling

+

+

++

+

+

−

−

−

−

+?

Expression

+

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Common features

Type of DNA damage

Type of mitochondrial damage

Autophagy

comprehend important mechanisms in normal cellular metabolism and some of the strategies cells have at their disposal to deal with outside stress. Most of the focus has been on unveiling mitochondrial and DNA damage pathways. The molecular mechanisms behind other aspects of cell death, such as how plasma membrane integrity is disrupted (a critical feature of cell death), are still not well understood.

GRANZYME A GzmA is a tryptase that induces caspaseindependent cell death, which is morphologically indistinguishable from apoptosis (125, 126). Tryptases with homology to GzmA have been found in cytotoxic cells as far back in evolution as bony ﬁsh (127). Although GzmA was the ﬁrst granzyme to be described and is

the most widely expressed, much less is known about it than about GzmB. Cells treated with GzmA and perforin die rapidly—within minutes they undergo membrane blebbing, lose plasma membrane integrity (take up propidium iodide), and have evidence of mitochondrial dysfunction [increased reactive oxygen species (ROS), loss of mitochondrial transmembrane potential ( m ), disruption of mitochondrial morphology] (128; D. Martinvalet, D.M. Dykxhoorn, R. Ferrini, J. Lieberman, manuscript submitted). Within an hour or two, the slower-onset hallmarks of apoptosis are apparent: externalization of phosphatidyl serine (measured by annexin V staining) and DNA damage. DNA is damaged by single-stranded cuts into megabase fragments that are much larger than the oligonucleosomal fragments generated during caspase- or GzmB-activated cell www.annualreviews.org • Granzyme-Mediated Cell Death

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death (129). Because the DNA fragments are too large to be released from the nucleus, assays that measure DNA release into culture supernatants are typically negative until many hours later. Until recently, this was incorrectly interpreted as meaning that GzmA induces a slow, nonapoptotic death. Indeed, the caspases are not activated, and cell death proceeds unabated in the presence of pancaspase inhibition or in cells overexpressing bcl-2 family members or other inhibitors of caspase-mediated apoptosis (125). Moreover, mitochondria are damaged without mitochondrial outer membrane permeabilization (MOMP) or release of proapoptotic mediators, such as cytochrome c, from the mitochondrial intermembrane space (128). The molecular basis for the parallel caspase-independent programmed cell death pathway activated by GzmA has begun to be elucidated. Triggering mitochondrial damage is key to cell death induction because treating target cells with superoxide scavengers completely blocks cell death (and also blocks cell death by CTLs expressing all granzymes) (128). From the cytosol, GzmA is transported by an unknown mechanism [that may involve its ability to bind to the mitochondrial chaperone heat shock proteins (130; D. Martinvalet, D.M. Dykxhoorn, R. Ferrini, J. Lieberman, manuscript submitted)] into the mitochondrial matrix, where it cleaves a component of the electron transport chain complex I to interfere with mitochondrial redox function, ATP generation, and maintenance of m and to generate superoxide anion (128, 131; D. Martinvalet, D.M. Dykxhoorn, R. Ferrini, J. Lieberman, manuscript submitted). The superoxide generated by damaged mitochondria drives an ER-associated oxidative stress response complex, called the SET complex, which plays a critical role in GzmA-induced nuclear damage, into the nucleus (128, 129). The SET complex contains three nucleases [the base excision repair (BER) endonuclease Ape1, an endonuclease NM23-H1, and a 5 -3 exonuclease Trex1]; the chromatin-

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modifying proteins SET and pp32, which are also inhibitors of the tumor suppressor protein phosphatase 2A; and a DNA-binding protein that recognizes distorted DNA, HMGB2 (132–136). One of the functions of the complex is to repair abasic sites in DNA generated by oxidative damage. GzmA, which trafﬁcs to the nucleus by an unknown mechanism, converts this DNA repair complex into an engine for DNA destruction by cleaving SET, an inhibitor of the endonuclease NM23H1 (134) (Figure 2). This allows NM23-H1 to nick DNA; the exonuclease Trex1 then extends the break (133) (Figures 3 and 4). At the same time, GzmA cleaves and inactivates HMGB2 and Ape1 to interfere with BER (135, 136). In addition to disabling BER, GzmA also interferes with DNA repair more generally by interfering with the recognition of damaged DNA by cleaving and inactivating Ku70 (137) and PARP-1 (P. Zhu, D. Martinvalet, D. Zhang, A. Schlesinger, D. Chowdhury, J. Lieberman, manuscript in preparation). Within the nucleus, GzmA also opens up chromatin by cleaving the linker histone H1 and removing the tails from the core histones, making DNA more accessible to any nuclease, and disrupts the nuclear envelope by cleaving lamins (139, 140). It is noteworthy that all the GzmA substrates mentioned above are cleaved both in vitro and in vivo. When a mutant form of the key substrates that lacks the GzmA cleavage site is expressed in target cells, the target cell is less susceptible to GzmA-mediated cell death.

GRANZYME B GzmB is unique among serine proteases because it cleaves after aspartic acid residues, like the caspases (reviewed in 141, 142). It induces target cell apoptosis by activating the caspases, particularly the key executioner caspase, caspase-3 (143, 144) (Figure 3). Human GzmB, but not the mouse enzyme, also activates cell death by directly cleaving the key caspase substrates, bid and ICAD, to activate the same mitochondrial and DNA damage

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NM23-H1

APE1

SET

pp32

TREX1

HMGB2

APE1

GzmA

ER

Nucleus

Histone tails

NM23-H1

SET pp32 TREX1

HMGB2

pp32

NM23-H1

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TREX1

Histone H1

Figure 4 The GzmA pathway of DNA damage. ROS generated by GzmA in mitochondria drives the ER-associated SET complex into the nucleus. GzmA also enters the nucleus by an unknown pathway. In the nucleus, GzmA cleaves three components of the SET complex (SET, HMGB2, and APE1) to activate two nucleases in the complex to make single-stranded DNA lesions—NM23-H1 makes a nick, which is extended by the exonuclease TREX1. GzmA also degrades the linker histone H1 and removes the tails from the core histones, opening up chromatin and making it more accessible to these nucleases.

pathways, respectively, as the caspases (53, 145–151) (Figure 3). As a consequence, caspase inhibitors have little effect on human GzmB-mediated cell death and DNA fragmentation, whereas the same inhibitors significantly block the action of the mouse enzyme. Therefore, human CTLs and NK cells may be more effective than mouse killer cells at eradicating virus-infected cells or tumors that have developed methods for evading the caspases. Both human and mouse enzymes cleave many of the same substrates as the caspases (including PARP-1, lamin B, NuMa, DNA-PKcs , tubulin) and have substrate speciﬁcity close to that of caspases-6, -8, and -9 (152). However, human GzmB cleaves optimally after the tetrapeptide IEPD, whereas mouse GzmB has somewhat different peptide speciﬁcity, preferring to cleave after IEFD (53, 145). Moreover, other regions, including the P’ region (C-terminal to the cleavage site) and more distal regions, contribute to substrate speciﬁcity. As a consequence of subtle differences in sequence, the human and mouse GzmB can differ in important ways with respect to sub-

strates and the efﬁciency with which they are cleaved. The GzmB (and caspase) mitochondrial pathway leads to ROS generation, dissipation of m and MOMP, with release of cytochrome c and other proapoptotic molecules from the mitochondrial intermembrane space. Human GzmB activates this pathway directly by cleaving bid, whereas mouse GzmB activates it indirectly. However, GzmB targets the mitochondria in other ways that remain to be worked out. Loss of m , but not cytochrome c release, occurs in the presence of pan-caspase inhibitors (even using mouse GzmB) and in mice genetically deﬁcient for bid, bax, and bak (the latter two bcl-2 family members are required for bidinduced mitochondrial damage) (153–156). Mitochondrial damage is key to cell death induction because treatment of target cells with superoxide scavengers that neutralize ROS completely blocks cell death by CTLs expressing all granzymes (128). DNA damage by GzmB is mediated primarily by the activation of the caspase-activated DNase (CAD) www.annualreviews.org • Granzyme-Mediated Cell Death

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following proteolytic cleavage of its inhibitor ICAD either directly by human GzmB or indirectly by executioner caspases, such as caspase-3. In humans, there is a common polymorphism of GzmB in which three amino acids (Q48 , P88 , Y245 ) are mutated to R48 A88 H24 (157). This polymorphism does not seem to affect cytotoxicity and probably does not have any clinical signiﬁcance (158).

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GRANZYMES C AND H Mouse GzmC and human GzmH, homologous granzymes that lie downstream from GzmB, are predicted to have chymotryptic activity and cleave substrates after aromatic residues (159, 160). GzmH is thought to have arisen during primate evolution, independently of GzmC, in an intergenic recombination event between GzmB and a mast cell chymase (161). Both induce caspaseindependent death with hallmarks of programmed cell death—ROS generation, dissipation of m , chromatin condensation, and nuclear fragmentation (159, 160). DNA destruction by GzmC (and probably GzmH as well) is via single-stranded nicks and does not involve CAD. Rapid mitochondrial swelling and disruption of mitochondrial ultrastructure are particularly striking in cells treated with GzmC. The mitochondrial pathways activated by GzmC and GzmH may be different; GzmC has been reported to trigger cytochrome c release, a sign of MOMP, whereas GzmH does not seem to cause cytochrome c release (159, 160). Although no normal cellular substrates have yet been identiﬁed for GzmC or GzmH, GzmH cleaves two adenoviral proteins— DBP (also a GzmB substrate) and the adenovirus 100K assembly protein, a previously described inhibitor of GzmB (121, 123). Cleavage of DBP interferes with viral DNA replication, whereas cleavage of 100K restores GzmB function in adenovirus-infected cells. Therefore, GzmH may play a special role in adenoviral immune defense. Because GzmH 404

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is expressed in NK cells, it may be used to eliminate adenovirus early in infection, before adaptive immunity has had a chance to develop.

GRANZYME K GzmK (also known as Gzm 3) is another tryptase found in mice, rats, and humans, encoded downstream close to GzmA on human 5q11–12 (or the syntenic region of mouse chromosome 13). It is expressed much less than GzmA and, unlike GzmA, is a monomer, not a dimer. Mice genetically deﬁcient in GzmA express GzmK, which may explain the lack of a signiﬁcant phenotype of GzmA−/− mice, except when challenged with some viruses (162, 163). Puriﬁed rat and recombinant human GzmK has been available for some time (164, 165), but little was known about its cell death activation until recently. Like GzmA, puriﬁed rat GzmK efﬁciently induces caspase-independent cell death, characterized by mitochondrial dysfunction without MOMP (ROS and loss of m , but without cytochrome c release) (154). However, unlike GzmA, rat GzmK–induced cell death was originally reported to be inhibited in cells overexpressing bcl-2 (154). This ﬁnding was surprising because bcl-2 inhibits MOMP, which leads to cytochrome c release, which was not detected in GzmK-treated cells. In fact, a more recent study found that cell death by recombinant human GzmK did not activate caspase-3 and was unaffected by caspase inhibitors or bcl-xL overexpression (166). GzmK mimics GzmA DNA damage (166)—it causes caspase-independent nuclear fragmentation and nuclear condensation and singlestranded DNA breaks by targeting the SET complex. Like GzmA, GzmK causes SET complex nuclear translocation and hydrolyzes and inactivates SET, Ape1, and HMGB2 in the SET complex (166). Presumably, cleavage of SET, the inhibitor of NM23-H1, triggers DNA damage by the GzmA-activated DNases, NM23-H1 and Trex1, in the SET complex (133, 134). The same group recently

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reported that GzmK causes mitochondrial damage that includes not only ROS generation and dissipation of m , but also bid cleavage (to a fragment that appears to be the same size as is generated by GzmB) and MOMP with release of cytochrome c and endoG (167). This ﬁnding needs to be veriﬁed because rat GzmK does not cause cytochrome c release (154), and this same group showed that caspases are not activated by GzmK, nor does overexpression of bcl-xL interfere with human GzmK–induced cell death (166), as would be expected if MOMP is triggered. Although GzmK appears to duplicate GzmA’s nuclear damage pathway, further studies are needed to determine whether the mitochondrial GzmK pathway resembles that activated by GzmA (no MOMP) or GzmB (bid cleavage, MOMP) or is a hybrid of both.

GRANZYME M GzmM is the most distinctive of the granzymes. It likely arose from a gene duplication of a neutrophil protease because it is encoded near a cluster of other neutrophil proteases in human chromosome 19p13.3 (or a synteic region of mouse chromosome 10) and is slightly more homologous to one of them (complement factor D) than to the other granzymes (168). Unlike the other granzymes, GzmM cuts after Met or Leu (169, 170). None of the serine protease inhibitors that block the other granzymes, including the pangranzyme inhibitor 3,4-dichloroisocoumarin, effectively inhibit GzmM (171). Moreover, GzmM appears to function primarily in innate immunity, as it is expressed mostly in NK cells and γδ T cells and only in the subset of CD56+ T cells (112). Until recently it was not clear whether GzmM induces cell death (172). GzmM−/− mice have unimpaired NK and T cell development and NK cell–mediated cytotoxicity but are less able to defend against mouse cytomegalovirus infection (173). The literature does not agree about the type of cell death activated by GzmM. On the one hand, Kelly et al. (172), using recom-

binant human GzmM expressed from baculovirus in insect cells, found that GzmM induced rapid, caspase-independent cell death that looked like autophagic death and did not ﬁnd evidence for DNA fragmentation, mitochondrial depolarization, phosphatidyl serine externalization, or caspase activation. On the other hand, using human GzmM expressed in yeast, the Fan laboratory (174, 175) argued that GzmM activated caspase-dependent cell death with phosphatidyl serine externalization, caspase activation, ICAD cleavage, and CAD activation with oligonucleosomal DNA laddering, PARP cleavage, and mitochondrial disruption with MOMP (mitochondrial swelling, dissipation of m , ROS generation, cytochrome c release). This group also provides evidence that another GzmM substrate may be TRAP75, a heat shock protein that inhibits GzmM-induced ROS generation (175). However, one aspect of this study that may not be completely consistent with what is known about GzmM is that the Fan paper (174, 175) claims that GzmM cleaves ICAD after a Ser residue, whereas peptides containing Ser at the P1 site are not substrates of GzmM expressed in yeast. Therefore, further work is needed to determine whether GzmM activates GzmB-like caspase-dependent cell death or a completely novel pathway distinct from any of the other granzymes. Examining cell death induced by native puriﬁed GzmM may be necessary to determine what type of cell death is induced by this enzyme. One other intriguing activity of GzmM may be to cleave and inactivate the GzmB serpin inhibitor PI-9, which it does in vitro (170). If this proves to be a physiologically relevant substrate in cells, then one function of GzmM may be to potentiate the activity of GzmB.

EXTRACELLULAR ROLES OF GRANZYMES Although most research has focused on the cell death–inducing properties of granzymes, there is an older and growing literature to suggest that granzymes may have extracellular www.annualreviews.org • Granzyme-Mediated Cell Death

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functions in promoting inﬂammation and degrading extracellular matrix, potentially to allow cytotoxic cells access to target cells within tissue or to induce death by anoikis of anchorage-dependent cells. It is unclear whether, during killer cell degranulation, the immune synapse forms a perfectly tight gasket that completely prevents granzymes from leaking out into the extracellular space. Moreover, increasing evidence suggests that granzymes may be expressed without perforin and secreted by other types of white blood cells during inﬂammation, including mast cells, neutrophils, activated macrophages, as well as potentially some nonhematopoietic cells, such as UV-damaged keratinocytes (7, 10, 11, 176–178). GzmB is even expressed by developing germ cells in the testes and by syncytial trophoblasts in the placenta (14). Low concentrations of GzmA, GzmB, and GzmK have been detected in the serum of healthy donors (179). During inﬂammation and infection, elevated levels of granzymes exist in both serum and other bodily ﬂuids. Examples in which extracellular granzymes have been detected include the serum of patients undergoing acute cytomegalovirus infection or chronic HIV infection, the joints of rheumatoid arthritis patients, and the bronchoalveolar lavage ﬂuid of allergenchallenged patients with asthma and patients with chronic obstructive pulmonary disease (9, 114, 179–183). Elevated granzyme levels also occur in the serum of patients with endotoxemia and bacteremia, supporting the idea that granzymes are expressed and secreted by activated leukocytes, not just by lymphocytes (184). In fact, in sepsis patients, not only is serum GzmK elevated, but its natural inhibitor (inter-α protein) is depleted, so the free active form of the enzyme is circulating and may cause damage (185). GzmB has also been detected in macrophages of atheromatous lesions and rheumatoid joints (176). Proteolysis by extracellular granzymes is inhibited to some extent by serum and extracellular protease inhibitors, such as the

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trypsin inhibitors antithrombin III and alpha2-macroglobulin (114). Some conditions that induce extracellular granzymes may also increase the release of intracellular serpins (186). Although these extracellular granzymes may not be able to get into the cytoplasm of cells to induce cell death without a high local concentration of perforin, they could proteolyze cell surface receptors or extracellular proteins. The extracellular functions of the granzymes that have been reported are summarized below, but these proteases, despite their high degree of substrate speciﬁcity, likely have multiple, as yet unappreciated, destructive effects, particularly if present at high concentrations at inﬂamed sites in the absence of natural inhibitors. The known extracellular activities of GzmA suggest a proinﬂammatory effect. GzmA can activate the proinﬂammatory cytokine IL-1β directly by cleaving its propeptide (187). Other reports suggest that GzmA may proteolytically activate macrophages to secrete cytokines (188). It can also cause neurite retraction on astrocytes and inhibit thrombin-induced platelet aggregation by cleaving the thrombin receptor (189, 190). One study suggests another anticoagulant effect via activating prourokinase to activate plasminogen (191). Other papers suggest possible roles in degrading extracellular matrix proteins, including heparin sulfate proteoglycans, collagen type IV, and ﬁbronectin (192– 194). Moreover, binding of GzmA to basement membrane proteoglycans may protect it from extracellular tryptase inhibitors and serve to release growth factors, such as bFGF, that are stored bound to the extracellular matrix (194). GzmB also remodels the extracellular matrix by direct cleavage of vitronectin, ﬁbronectin, and laminin (195). In fact, GzmB can cleave after the RGD integrin-binding domain of vitronectin. Proteolysis of the extracellular matrix may cause anchorageindependent cell death, restrict tumor cell

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invasion, facilitate lymphocyte migration to sites of infection or inﬂammation, or cause tissue destruction at sites of inﬂammation (195, 196). In addition, GzmB can degrade cartilage proteoglycans potentially to exacerbate autoimmune or inﬂammatory arthritis (197, 198). In the central nervous system, GzmB cleaves a glutamate receptor (GluR3), potentially contributing to immunoneurotoxicity, excitation, and autoimmunity in the brain (199, 200). GzmB on its own causes death of neurons in a pertussis toxin–sensitive manner, suggesting possible cleavage or involvement of G protein–coupled receptors (201). Other potential GzmB receptor targets are Notch1 and FGFR1, which may inhibit growth signals to developing or malignant cells (202).

LESSONS FROM KNOCKOUT MICE GzmA-, GzmB-, and perforin-deﬁcient mice were generated a decade ago, and early studies have been extensively reviewed (1). Although perforin-deﬁcient mice are severely immunodeﬁcient and compromised in their ability to defend against viruses and tumors, mice deﬁcient in any one of the ten granzymes, or even of the GzmB cluster, have only subtle differences compared with wild-type animals. These experiments highlight the functional redundancy of the granzymes. Although only one molecule (perforin) effectively delivers the granzymes into target cells, each of the granzymes can trigger cell death. However, target cells may be selectively resistant to one or another of the granzymes, i.e., by bcl-2 overexpression or by expression of viral serpins, and granzyme expression also varies in different types of immune responders. Speciﬁc requirements for a single granzyme have been shown in some cases by speciﬁc immune challenges. For example, GzmA-deﬁcient mice are particularly susceptible to the pox virus ectromelia (203), and GzmB-deﬁcient mice have a markedly attenuated incidence of graft-versus-host disease (204). In construct-

ing genetically deﬁcient mice, genetic alterations of one gene can affect the expression of nearby granzyme genes. In the original GzmB-knockout mice, the presence of a phosphoglycerate kinase promoter-neomycin resistance gene cassette in the GzmB locus impedes the expression of the GzmB-proximal genes (GzmC, D, and F). The GzmB gene has also been deleted while keeping the expression of GzmC, D, and F intact (205). CTL from the GzmB-speciﬁc deletion mouse are significantly more effective at inducing apoptosis than the earlier GzmB cluster–knockout animal, underlining the importance of the other GzmB cluster granzymes. Because GzmA and GzmB are the most abundantly expressed granzymes in T cells, GzmA/B doubly deﬁcient mice are more immunodeﬁcient than the single knockouts (206–208). CTLs from GzmA/B-deﬁcient mice, although somewhat impaired in cytotoxicity relative to wild-type cells, nonetheless largely retain the ability to kill target cells (131, 209, 210). However, the timing of key molecular events during apoptosis, such as membrane rufﬂing and externalization of phosphatidylserine (annexin V staining), is markedly different during cell death induced by wild-type CTLs versus GzmA/Bdeﬁcient CTLs (210). CTLs lacking GzmA and GzmB induce a modiﬁed form of cell death that is morphologically distinct from either perforin-mediated necrosis or wildtype CTL-mediated apoptosis (210). These differences could potentially be physiologically signiﬁcant. In contrast to perforindeﬁcient mice, GzmA/B-deﬁcient animals develop normally, do not develop spontaneous tumors, and clear many viruses normally. The most economic explanation of these results is that the other death-inducing, perforindependent granzymes (particularly C, K, and M) (154, 159, 172) substitute for GzmA and GzmB. GzmM, abundantly expressed in NK cells, may be particularly important in early innate defense to contain the spread of infection or recognize transformed cells.

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SUMMARY: WHY SO MANY GRANZYMES?

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Recent studies have begun to deﬁne multiple death pathways activated by individual granzymes and potentially important extracellular roles of these enzymes. The granzymes can trigger at least three distinct cell death pathways, which are just being elucidated with the recent availability of recombinant active forms of many of the granzymes. The immune system needs to contend with a wide variety of tumors and infections, which have elaborated multiple strategies to evade apoptosis and immune destruction. Moreover, the granzyme-perforin system may also play a signiﬁcant role in regulating immune cell numbers and function and disarm-

ing speciﬁc intracellular pathogens. The redundancy of granzymes makes sense, given the variety of tasks they need to accomplish. The example of the interplay between GzmB and GzmH and adenovirus illustrates why multiple granzymes may have evolved to eliminate important pathogens (121–123). Although both enzymes can cleave and inactivate at least two adenoviral proteins, the virus has also developed a way of inactivating GzmB. It appears that GzmH can potentiate the effect of GzmB by destroying the GzmB inhibitor. In the future, careful in vitro and in vivo studies of immune protection from important pathogens will likely deﬁne the speciﬁc role of each of the granzymes and help us understand their evolution.

ACKNOWLEDGMENTS We thank members of the Lieberman laboratory and Mark Smyth for useful discussions and Rohit Panchakshari for help with Figure 1. This work was supported by NIH grants AI45587 and AI63430 ( J.L.) and a Leukemia and Lymphoma Society fellowship (D.C.).

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202. Loeb CR, Harris JL, Craik CS. 2006. Granzyme B proteolyzes receptors important to proliferation and survival, tipping the balance toward apoptosis. J. Biol. Chem. 281:28326– 35 203. Mullbacher A, Ebnet K, Blanden RV, Hla RT, Stehle T, et al. 1996. Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia. Proc. Natl. Acad. Sci. USA 93:5783–87 204. Graubert TA, Russell JH, Ley TJ. 1996. The role of granzyme B in murine models of acute graft-vs-host disease and graft rejection. Blood 87:1232–37 205. Revell PA, Grossman WJ, Thomas DA, Cao X, Behl R, et al. 2005. Granzyme B and the downstream granzymes C and/or F are important for cytotoxic lymphocyte functions. J. Immunol. 174:2124–31 206. Davis JE, Smyth MJ, Trapani JA. 2001. Granzyme A and B-deﬁcient killer lymphocytes are defective in eliciting DNA fragmentation but retain potent in vivo antitumor capacity. Eur. J. Immunol. 31:39–47 207. Pardo J, Balkow S, Anel A, Simon MM. 2002. Granzymes are essential for natural killer cell-mediated and perf-facilitated tumor control. Eur. J. Immunol. 32:2881–87 208. Simon MM, Hausmann M, Tran T, Ebnet K, Tschopp J, et al. 1997. In vitro- and ex vivoderived cytolytic leukocytes from granzyme A × B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells. J. Exp. Med. 186:1781–86 209. Krenacs L, Smyth MJ, Bagdi E, Krenacs T, Kopper L, et al. 2003. The serine protease granzyme M is preferentially expressed in NK-cell, γδ T-cell, and intestinal T-cell lymphomas: evidence of origin from lymphocytes involved in innate immunity. Blood 101:3590–93 210. Waterhouse NJ, Sutton VR, Sedelies KA, Ciccone A, Jenkins M, et al. 2006. Cytotoxic T lymphocyte-induced killing in the absence of granzymes A and B is unique and distinct from both apoptosis and perforin-dependent lysis. J. Cell Biol. 173:133–44

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:389-420. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Annu. Rev. Immunol. 2008.26:421-452. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer Infectious Diseases Service, Department of Medicine, Immunology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021; email: [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:421–52

Key Words

First published online as a Review in Advance on December 3, 2007

inﬂammation, monocyte differentiation, chemokines, microbial pathogens

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090326 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0421$20.00

Abstract Circulating blood monocytes supply peripheral tissues with macrophage and dendritic cell (DC) precursors and, in the setting of infection, also contribute directly to immune defense against microbial pathogens. In humans and mice, monocytes are divided into two major subsets that either speciﬁcally trafﬁc into inﬂamed tissues or, in the absence of overt inﬂammation, constitutively maintain tissue macrophage/DC populations. Inﬂammatory monocytes respond rapidly to microbial stimuli by secreting cytokines and antimicrobial factors, express the CCR2 chemokine receptor, and trafﬁc to sites of microbial infection in response to monocyte chemoattractant protein (MCP)-1 (CCL2) secretion. In murine models, CCR2mediated monocyte recruitment is essential for defense against Listeria monocytogenes, Mycobacterium tuberculosis, Toxoplasma gondii, and Cryptococcus neoformans infection, implicating inﬂammatory monocytes in defense against bacterial, protozoal, and fungal pathogens. Recent studies indicate that inﬂammatory monocyte recruitment to sites of infection is complex, involving CCR2-mediated emigration of monocytes from the bone marrow into the bloodstream, followed by trafﬁcking into infected tissues. The in vivo mechanisms that promote chemokine secretion, monocyte differentiation and trafﬁcking, and ﬁnally monocyte-mediated microbial killing remain active and important areas of investigation.

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INTRODUCTION

HUMAN MONOCYTE SUBSETS

The mammalian immune system defends against a spectrum of microbial pathogens that, in terms of environmental prevalence, range from common to rare. Invasion by common environmental microbes is prevented by constitutive innate immune defenses in mucosal and epithelial tissues. On the one hand, the metabolic costs of establishing and maintaining constitutive innate defenses against ubiquitous microbes are easily justiﬁed. Highly virulent pathogens, on the other hand, are generally less prevalent and have evolved mechanisms to circumvent constitutive immune barriers. Upon infection with these organisms, auxiliary innate defenses are induced to combat the pathogen. Neutrophils, macrophages, and dendritic cells (DCs) are important cellular mediators of innate immune defense. Circulating monocytes, however, are increasingly implicated as essential players in defense against a range of microbial pathogens. Most cellular components of the mammalian immune system derive from progenitors in the bone marrow. The typical developmental pathway begins with pluripotent bone marrow stem cells that give rise to progenitors that follow a variety of differentiation pathways to become mature cells with deﬁned effector functions. Mammalian monocytes, a pleomorphic and pleiotropic population of circulating mononuclear cells, contribute to antimicrobial defense by supplying tissues with macrophage and DC precursors (1–4). When the mammalian host is confronted with a virulent pathogen, however, the normal, homeostatic differentiation pathway of monocytes is temporarily refocused, and bone marrow and blood monocytes differentiate into a spectrum of effector cells with distinct antimicrobial activities. This review focuses on the contributions of monocyte subsets to immune defense against microbial pathogens.

In humans, circulating monocytes are divided into two subsets on the basis of the expression of CD14, a component of the lipopolysaccharide (LPS) receptor complex, and CD16, the FcγRIII immunoglobulin receptor (5). These monocyte subsets express distinct chemokine, immunoglobulin, adhesion, and scavenger receptors (3) (Table 1). CD14high CD16− monocytes (henceforth referred to as CD14+ monocytes) are large, ∼18 μm in diameter, and represent ∼80%– 90% of circulating monocytes. In contrast, CD14low CD16+ monocytes (referred to as CD16+ monocytes) are smaller, ∼14 μm in diameter, and constitute ∼10% of circulating monocytes. CD16+ monocytes increase in frequency during infections (6, 7), produce high levels of tumor necrosis factor (TNF) and low levels of IL-10 upon stimulation with Toll-like receptor (TLR) agonists (8), and therefore are also referred to as proinﬂammatory monocytes. CD14+ and CD16+ monocytes respond to distinct trafﬁcking cues. CD14+ monocytes express high levels of CCR1, CCR2, and CXCR2 and low levels of CX3CR1, whereas CD16+ monocytes express high levels of CX3CR1 and low levels of CCR2 (9, 10). Accordingly, CD14+ monocytes respond to monocyte chemoattractant protein (MCP)-1 (CCL2), whereas CD16+ monocytes respond to fractalkine (CX3CL1) in transendothelial migration assays (10). CD14+ monocytes express higher levels of CD62L (L-selectin) and CD11b (also referred to as Mac-1 or CR3) and lower levels of MHC class II than do CD16+ monocytes (11). A detailed review describing the differences between human monocyte subsets has been published recently (3). Additional, albeit smaller, monocyte subsets can also be distinguished by surface molecule expression. For example, a population of CD14+ CD16+ CD64+ monocytes is highly phagocytic, like CD14+ monocytes,

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Surface antigen expression on the principal circulating murine and human monocyte subsetsa Murine Ly6C+

Murine CX3CR1+

Human CD14+

Human CD16+

monocytes

monocytes

monocytes

monocytes

References

Monocyte markers CD11b

+

+

+

+

16

CD14

ND

ND

++

+/−

5

CD16 (FcγRIII)

ND

ND

−

+

5

+

+

ND

ND

21

CD115

+

+

ND

ND

16

Gr-1 (Ly6C/Ly6G)

++

+/−

ND

ND

16

Ly6C

++

+/−

ND

ND

21

Ly6G

−

−

ND

ND

197

CCR1

ND

ND

+

−

16, 10

CCR2

+

−

+

−

17, 9, 10

CCR4

ND

ND

+/−

−

16

CCR5

ND

ND

+/−

+/−

9, 16, 10, 198

CCR7

ND

ND

+/−

−

16

CXCR1

ND

ND

+/−

−

16, 10

CXCR2

ND

ND

+

−

16, 10

CXCR4

ND

ND

+/−

+

16, 10

CX3CR1

+/−

++

+/−

++

16, 10

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F4/80

Chemokine receptors

Other receptors and lineage markers +

−

ND

ND

18

ND

ND

+

+

199

CD11a

+

++

ND

ND

16, 10

CD11c

−

+

+

+

21, 16, 198, 199

7/4 CD4

+

+

+

+

16

CD32 (FcγRII)

ND

ND

+

++

13

CD33

ND

ND

++

+

13

CD43

−

+

++

+

21

CD49b

+

−

ND

ND

16

CD62L (L-selectin)

+

−

+

−

17, 16, 10

CD64 (FcγRI)

ND

ND

−

+

12

CD86

ND

ND

+

++

13

Inducibleb

Inducibleb

+

++

16

−

−

ND

ND

16

ND

ND

+/−

+

200

CD31

MHC class II NK1.1 Scavenger receptor a

Surface expression levels have been arbitrarily designated as undetectable (−), marginal (+/−), positive (+), and high (++) based on ﬂow cytometric analysis; ND, not determined. b MHC class II levels are marginal under homeostatic conditions but increase rapidly with infectious or inﬂammatory stimulation (16).

but expresses high levels of MHC class II, like CD16+ monocytes (12, 13). This subset, referred to as transitional monocytes, can activate T cells. Their developmental relationship

to CD14+ and CD16+ monocytes remains unclear. Another small subset, constituting ∼1%–2% of mononuclear cells (14), expresses CD56, a neural cell adhesion molecule www.annualreviews.org • Monocyte-Mediated Defense

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isoform. The frequency of CD16+ CD56+ monocytes is increased in patients with inﬂammatory bowel disease (15).

MURINE MONOCYTE SUBSETS

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Owing to possible species-speciﬁc differences in receptor expression and the absence of useful monoclonal antibody reagents, murine monocyte subsets are not distinguished by CD14 and CD16 expression. Murine blood monocytes express CD115 [the receptor for macrophage colony stimulating factor (CSF1R)], CD11b, and low levels of the F4/80 antigen. Murine monocyte subsets are distinguished by differential Ly6C, CX3CR1 (16), CCR2 (17), and 7/4 (18) expression (Table 1). Engineered expression of green ﬂuorescent protein (GFP) from the CX3CR1 locus (termed CX3CR1gfp/+ mice) (19) has enabled monocyte subset isolation and adoptive transfer studies (16). GFPdim monocytes express low levels of CX3CR1 and high levels of CCR2 and Ly6C and are most similar to human CD14+ monocytes. GFPbright monocytes express high levels of CX3CR1 and low levels of Ly6C and do not express CCR2; they are most similar to human CD16+ monocytes. CX3CR1low CCR2+ Ly6C+ monocytes (henceforth referred to as Ly6C+ monocytes) are granular and larger than CX3CR1+ CCR2− Ly6Clow monocytes (referred to herein as CX3CR1+ monocytes), with typical diameters of 10–14 μm and 8– 12 μm, respectively (16). In adoptive transfer experiments, Ly6C+ monocytes home to peripheral tissues in response to inﬂammatory stimuli, prompting their designation as inﬂammatory monocytes. Following recruitment to the inﬂamed peritoneum, Ly6C+ monocytes upregulate CD11c and MHC class II and migrate to draining lymph nodes, where they can promote T cell proliferation, suggesting that this monocyte subset differentiates into DCs (16). Ly6C+ monocytes have a short transit time in the bloodstream and are not recovered from peripheral tissues in the absence of 424

Serbina et al.

inﬂammation (16) but instead home to the bone marrow (20). CX3CR1+ monocytes remain in the circulation for longer periods and trafﬁc into peripheral tissues under noninﬂammatory conditions (16). These cells reconstitute tissue macrophages and DCs and are referred to as resident monocytes. A third subset, constituting only ∼5% of circulating murine monocytes, expresses intermediate levels of Ly6C (21) and may correspond to human CD14+ CD16+ CD64+ monocytes. Murine Ly6Cint monocytes and human CD14+ CD16+ CD64+ monocytes express a broader array of chemokine receptors than do CD14+ or CD16+ monocytes (22, 23). Although circulating human and murine monocytes have been divided into two principal and several minor subsets, there are important species-speciﬁc differences. First, the relative frequencies of the two major subsets are different in mice and humans. Under resting conditions, CD14+ monocytes predominate in the bloodstream of humans, whereas Ly6C+ and CX3CR1+ monocytes in mice are present in roughly similar proportions. Second, human CD16+ monocytes synthesize high levels of inﬂammatory cytokines following TLR stimulation, whereas murine Ly6C+ monocytes, which in terms of chemokine receptor expression are more similar to human CD14+ monocytes, are more responsive to TLR stimulation. Thus, the designation of human CD16+ monocytes as proinﬂammatory and murine Ly6C+ monocytes as inﬂammatory can create confusion. To some extent, identiﬁcation of monocyte subsets in different species using distinct surface markers likely accounts for some of these disparities.

MONOCYTE DEVELOPMENT AND DIFFERENTIATION Progenitors in the Bone Marrow Circulating murine monocytes descend from self-renewing hematopoietic stem cells that initiate myeloid differentiation and give rise to

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multipotent precursors (24, 25). These multipotent cells are lineage-associated marker negative (Lin− ), Sca-1+ , and CD117+ (ckit) and give rise to lineage-restricted common myeloid progenitor cells (CMPs) (26) and common lymphoid progenitor cells (CLPs) (27). Granulocyte macrophage progenitors descend from CMPs. Recently, myeloid lineage macrophage-DC progeni-

tors (MDPs) were isolated from bone marrow suspensions of CX3CR1gfp/+ mice (28) as GFP+ CD117+ Lin− cells that give rise to macrophages and DCs, but not to neutrophils. When introduced into bone marrow, MDPs give rise to Ly6C+ and CX3CR1+ bone marrow monocytes, which give rise to the two principal circulating subsets (20, 28) (Figure 1).

Ly6C+ monocytes

CX3CR1+ monocytes

Bone marrow Ly6C+ monocytes

Bone marrow CX3CR1+ monocytes

CD11b+ CD11c int DC

cDC

Skin

Inflammation

Spleen

Pre-DC

Cir

UV

Langerhans cell

culation

Intestine

MΦ Lung MΦ Lung DC Lamina propria

LPS, tion depletion

Lung

DC

Alveolar DC

MDP Figure 1 Monocyte differentiation into DCs and tissue macrophages. Macrophage-DC progenitors (MDPs) give rise to Ly6C+ bone marrow monocytes, which exit the bone marrow, in part guided by CCR2-dependent signals. Ly6C+ monocytes convert into CX3CR1+ monocytes, although the location of this event, in the circulation or bone marrow, remains incompletely understood. Black arrows indicate differentiation steps into tissue DCs and macrophages that occur under homeostatic conditions. Red arrows indicate differentiation steps that occur under inﬂammatory conditions (UV-induced skin injury, intratracheal LPS administration, or depletion of autologous CD11c+ cells). Dashed arrows represent steps that remain uncertain. In the case of splenic cDCs, splenic pre-DCs are the most signiﬁcant upstream precursor in numeric terms (bold arrow), although MDPs and CX3CR1+ monocytes may contribute as well. www.annualreviews.org • Monocyte-Mediated Defense

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Relationship Between Murine CX3CR1+ and Ly6C+ Blood Monocytes

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Exit of Ly6C+ murine monocytes from the bone marrow is driven, at least in part, by CCR2-mediated signals. The number of circulating Ly6C+ murine monocytes in CCR2−/− mice under homeostatic conditions and following systemic microbial infection (29, 30) is markedly diminished. In contrast, the frequency of circulating CX3CR1+ monocytes is similar in CCR2+/+ and CCR2−/− mice (23). Following depletion of circulating monocytes, Ly6C+ monocytes reach pretreatment levels in the bloodstream in three to four days (21). In contrast, CX3CR1+ monocytes return to the circulation seven days after depletion (21). To determine whether CX3CR1+ monocytes descend from Ly6C+ monocytes, the latter cells were labeled with ﬂuorescent liposomes or latex microspheres following systemic depletion (21) or under steady-state conditions (31). In both cases, labeled monocytes converted from a Ly6C+ to a Ly6Clow phenotype, indicating that Ly6C+ monocytes mature into CX3CR1+ monocytes. Adoptive transfer studies in rats give similar results (32).

MONOCYTE DIFFERENTIATION IN VIVO Circulating monocytes are precursors for tissue macrophages and many DC subsets (3). Monocytes give rise to DCs in vitro (33) and in vivo (16, 34–36), and microbial infection triggers in vivo monocyte differentiation into specialized DC populations that enhance microbial clearance (37, 38). The developmental pathways for DC sublineages in mice and humans are complex and have been reviewed comprehensively (39, 40). The contribution that circulating monocytes make to the formation and replenishment of speciﬁc DC subsets and tissue macrophages has been the focus of a number of interesting recent experiments. Many of these studies used adoptive 426

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cell transfer to investigate monocyte trafﬁcking and differentiation. Several themes concerning the role of circulating monocytes in the repopulation of tissue macrophages and DCs are emerging.

Monocyte Differentiation into Splenic Macrophage and DC Subsets The major splenic conventional DC (cDC) subsets (CD8+ CD4− , CD8− CD4− , and CD8− CD4+ ) turn over rapidly, with half-lives that range from one and a half to seven days (41, 42). To maintain steady-state splenic cDC populations in mice, a daily inﬂux of ∼105 circulating progenitor cells is required (42). Previous studies have identiﬁed several candidate circulating DC precursors that appear to be distinct from monocytes (43, 44) or that are monocytic in origin (45). In addition, nonmonocytic intrasplenic DC precursors with limited potential for cell division, termed preDCs, contribute to the maintenance of all cDC subsets (46, 47). Under homeostatic conditions, MDPs contribute to the steady-state splenic mononuclear phagocyte pool because adoptively transferred cells give rise to CD8+ and CD8− cDCs as well as to splenic marginal zone and marginal sinus macrophages in nonirradiated recipient mice (28). In contrast, more differentiated bone marrow cell populations were much less efﬁcient than MDPs in generating DCs (28) (20). In a separate study, splenic-resident pre-DCs gave rise to all CD8+ and CD8− DC subsets and were 50-fold more efﬁcient in generating CD8− cDCs than were puriﬁed blood Ly6Clow monocytes (46). Thus, in the steady state, MDPs and splenic pre-DCs, rather than bone marrow and circulating monocytes, appear to reconstitute cDCs most effectively (Figure 1). If recipient mice are irradiated, adoptively transferred Ly6C+ bone marrow monocytes readily give rise to splenic CD8+ and CD8− cDCs as well as F4/80+ splenic macrophages (45). In the setting of systemic inﬂammation,

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Ly6C+ monocytes differentiate into splenic DCs with a CD11b+ CD11cint Mac-3+ phenotype, distinct from the major cDC subsets (46). The phenotype of these inﬂammationinduced DCs is similar to TNF- and inducible nitric oxide synthase (iNOS)-producing DCs (TipDCs) that inﬁltrate the spleen during systemic listeriosis (37). In mice depleted of CD11c+ cells (48), adoptive transfer of MDPs or Ly6C+ bone marrow monocytes yields a similar population of CD11b+ CD11cint splenic cells (20). Thus, both Ly6C+ and Ly6Clow monocytes have the capacity to differentiate into splenic DC subsets under speciﬁc host circumstances, although their contribution in the steady state may be limited (Figure 1).

Monocyte Differentiation into Intestinal and Pulmonary Mononuclear Phagocytes The intestinal lumen and bronchoalveolar space represent major portals of entry for pathogenic microbes. Monocyte descendants play a major role in surveillance and immune defense in these tissues. In the intestinal lamina propria, intravenously transferred MDPs or Ly6C+ bone marrow monocytes differentiate into CX3CR1high CD11c+ DCs and CX3CR1low CD11c+ macrophages (20) (Figure 1). The respiratory tract and lung contain a number of resident macrophage and DC subsets that can be distinguished by surface antigen expression and localization (49–51). Major subsets include alveolar and lung macrophages, with a CD11c+ CD11b− CX3CR1− surface phenotype, and CD11c+ CD11b+ CX3CR1+ lung DCs (49, 52). Adoptively transferred CX3CR1+ monocytes trafﬁc to the lungs of recipient mice in the steady state (16), and these cells, along with Ly6C+ monocytes, give rise to pulmonary DCs (52). In the setting of local inﬂammation (via intratracheal LPS administration) or depletion of autologous respiratory tract CD11c+ cells, CX3CR1+ monocytes give

rise to lung macrophages and alveolar DCs as well (52) (Figure 1). In the steady state, alveolar macrophages are long-lived cells that turn over slowly (40%–60% replacement in one year) in bone marrow chimeric mice (53, 54). Although induction of local inﬂammation accelerates their turnover (54), the role of monocytes and local precursors in their replenishment remains unresolved.

Monocyte Differentiation into Langerhans Cells, Dermal DCs, and Lymph Node DCs Dermal DCs and Langerhans cells (LCs) contribute to skin immunity by forming a cellular surveillance system throughout the epidermis and delivering foreign antigens to draining lymph nodes. To maintain steady-state numbers, LCs are replenished by local precursors. Upon tissue damage, such as after intense UV irradiation, circulating precursors are required for replenishment. Although both myeloid and lymphoid progenitors (particularly fetal liver kinase 2+ cells) can yield LCs, CMPs are ∼20-fold more efﬁcient in this process than are CLPs (55). In vitro, circulating human CD14+ monocytes differentiate into LCs through a CD14+ CCR6+ langerin+ dermal precursor (56). In a murine model of UV-induced skin injury, Ly6C+ murine monocytes labeled with latex particles enter the skin within four days, proliferate, and differentiate into MHC class II+ langerin+ LCs (36) (Figure 1). F4/80+ CD68+ dermal macrophages descend from inﬁltrating Ly6C+ monocytes as well. Inﬂammation in the skin also promotes Ly6C+ monocyte trafﬁcking from the bloodstream to skindraining lymph nodes via high endothelial venules (HEVs) (17). In this setting, MCP1, a chemokine that triggers CCR2 signaling, is transported from the inﬂammatory focus in the skin to the draining lymph node, where it binds to the luminal surface of HEVs and mediates entry of CCR2-expressing Ly6C+ monocytes into the lymph node (17). www.annualreviews.org • Monocyte-Mediated Defense

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Under infectious or inﬂammatory conditions, skin-draining lymph nodes contain a number of non-LC DC populations that have been implicated in antigen presentation and T cell priming (57). CD11b+ F4/80− monocytic cells injected subcutaneously acquire ﬂuorescent latex particles and mature into CD11cdim MHC class II+ cells (35). In this experimental model, the Ly6Cint murine monocyte subset may represent the relevant DC precursor (23).

MONOCYTES IN MUCOSAL IMMUNITY The mammalian intestine is home to complex microbial populations and also serves as a portal of entry for a wide range of pathogens. Many intestinal pathogens penetrate the intestinal mucosa and thereby come in direct contact with the extensive network of DCs in submucosal tissues (58). In most cases, entry of pathogens through the intestinal mucosa occurs via epithelial M cells, which overlay Peyer’s patches (59–62), and requires expression of a specialized set of bacterial virulence factors. For example, the enteric pathogen Salmonella typhimurium traverses M cells by expressing a family of genes encoded in the Salmonella pathogenicity island (SPI)-1 locus that injects virulence factors into epithelial cells (63). S. typhimurium, however, can also traverse the intestinal epithelium by an alternate route. SPI-1-deﬁcient S. typhimurium, for example, is taken up by circulating CD18-expressing monocytes and disseminates from the intestine to spleen and liver (64). Remarkably, although CD18deﬁcient mice are more susceptible to splenic and hepatic infections following the intraperitoneal challenge, they are more resistant to intestinal infection by SPI-1deﬁcient S. typhimurium than are wild-type mice. Although intestinal epithelial cells form a tight barrier separating bowel contents from submucosal tissues, recent studies indicate that DCs in the submucosa can extend den428

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drites into the bowel lumen and interact with intestinal bacteria. Rescigno et al. (65) demonstrated that CD11c-expressing cells are rapidly recruited to intestinal loops following S. typhimurium infection. Intraepithelial CD11c+ (65) and CD11b+ CD8α− (66) DCs express tight junction proteins that interact with epithelial cell tight junctions, thereby allowing dendrites to pass between intestinal epithelial cells without disrupting the integrity of the barrier. Intravital microscopy of small bowel explants (67) demonstrated that formation of DC extensions requires MyD88mediated signals in nonhematopoietic cells, presumably intestinal epithelial cells. Sampling of luminal bacteria by CD11c+ DCs can lead to direct infection of these cells (65, 67, 68) and transport of bacteria from the intestinal tract to mesenteric lymph nodes (MLNs) (68, 69). Intestinal DCs promote IgA synthesis by B cells and may prevent mucosal dissemination by commensal bacteria (68, 195). Intestinal TipDCs induce mucosal IgA secretion, and TipDC-derived nitric oxide (NO) is essential for this function (196). The lamina propria of the small and large intestine contains extensive networks of CX3CR1-expressing DCs (70), which may originate from circulating CX3CR1high monocytes (16). CX3CL1/fractalkine, the ligand for CX3CR1, is expressed by intestinal epithelial and endothelial cells (71–73) and is required for formation of transepithelial DC extensions. CX3CR1-expressing cells can engulf both nonpathogenic and pathogenic bacteria and transport them to MLNs (70). CX3CR1-deﬁcient mice are more susceptible to infection with S. typhimurium, suggesting that CX3CR1 signaling contributes to antimicrobial responses. It remains unclear whether enhanced susceptibility results from impaired induction of systemic immune responses or whether CX3CR1expressing cells directly exert bactericidal activity. Microbial infection induces the recruitment of monocytes to mucosa-associated

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lymphoid tissues. Following oral infection with S. typhimurium, CD11c+ CD11b+ monocytes accumulate in infected organs (74). A recent report demonstrated increased frequencies of monocytes in blood and their recruitment to Peyer’s patches and MLN following oral S. typhimurium innoculation (75). These recruited monocytes resemble Ly6C+ monocytes because they express F4/80, CD11b, CCR2, and CD68, and they are the main producers of TNF and iNOS during early S. typhimurium infection (Figure 2). Although protection against Salmonella requires secretion of TNF and reactive nitrogen intermediates (RNI), it is not known whether recruitment of inﬂammatory monocytes is critical for host survival during infection (Table 2).

MONOCYTES IN THE IMMUNE RESPONSE TO LISTERIA MONOCYTOGENES Innate Immune Responses to Listeria monocytogenes Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that infects a wide range of invertebrate and vertebrate hosts. One of the ﬁrst manuscripts describing infection with this pathogen noted an increased number of monocytes in tissues of infected rabbits, leading to the species name “monocytogenes” (76). L. monocytogenes is acquired via the gastrointestinal tract. Successful clearance of L. monocytogenes requires activation of innate and adaptive immune responses. Innate immunity is rapidly triggered following infection and restricts in vivo bacterial growth (77). A summary of bacterial pathogenesis and innate immune defenses is shown in Table 2. Innate immune responses to L. monocytogenes infection require synthesis of TNF, IFN-γ, IL-12, and IL-18, whereas deﬁciency in type I interferon (IFN) receptors or the IFN regulatory factor (IRF)-3 transcription factor renders mice more resistant to infection (77).

Monocyte Function During Innate Immune Responses Cells of myeloid lineage play a key role in defense against L. monocytogenes infection (77). Infection with L. monocytogenes induces an inﬂux of monocytes and macrophages to sites of infection (78). Maximum recruitment of monocytes occurs 72 to 96 h following infection and thus is delayed relative to granulocyte recruitment (78). In vivo administration of RB6-8C5 antibody speciﬁc for Gr1, an epitope that is expressed on Ly6G and Ly6C antigens (79), leads to depletion of granulocytes and a subset of monocytes and renders mice highly susceptible to L. monocytogenes infection (80, 81). Gr1-expressing cells are most critical during the ﬁrst 24 h of the innate immune response (81, 82). In vivo administration of the 5C6 antibody, which blocks CD11b, renders mice highly susceptible to L. monocytogenes infection (83). Blocking CD11b abrogates monocyte and granulocyte accumulation in spleen and liver but, as with Gr1-depletion, only enhances susceptibility to L. monocytogenes infection during the ﬁrst 24 h of infection. In the absence of CD11b-mediated granulocyte and monocyte recruitment, L. monocytogenes replicates within nonphagocytic cells, such as hepatocytes, and also extracellularly. Thus, CD11b-mediated recruitment of inﬂammatory cells is essential for bacterial containment and killing early during infection.

Role of Monocytes in Bacterial Dissemination In humans, infection with L. monocytogenes is often associated with bacteremia and frequently results in the development of meningitis (84, 85). Although mouse models of systemic L. monocytogenes infection do not precisely replicate the course of human meningeal infection, infection of mice with very high inocula of bacteria has been used to investigate the pathogenesis of L. monocytogenes meningitis. In these settings, bloodstream L. monocytogenes is found www.annualreviews.org • Monocyte-Mediated Defense

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Red pulp

a

White pulp

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Spleen

L. monocytogenesinfected macrophages

Bone marrow CCR2+ monocytes Differentiated CCR2+ monocytes TNF/iNOS-producing CCR2+ DC (TipDC) Infected macrophages TNF NO

CNS

b

S. typhimurium (gut)

c

M. tuberculosis (lung)

IL-12

d

T. gondii (peritoneum)

CNS

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Microbial pathogenesis in the murine models of infectious disease

Microbe

Route of infection

Target cell/site and localization

Innate immune effector molecules required for resistance

Impact of CCR2 deficiency

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Bacteria L. monocytogenes

Gastrointestinal

Intestinal epithelium, hepatic and splenic macrophages, hepatocytes (intracellular, cytoplasmic)

TNF, IFN-γ, RNI, ROI

↑ susceptibilitya

M. tuberculosis

Inhalation

Macrophages (intracellular, vacuolar)

TNF, IFN-γ, IL-12, RNI

↑ susceptibility (high-dose iv infection)a

S. typhimurium

Gastrointestinal

Intestinal epithelium, macrophages (intracellular, vacuolar)

TNF, IFN-γ, IL-12, ROI, RNI

Not determined

Gastrointestinal

Macrophages and many nucleated cells (intracellular, vacuolar)

TNF, IFN-γ, IL-12

↑ susceptibilitya

Inhalation

Alveolar macrophages (intracellular conidia)

ROI

↑ pulmonary allergic responses

Protozoa T. gondii Fungi A. fumigatus

Alveolar spaces and lung tissue (extracellular conidia and hyphae) a b

↓ fungal clearanceb

monocytes implicated. role of monocytes not known.

predominantly in circulating monocytes (21, 86). The majority of infected monocytes express CD11b and high levels of Ly6C (87) and thus resemble inﬂammatory monocytes (16). Monocytes may also be infected in bone marrow prior to entering peripheral circulation (88).

Following infection with L. monocytogenes, Ly6C+ monocytes are recruited into the brain parenchyma, supporting the notion that these monocytes carry bacteria into the central nervous system (CNS) (87). Adoptive transfer of L. monocytogenes–infected Ly6C+ CD11b+ bone marrow monocytes leads to CNS

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 2 Effector functions of inﬂammatory monocytes. In the absence of inﬂammation, bone marrow CCR2+ monocytes have an immature phenotype and are characterized by low levels of expression of MHC class II and costimulatory molecules. Following infection, monocytes are released into the peripheral circulation and migrate to sites of inﬂammation, where they express distinct effector phenotypes and undergo differentiation into DCs. The effector functions of CCR2+ monocytes are dictated by the inﬂammatory context and by the nature of the invading pathogen. (a) Following infection with L. monocytogenes, monocytes are ﬁrst present in the marginal zone area of the spleen and subsequently migrate to the white pulp area, where bacterial lesions are established. Monocytes undergo differentiation into TipDCs and surround infected cells, thus preventing bacterial dissemination from the lesion. While most CCR2+ monocytes are not infected in vivo, monocytes in the peripheral circulation may become infected and transport bacteria to the CNS. (b) In the gastrointestinal tract, infection with S. typhimurium induces inﬂux of inﬂammatory monocytes and their differentiation into TipDCs. (c) Although less is known regarding the function of monocytes during M. tuberculosis infection, they are recruited to the lung and may function as a source of nitric oxide (NO). (d ) During infection with T. gondii, inﬂammatory monocytes become directly infected and secrete IL-12 and NO and kill parasites. T. gondii–infected monocytes may also be involved in transport of parasites to the brain. www.annualreviews.org • Monocyte-Mediated Defense

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infection as early as 6 h post-transfer. Because dissemination of L. monocytogenes to the brain occurs in mice treated with gentamicin, an antibiotic that kills extracellular but not intracellular bacteria (87, 89), it has been argued that L. monocytogenes uses monocytes as a Trojan horse to enter the CNS.

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Monocytes kill bacteria by producing reactive nitrogen intermediates (RNIs) and reactive oxygen intermediates (ROIs) (90) and through the action of phagolysosomal enzymes (91). Administration of iNOS inhibitor aminoguanidine and genetic deﬁciency in iNOS, gp91, or p47phox renders mice more susceptible to L. monocytogenes infection (92– 94), implicating these mechanisms in bacterial clearance (Table 2). Signaling through TLR molecules is essential for protection during L. monocytogenes infection, and mice deﬁcient in the TLR adaptor molecule, MyD88, are highly susceptible to infection (95, 96). MyD88 deﬁciency is associated with diminished IL-12, IFN-γ, TNF, and NO responses (95, 96), and thus MyD88-deﬁcient mice are more susceptible to L. monocytogenes infection than mice lacking IFN-γ or both IL-12 and IFN-γ (96). Although the preceding studies demonstrated the importance of TLR signaling in defense against L. monocytogenes, it remains unclear in which cell population TLR signaling is most critical. A recent study took advantage of the essential role of gp96, an endoplasmic reticulum chaperone, in the folding of TLR molecules. gp96−/− macrophages fail to respond to intracellular and cell surface TLR ligands but respond normally to activation by IFN-γ, TNF, and IL-1. Using mice with monocyte- and macrophage-speciﬁc deletion of gp96, these investigators demonstrated that TLR signaling in these cells is essential for defense against L. monocytogenes despite intact signaling via IFN-γ, TNF, and IL-1 receptors (97). 432

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Recruitment of TNF- and iNOS-Producing Monocytes During L. monocytogenes Infection Mice lacking CCR2 are highly susceptible to L. monocytogenes and succumb to infection within four days, a time frame that indicates failure of innate immune defenses (98). L. monocytogenes–infected, CCR2-deﬁcient mice have normal levels of IL-12 and IFN-γ but markedly diminished levels of TNF and iNOS in infected spleens (37). During the ﬁrst three days of systemic infection, TNF and iNOS are predominantly expressed by a population of monocyte-derived TipDCs that are recruited to foci of infection (Figure 2). Recruitment of TipDCs to spleen does not occur in CCR2deﬁcient mice. In infected spleens, TipDCs express CD11b, low or intermediate levels of CD11c, high levels of intracellular Mac3, high levels of Ly6C, and variable levels of F4/80 (29, 37) and are distinct from conventional and plasmacytoid DCs. TipDCs express high levels of MHC class II and costimulatory molecules during L. monocytogenes infection. TipDCs recruited to infected spleens are derived from bone marrow monocytes. Although Ly6C+ monocytes are present in the peripheral circulation and spleen of L. monocytogenes–infected wild-type mice, they are absent from the blood and spleen of infected CCR2-deﬁcient mice but instead accumulate in the bone marrow (29). In the absence of infection, Ly6C+ cells in the bone marrow do not express MHC class II or CD11c. In vitro culture of these progenitors with listerial antigens and IFN-γ leads to upregulation of MHC class II and CD11c molecules and induces iNOS, recapitulating the TipDC phenotype seen during in vivo infections. Interestingly, Ly6C+ monocytes accumulating in the bone marrow of CCR2deﬁcient mice during infection express MHC class II and costimulatory molecules but not CD11c, suggesting that the bone marrow environment only induces partial monocyte differentiation. It is not clear whether Ly6C+ monocytes accumulating in the bone marrow

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contribute to antimicrobial responses. However, despite uncontrolled bacterial growth in spleen and liver of CCR2-deﬁcient mice, bacterial numbers are comparable or slightly lower in bone marrow of CCR2-deﬁcient mice compared with wild-type mice, suggesting that monocytes retained in the bone marrow of CCR2-deﬁcient mice mediate antimicrobial effects.

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Antimicrobial Function of TipDCs Failure to recruit TipDCs to sites of infection diminishes clearance of L. monocytogenes (Table 2). TipDCs produce the highest amounts of TNF of any cell population in the L. monocytogenes–infected spleen. However, because many hematopoietic and nonhematopoietic cells can secrete TNF, it remains unclear which source of TNF is essential for bacterial clearance. One recent study demonstrated that mouse strains with selective depletion of TNF in monocytes and macrophages are highly susceptible to infection, suggesting that these cell populations are the relevant source of TNF in defense against L. monocytogenes infection (99). Interestingly, deﬁciency in type I IFN signaling leads to increased numbers of TNFproducing mononuclear cells (100), although the mechanisms that lead to this increase remain incompletely deﬁned. NO production is also diminished in spleens of infected CCR2-deﬁcient mice, but whether iNOS expression by TipDCs contributes directly to in vivo microbial killing remains unclear. TipDCs do not appear to be directly infected in vivo, suggesting that NO produced by these cells may act on local cells to enhance microbicidal activity. Alternatively, bacteria may be very rapidly killed and degraded by infected TipDCs, so that infected cells are not readily detected. Although recruitment of TipDCs to spleen is MyD88independent, TNF secretion by TipDCs requires MyD88-mediated signals (101). TNF and iNOS production by recruited CCR2expressing monocytes may be important in

immune protection against some but not all bacterial pathogens. During Escherichia coli– induced urinary tract infection, TipDCs are rapidly recruited to the infected bladder but do not appear to participate in protective immune responses (102).

MONOCYTE RECRUITMENT DURING MYCOBACTERIUM TUBERCULOSIS INFECTION Mycobacterium tuberculosis is an inhaled intracellular bacterial pathogen that persists in macrophages of infected organs. Protective immunity to tuberculosis is T cell– mediated and requires secretion of IFN-γ, TNF, and IL-12 and production of RNIs (103). Successful activation of immune responses against mycobacteria requires signaling through MyD88 (104, 105). Pathogenesis of M. tuberculosis infection is summarized in Table 2. A number of recent studies have focused on the recruitment and function of circulating monocytes during tuberculosis. Following aerosol infection of mice with M. tuberculosis, DCs, monocytes, macrophages, and granulocytes trafﬁc into the bronchoalveolar space (49). Recruited monocytes express CD11b, F4/80, and CCR2 and thus are characterized as inﬂammatory monocytes. The frequency of F4/80+ monocytes is signiﬁcantly reduced in CCR2−/− mice following M. tuberculosis challenge (106). The susceptibility of CCR2-deﬁcient mice is inﬂuenced by the dose of M. tuberculosis infection. CCR2 deﬁciency results in early mortality following high-dose intravenous (iv) challenge and aerosol challenge (107, 109). In this setting, CCR2 deﬁciency also leads to delayed T cell priming and a reduction in the number of IFN-γ-secreting CD4 T cells in the lung (107, 108). In contrast to high-dose iv infection, CCR2-deﬁcient mice survive low-dose aerosol infection despite reduced recruitment of alveolar macrophages, diminished iNOS levels, and delayed T cell inﬂux (109). Thus, CCR2+ monocytes may www.annualreviews.org • Monocyte-Mediated Defense

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be required for protection when bacterial burdens are high, such as during systemic M. tuberculosis infection. In this setting, recruited monocytes may provide a source of iNOS (Figure 2). In contrast, the bactericidal functions of resident alveolar macrophages may be sufﬁcient to control bacterial growth following low-dose aerosol infection with M. tuberculosis. How much human CCR2-expressing monocytes participate in immune responses during human mycobacterial diseases is not known. CCR2-expressing cells are detected in human skin lesions caused by Mycobacterium leprae, the cause of leprosy (110). In vitro, human monocytes can be differentiated into two distinct subsets, DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN− DCs. DC-SIGN+ CD16+ and CD1b+ DC-SIGN− cells can be detected in leprosy skin lesions (111), suggesting that monocyte recruitment and differentiation occur in the setting of human disease. A promoter polymorphism that induces hyperproduction of MCP-1 is associated with increased susceptibility to pulmonary tuberculosis (112). High levels of circulating MCP-1 may lead to desensitization of CCR2, thereby limiting the recruitment of monocytes to sites of lesions. This mechanism has been proposed as an explanation for the increased susceptibility of MCP-1 transgenic mice to infection with L. monocytogenes (113). Generation of NO by human macrophages in vitro is difﬁcult to demonstrate, and many different experimental strategies have rendered inconsistent results. However, iNOS expression in human monocytes can be induced in vitro in response to M. tuberculosis lipoproteins (114) and in vivo in the lungs of patients with active M. tuberculosis (115), suggesting that the RNI-mediated pathway may be operative in human infection. RNI-independent mycobacterial killing by human monocytes and macrophages has also been reported. Human monocytes can kill M. tuberculosis in a TLR-dependent and NO-independent manner (116).

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MONOCYTE RECRUITMENT DURING TOXOPLASMA GONDII INFECTION Toxoplasma gondii is a protozoan pathogen that infects a wide range of mammals including humans. T. gondii is an obligate intracellular parasite and resides in a vacuole in many different nucleated cell populations. Recent studies have demonstrated that monocyte recruitment is essential for initial restriction of T. gondii growth in the murine mouse model of toxoplasmosis (117, 118). Protection against T. gondii requires MyD88-mediated signaling and induction of IL-12, IFN-γ, and IFN-γinducible p47 GTPase but is independent of RNI production (119, 120). Table 2 depicts the summary of pathogenesis and innate immune responses during toxoplasmosis. Gr-1-expressing monocytes are recruited to the peritoneum four to ﬁve days following intraperitoneal infection of mice with an attenuated strain of Toxoplasma gondii (117). Recruited monocytes express CD68, CD11b, F4/80, and iNOS and secrete IL-12p40 in vivo. T. gondii infects monocytes in the peritoneum, stimulating upregulation of MHC class II and costimulatory molecules and differentiation into DCs. Monocytes puriﬁed from peritoneum of infected mice inhibit parasite replication in vitro by NOindependent mechanisms. Recently, Ling et al. (121) demonstrated that in vivo destruction of T. gondii by peritoneal CD11b+ F4/80+ macrophages occurs via stripping of the parasite plasma membrane followed by fusion with autophagosomes. CCR2-deﬁcient mice are more susceptible to T. gondii infection, a phenotype that correlates with diminished recruitment of Gr1-expressing monocytes to sites of infection (118). Diminished recruitment of inﬂammatory monocytes to sites of T. gondii infection and increased in vivo parasite growth occur despite normal levels of IFN-γ and TNF. However, IL-12p70 levels are diminished in the serum of infected CCR2-deﬁcient mice, suggesting that IL-12 secretion by

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inﬂammatory monocytes contributes to protection (Figure 2). Inﬂammatory monocytes implicated in resistance to T. gondii infection resemble L. monocytogenes–induced TipDCs. However, they appear to mediate immune protection in a manner distinct from that of TipDCs. Monocytes elicited by T. gondii and L. monocytogenes infections may originate from the same circulating progenitor and be driven to differentiate along similar pathways, but, given the different molecular composition of these two pathogens, the inﬂammatory cues driving differentiation are likely distinct. Identifying the signals that drive monocyte trafﬁcking and differentiation in these two settings will require additional experimentation. Monocytes may be involved in the transport of T. gondii tachyzoites to the brain during infection (122). Following intragastric inoculation with T. gondii, parasites circulating in the bloodstream reside within monocytes. Adoptive transfer of T. gondii–infected monocytes results in the appearance of parasites in the brains of recipient mice.

ROLE OF MONOCYTES IN HOST DEFENSE AGAINST FUNGAL PATHOGENS The ﬁlamentous mold Aspergillus fumigatus (see Table 2) and the encapsulated yeast Cryptococcus neoformans are ubiquitous in the environment, and mammalian infections are acquired via the respiratory route (123, 124). The fungal dimorph Candida albicans causes mucosal disease as well as systemic infections. Tissue macrophages and neutrophils play critical roles in defense against fungal infection (123, 124). Although recruitment of monocytes to sites of fungal infection has been demonstrated in vivo (125), their role in fungal killing remains unclear. However, puriﬁed murine and human monocytes or cultured macrophages have been studied in vitro to characterize the induction of inﬂammatory and fungicidal mediators, rates of fungal killing (126, 127), and host cell and

fungal transcriptional responses (128–130). Whether in vitro–deﬁned mechanisms of fungal inactivation are operative in vivo and contribute to fungal clearance, however, will require further studies. Recent studies in CCR2-deﬁcient mice indicate that inﬂammation-induced monocyte recruitment contributes to host antifungal immune responses. In murine pulmonary cryptococcosis, CCR2−/− mice are unable to control fungal growth. Increased susceptibility is associated with diminished pulmonary macrophage recruitment and the induction of maladaptive Th2-biased T cell responses (131, 132). Although these defects are likely related to impaired recruitment of inﬂammatory monocytes to sites of C. neoformans infection, it is possible that CCR2-expressing natural killer (NK) and T cell subsets (133) contribute to the phenotype of CCR2−/− mice. In a murine model of allergic disease associated with A. fumigatus antigen exposure, CCR2−/− mice exhibit prolonged pulmonary allergic responses, airway inﬂammation, and delayed clearance of fungal antigens, suggesting that CCR2 signaling restricts the development of fungus-associated asthmatic disease (134, 135). One hypothesis to explain this ﬁnding is that CCR2-mediated recruitment of monocytes sways A. fumigatus–speciﬁc CD4 T cell responses toward a Th1 as opposed to a Th2 phenotype. Alternatively, CCR2mediated recruitment of cells other than inﬂammatory monocytes may be critical in defense against A. fumigatus infection. In an experimental setting of invasive aspergillosis, CCR2+ NK cells mediate protective effects (136).

MONOCYTES IN REGULATION OF ADAPTIVE IMMUNE RESPONSES Given the capacity of monocytes to differentiate into DCs upon in vitro culture with GMCSF and IL-4, it is reasonable to speculate that monocytes contribute to the initiation www.annualreviews.org • Monocyte-Mediated Defense

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and differentiation of T cell responses (45, 137, 138). Although inﬂammatory stimuli generally promote monocyte differentiation into DCs and their migration to lymph nodes (35), in some settings TLR-mediated signals block monocyte differentiation into DCs (139). Nevertheless, increasing experimental evidence supports the notion that circulating monocytes impact T cell responses. Regulation of T cell responses by monocytes in the setting of microbial infection is complex, with positive and negative contributions to T cell proliferation and differentiation. CD8 T cell responses to an attenuated strain of L. monocytogenes, for example, are enhanced in CCR2-deﬁcient mice, suggesting that CCR2+ monocytes negatively regulate CD8 T cell proliferation or survival (37). Because NO inhibits proliferation of T cells (140, 141), iNOS expression by CCR2+ monocytes may dampen T cell responses. However, it is possible that other effector functions of CCR2+ cells inhibit T cell responses. For example, monocytes and their derivative cells secrete IL-10 in response to some microbial stimuli (142–148), and Ly6C+ monocytes puriﬁed from bone marrow secrete IL-10 in response to stimulation with heat-killed L. monocytogenes (N.V. Serbina & E.G. Pamer, unpublished results). T cell suppression by IL-10-secreting Gr-1+ CD11b+ cells during polymicrobial sepsis has also been reported (149). In this experimental system, Gr-1+ CD11b+ cells expand in spleen, lymph nodes, and bone marrow during sepsis in a MyD88-dependent manner. In contrast to L. monocytogenes infection, during M. tuberculosis infection CCR2+ monocytes enhance T cell priming and Th1 differentiation (106–108). Although T cells can express CCR2 (133, 150), defects in T cell recruitment during tuberculosis in CCR2deﬁcient mice are attributed to impaired monocyte and DC trafﬁcking and are independent of T cell CCR2 expression (106). In the setting of cutaneous Leishmania major infection, CCR2+ monocytes are recruited to skin lesions (38, 151). In L. major–infected

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CCR2-deﬁcient mice, protective Th1 responses are attenuated, whereas Th2 responses are enhanced, which impairs microbial clearance. Following cutaneous infection, monocytes enter draining lymph nodes via afferent lymphatics and HEVs and give rise to functionally distinct DC subsets that are not present in the steady state (38). Within the infected dermis, monocytes differentiate into dermal DCs with a CD11cint Ly6Chigh MHC classIIint CD86low phenotype and mature into CD11cint Ly6Clow MHC class IIhigh CD86high DCs upon transit to the lymph node (38). Intravenously or subcutaneously transferred monocytes differentiate into this DC subset, which exhibits high T cell stimulatory capacity ex vivo. In contrast, a second CD11cint Ly6Chigh MHC class IIint CD86− DC subset enters popliteal lymph nodes via HEVs because intravenous, but not subcutaneous, monocyte transfer results in their appearance. This DC subset is phenotypically less mature and primes CD4 T cells less efﬁciently than does the dermal-derived DC subset described above. Direct, monocyte-mediated priming of T cell responses was demonstrated using OVAconjugated particles. In this system, circulating B cells and neutrophils transferred antigens to immature monocytes in bone marrow (152), which then trafﬁc to spleen and lymph nodes and prime OVA-speciﬁc T cells. Although it is unclear whether this route of antigen presentation occurs during microbial infections, circulating CD11b+ Gr-1+ monocytes can internalize bacterial antigens in blood and trafﬁc to splenic marginal zones where they interact with B cells and induce differentiation into plasmablasts and T cell– independent antibody responses (153).

CHEMOKINES AND CHEMOKINE RECEPTORS Optimal immune responses to infection depend on chemokine networks to facilitate recruitment of speciﬁc leukocytes to sites of infection. Chemokine-mediated monocyte

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recruitment is pivotal for immune control of a variety of microbial infections. Chemokines are divided into four groups on the basis of the position of cysteine residues: C chemokines have one cysteine, CC chemokines have two adjacent cysteines near the amino terminus, CXC chemokines have an amino acid separating two cysteines, and CX3C chemokines have three amino acids located between two cysteines (154). CC chemokines trigger chemokine receptors on monocytes, basophils, eosinophils, T cell subsets, and DCs. The CCR2-binding chemokines MCP1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), and MCP-5 (CCL12) belong to this family. The only known member of the CX3C chemokine family is fractalkine (FKN, or CX3CL1). The soluble form of FKN is a potent chemoattractant for subsets of monocytes, T cells, and NK cells (155).

ROLE OF MCPs AND CCR2 IN MONOCYTE RECRUITMENT DURING INFECTION MCP-1−/− mice are not as susceptible to L. monocytogenes infection as CCR2−/− mice (37, 101, 118), suggesting that MCP-1 is not the sole CCR2 ligand responsible for monocyte recruitment and that other CCR2 ligands are induced during infection. Recently, MCP3−/− and MCP-2/5−/− mice have been generated and examined for monocyte trafﬁcking (30). MCP-3−/− mice, like CCR2−/− and MCP-1−/− mice, have diminished numbers of inﬂammatory monocytes in peripheral blood. In contrast, MCP-2/5−/− mice have normal circulating monocyte numbers, indicating that MCP-1 and MCP-3 are the predominant CCR2 ligands maintaining homeostatic numbers of circulating inﬂammatory monocytes (30). The role of MCP-3, MCP-2, and MCP-5 in antimicrobial defense is not known.

Induction of MCPs by Infection and Inflammation MCP-1 is induced during L. monocytogenes infection, with detectable levels in spleen

6 h after bacterial inoculation (98, 101). The source of MCP-1 during in vivo bacterial infection, however, remains unclear. Because γδ T cell–deﬁcient mice have diminished MCP-1 mRNA levels in the liver following infection compared with wild-type mice, investigators have suggested that γδ T cells produce MCP-1 (156). In vitro, infection of murine macrophages and hepatocytes with L. monocytogenes rapidly induces MCP-1 expression (157). T. gondii infection also induces MCP-1 expression in vivo and increases MCP-1 mRNA expression in peritoneal exudate cells (118). In vitro, T. gondii infection induces MCP-1 secretion in human ﬁbroblasts, epithelial cells, and astrocytes (158–161). MCP-1 secretion by ﬁbroblasts following infection depends on the stage of the parasite (160). Infection with fungal pathogens, such as Aspergillus fumigatus, also induces in vivo MCP-1 expression, but the level of induction differs depending on the level of preexisting immunity (134). MCP-1 production following M. tuberculosis infection has been examined in several different cell types in vitro. CD14+ blood monocytes from patients with active tuberculosis express higher levels of MCP-1 mRNA and protein than do CD14+ monocytes from healthy individuals with latent, inactive tuberculosis (162). The human A549 alveolar epithelial cell line infected with M. tuberculosis also expresses elevated MCP-1 mRNA and protein levels. Intracellular growth is necessary for M. tuberculosis to induce MCP-1 in alveolar epithelial cells, but neither mycobacterial virulence nor the rate of intracellular growth correlates with the level of chemokine production (162). Although MCP-1 expression is commonly measured following infections or in an inﬂammatory setting, much less is known about the expression and regulation of the other major CCR2 ligands, MCP-2, MCP-3, and MCP5. Whereas in some circumstances MCP-1 and MCP-3 expression are coordinately regulated, in others their expression levels differ (163, 164). MCP-3 expression by different cell types appears to be more restricted www.annualreviews.org • Monocyte-Mediated Defense

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than MCP-1 expression. Murine MCP-5, the closest homolog of human MCP-1 in terms of amino acid sequence (165), is expressed in lymphoid tissues and the lung and attracts human monocytes in chemotaxis assays (165). Although its role in defense against infections is unclear, MCP-5 has been implicated in the migration of leukocytes through the lung interstitium (166) and also in the recruitment of ﬁbrocytes to the lung in the setting of pulmonary ﬁbrosis (167).

Induction and Regulation of MCP-1 Expression In vitro studies characterizing MCP-1 induction have been performed in a range of different cell types [monocytes (168), ﬁbroblasts (169), epithelial cells (169, 170), endothelial cells (171, 172), vascular smooth muscle cells (173)] under different conditions (170). These studies have generated a complex picture with many consistent themes, but they have also produced a number of contradictory ﬁndings. The contradictions likely reﬂect real differences in MCP-1 induction pathways in distinct cell types. A summary of surface receptors and downstream signaling molecules involved in MCP-1 production in different cell types is provided in Table 3. TLRs and Nod molecules recognize bacterial ligands and initiate immune responses. Stimulation of TLR-2 and TLR-4 in mouse renal tubular epithelial cells (MTECs) and stimulation of TLR-1, TLR-2, TLR-3, TLR4, and TLR-9 in macrophages induce MCPTable 3

1 (170). MCP-1 induction by Nod stimulation differs depending on the cell type being investigated. Although stimulation of bone marrow–derived macrophages and DCs with the synthetic Nod1 agonist KF1B does not induce MCP-1 production (174), stimulation of mouse mesothelial cells does induce MCP-1 production (175). The contributions of innate immune signaling adaptor molecules and downstream kinases in MCP-1 regulation have been investigated in several systems. Induction of MCP-1 in bone marrow–derived macrophages following L. monocytogenes infection is MyD88-independent but requires bacterial invasion of the cytoplasm (101). In mesothelial cells, Nod1-mediated MCP-1 production, but not TLR-mediated MCP-1 production, requires RIP2-mediated signaling (175). Induction of MCP-1 in MTECs by TLR stimuli requires NF-κB activation but not p38 mitogen-activated protein kinase (MAPK) or c-Jun N-terminal kinase ( JNK) activity (170). In HeLa cells, however, induction of MCP-1 by T. gondii requires ERK1/2 and JNK MAPK activation, but it is independent of p38 MAPK (176). Induction of MCP-1 in human monocytes by M. tuberculosis requires NF-κB, ERK, and p38 MAPK signaling (177). In addition to direct induction of MCP1, stimulation of TLRs and Nods during infection also induces inﬂammatory cytokines, such as TNF and type I IFN. One interesting question is whether these inﬂammatory mediators also regulate MCP-1 production

Receptors and signaling molecules in MCP-1 production pathway

MTECs

• Response to TLR-2 and TLR-4 stimulation • MCP-1 production requires NF-κB activation but not p38 and JNK MAPKs

Mesothelial cells

• Response to TLR-2, TLR-3, TLR-4, and TLR-5 stimulation • Response to Nod1 stimulation through RIP-2-mediated signaling

Macrophages

• Response to TLR-1, TLR-2, TLR-3, TLR-4, and TLR-9 stimulation • No response to Nod1 stimulation • MCP-1 production following L. monocytogenes infection is MyD88-independent

Monocytes

• MCP-1 production following M. tuberculosis infection requires NF-κB, ERK, and p38 MAPK signaling

HeLa cells

• MCP-1 production following M. tuberculosis infection requires NF-κB, ERK, and p38 MAPK signaling

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in vivo. In vitro, MCP-1 induction by TNF and type I and type II IFNs has been examined (169, 172, 178). TNF-mediated induction and regulation of MCP-1 have been investigated in ﬁbroblasts and involve distal and proximal regulatory regions (178–184). The two functional κB sites located in the distal regulatory region and a GC-box in the proximal regulatory region are critical for TNFmediated induction of MCP-1 (178). Sp1, a DNA sequence–speciﬁc transcription factor, is essential for MCP-1 promoter assembly and molecular communication between the two NF-κB-dependent sites (182). More recent studies demonstrated that Sp1 and NFκB are required for histone acetylation within the MCP-1 promoter (179). Although TNF stimulates MCP-1 production in ﬁbroblasts, TNF-mediated signaling is not required in vitro for MCP-1 production by macrophages or MTECs (170) or in vivo during L. monocytogenes infection (101). Anti-inﬂammatory factors also modulate MCP-1 production. Glucocorticoids inhibit MCP-1 synthesis in a variety of cell types (185–187), a process that involves changes in MCP-1 mRNA stability. Steroid-induced mRNA degradation is attributed to a 224 nucleotide dexamethasone-sensitive region within the coding region of the MCP-1 message (173). Stabilization of MCP-1 mRNA is mediated by direct association with the glucocorticoid receptor (188).

MCP Structures and Functions: Oligomerization and Binding to GAGs The structure of human MCP-1 has been extensively investigated. Two structural features of MCP-1 appear to be particularly important for its in vivo activity (189). The ﬁrst relates to residues that enable dimerization. Point mutations in MCP-1 that prevent dimerization, but not association with CCR2, markedly abrogate in vivo inﬂammatory cell recruitment in mice (189). Interestingly, the same mutant forms of MCP-1

remain active in in vitro chemotaxis assays, indicating that the rules governing chemotaxis in vivo differ from those required for conventional in vitro chemotaxis assays. However, human MCP-3 is active in vivo despite the fact that it does not oligomerize (189), suggesting that MCP-3 and MCP-1 function differently. The second feature of MCP-1 that affects its in vivo activity relates to residues that associate with glycosaminoglycans (GAGs) (190). As with dimerization, point mutations that diminish association with GAGs abrogate in vivo inﬂammatory cell recruitment, whereas in vitro chemotaxis assays remain intact (191– 193). In addition, the process of dimerization and GAG binding can be interdependent, as MCP-1 is induced to oligomerize when it binds to GAGs. Thus, both dimerization and association with GAGs are essential for in vivo MCP-1 activity. These two structural features are thought to facilitate the formation of highly localized foci of MCP1, which in turn may generate chemotactic gradients that enable monocyte migration (194).

Models of MCP Function in vivo During Infection Microbial infection induces MCP-1 production by a wide variety of cells. Whether MCP1 is principally produced by cells that are directly infected or by bystander cells that respond to inﬂammatory cytokines or microbial molecules that are released into extracellular ﬂuids is unresolved. Although many studies have measured chemokine levels in serum and in tissues such as spleen, liver, kidneys, and brain, it remains unknown whether the chemokines that are detected in these assays contribute to monocyte recruitment and antimicrobial defense. It is possible, however, that MCP-1 levels in serum promote monocyte emigration from bone marrow. Alternatively, serum MCP-1 may be irrelevant, and only MCP-1 produced within the bone marrow, potentially by uninfected cells responding to TNF, IFN-γ, or type I IFNs, www.annualreviews.org • Monocyte-Mediated Defense

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a

L. monocytogenes MCP-1 Glycosaminoglycans (GAGs) MCP-1-producing cells

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CCR2+ monocytes

Intermediate

Early

Late

b

Bone marrow

Bone marrow

Bone marrow

Figure 3 Models of in vivo MCP-1-mediated monocyte recruitment. During infection, MCP-1 is produced and secreted by microbially infected or by cytokine-stimulated uninfected cells. In the ﬁrst model (a), secreted MCP-1 establishes a gradient across the distance from infection site and attracts monocytes to infection sites. In an alternative model (b), the MCP-1 gradient is established not by distance from chemokine production site but rather by chemokine binding with speciﬁc GAGs. Association with GAGs increases MCP-1 concentration in speciﬁc regions and further facilitates oligomerization of MCP-1.

may promote the emigration of monocytes from bone marrow into the circulation. Several models can be proposed to explain MCP-1-mediated recruitment of monocytes to sites of infection. In the simplest model, MCP-1 is produced and released by microbially infected cells, establishing a chemokine gradient that guides responding monocytes to the site of infected cells (Figure 3a). While attractive for its simplicity, this model does not explain how monocyte emigration from the bone marrow is induced by infection in spleen or other tissues, and it suggests that once serum chemokine levels increase to the high levels seen in many infections, MCP-1 440

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gradients and the ability to guide monocytes to sites of infection would be lost. An alternative model for in vivo MCP-1 function is that chemokines bind to tissue-speciﬁc GAGs, possibly in bone marrow and also at sites of infection, and in this way guide monocytes out of the bone marrow into the bloodstream and then into infected tissues (Figure 3b). This model provides a mechanism by which MCP1 produced in infected tissues can circulate to bone marrow, bind GAGs, oligomerize, and promote monocyte emigration. Although both models are supported by a number of in vitro studies and some in vivo experiments, they remain unproven. Further studies that

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elucidate the in vivo processes that promote monocyte recruitment in the setting of microbial infection will be exciting and may provide important opportunities to improve immune defense in the immunocompromised host.

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SUMMARY Inﬂammatory monocytes play an essential role in innate immune defense against microbial infection and also contribute to adaptive immune responses and long-term immunity. Recent investigations have started to reveal how the constitutive pathway of monocyte maturation and differentiation into tissue macrophages and DCs is redirected in

the setting of microbial infection. Infections with a diversity of pathogens, including Listeria monocytogenes, Toxoplasma gondii, and Cryptococcus neoformans, require CCR2-mediated recruitment of monocytes to sites of infection, where they restrict further microbial growth and invasion. In the absence of infection, circulating inﬂammatory monocytes return to the bone marrow and differentiate into monocytes that constitutively supply peripheral tissues with macrophages and DCs. Much remains to be learned about the trafﬁcking cues that ﬁnely control the numbers of macrophages and DCs in various tissues and the stimuli that redirect trafﬁcking and monocyte differentiation in the setting of microbial infection.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS The authors’ research is supported by the National Institutes of Health (AI39031, E.G.P.; K12 CA120121, N.V.S.; T32 AI055409, T.M.H.), Irvington Institute for Immunological Research (N.V.S.), and Charles H. Revson Foundation (T.M.H.).

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186. Mukaida N, Zachariae CC, Gusella GL, Matsushima K. 1991. Dexamethasone inhibits the induction of monocyte chemotactic-activating factor production by IL-1 or tumor necrosis factor. J. Immunol. 146:1212–15 187. Paine R 3rd, Rolfe MW, Standiford TJ, Burdick MD, Rollins BJ, Strieter RM. 1993. MCP-1 expression by rat type II alveolar epithelial cells in primary culture. J. Immunol. 150:4561–70 188. Dhawan L, Liu B, Blaxall BC, Taubman MB. 2007. A novel role for the glucocorticoid receptor in the regulation of monocyte chemoattractant protein-1 mRNA stability. J. Biol. Chem. 282:10146–52 189. Proudfoot AE, Handel TM, Johnson Z, Lau EK, LiWang P, et al. 2003. Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. USA 100:1885–90 190. Johnson Z, Proudfoot AE, Handel TM. 2005. Interaction of chemokines and glycosaminoglycans: a new twist in the regulation of chemokine function with opportunities for therapeutic intervention. Cytokine Growth Factor Rev. 16:625–36 191. Lau EK, Paavola CD, Johnson Z, Gaudry JP, Geretti E, et al. 2004. Identiﬁcation of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo. J. Biol. Chem. 279:22294–305 192. Chakravarty L, Rogers L, Quach T, Breckenridge S, Kolattukudy PE. 1998. Lysine 58 and histidine 66 at the C-terminal alpha-helix of monocyte chemoattractant protein-1 are essential for glycosaminoglycan binding. J. Biol. Chem. 273:29641–47 193. Yu Y, Sweeney MD, Saad OM, Crown SE, Hsu AR, et al. 2005. Chemokineglycosaminoglycan binding: speciﬁcity for CCR2 ligand binding to highly sulfated oligosaccharides using FTICR mass spectrometry. J. Biol. Chem. 280:32200–8 194. Allen SJ, Crown SE, Handel TM. 2007. Chemokine: receptor structure, interactions, and antagonism. Annu. Rev. Immunol. 25:787–820 195. Mora JR, Iwata M, Eksteen B, Song S-Y, Junt T, et al. 2006. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science 314:1157–60 196. Tezuka H, Abe Y, Iwata M, Takeuchi H, Ishikawa H, et al. 2007. Regulation of IgA production by naturally occurring TNF/iNOS-producing dendritic cells. Nature 448:929–33 197. Taylor PR, Brown GD, Geldhof AB, Martinez-Romares LM, Gordon S. 2003. Pattern recognition receptors and differentiation antigens deﬁne murine myeloid cell heterogeneity ex vivo. Eur. J. Immunol. 33:2090–97 198. Tacke F, Alvarez D, Kaplan TJ, Jakubzick C, Spanbroek S, et al. 2007. Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques. J. Clin. Invest. 117:185–94 199. Grage-Griebenow E, Flad H, Ernst M. 2001. Heterogeneity of human peripheral blood monocyte subsets. J. Leukoc. Biol. 69:11–20 200. Draude G, von Hundelshausen P, Frankenberger M, Ziegler-Heitbrock HW, Weber C. 1999. Distinct scavenger receptor expression and function in the human CD14+ /CD16+ monocyte subset. Am. J. Physiol. 276:H1144–49

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:421-452. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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The Biology of Interleukin-2 Thomas R. Malek Department of Microbiology and Immunology and the Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, Florida 33101; email: [email protected]

Annu. Rev. Immunol. 2008. 26:453–79

Key Words

First published online as a Review in Advance on December 6, 2007

IL-2, immune tolerance, T regulatory cell, T effector cell, immune memory

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090357 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0453$20.00

Abstract Much data support an essential role for interleukin (IL)-2 in immune tolerance. This idea is much different from the early paradigm in which IL-2 is central for protective immune responses. This change in thinking occurred when a T regulatory cell defect was shown to be responsible for the lethal autoimmunity associated with IL-2/IL-2R deﬁciency. This realization allowed investigators to explore immune responses in IL-2-nonresponsive mice rendered autoimmune-free. Such studies established that IL-2 sometimes contributes to optimal primary immune responses, but it is not mandatory. Emerging ﬁndings, however, suggest an essential role for IL-2 in immune memory. Here, the current understanding of the dual role of IL-2 in maintaining tolerance and contributing to immunity in vivo is reviewed with some emphasis on T regulatory cell production and homeostasis. Also discussed are implications of this new appreciation concerning the immunobiology of IL-2 with respect to targeting IL-2 or its receptor in immunotherapy.

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INTRODUCTION More than 30 years ago, Doris Morgan, Francis Ruscetti, and Robert Gallo (1) and Steven Gillis and Kendall Smith (2) established that culture ﬂuids of activated T cells contain mediators that induce the proliferation of antigen-activated T cells. In the ensuing years, the protein responsible for this T cell growth factor activity was puriﬁed and its gene cloned to establish that this activity is due to a single molecular species (3). T cell growth factor was assigned the more generic nomenclature of interleukin (IL)-2, as lymphokines typically mediate diverse biological functions. Work during the 1980s, which largely relied on in vitro systems, showed that T cell responses require IL-2, leading to a widely accepted model. Activation of T cells through the T cell receptor (TCR) and costimulatory molecules such as CD28 causes production of IL-2 and expression of the IL-2 receptor (IL-2R). The IL-2/IL-2R interaction then drives extensive clonal expansion and effector development. This model places IL2 as a central player for T cell–dependent immune responses. It was quite surprising, therefore, that after the genes for IL-2 or two subunits of the IL-2R, IL-2Rα (CD25) and IL-2Rβ (CD122), were individually inactivated in mice by gene targeting, the resulting phenotype is not immunodeﬁciency, as predicted, but rather a very serious lymphoproliferative and autoimmune disorder (4–7). The progression of this disease in IL-2Rβ−/− mice is shown in Table 1. There is now much evidence that a defect in CD4+ CD25+ Foxp3+ T regulatory (Treg)

Treg cell: T regulatory cell

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WT: wild-type

Table 1

cell production is the main reason for autoimmunity associated with IL-2/IL-2R deﬁciency. This cell population suppresses potentially autoreactive peripheral T cells that escape thymic negative selection (8). Initially, before it was appreciated that CD25 was a good marker for Treg cells, IL-2- and lL2Rα-deﬁcient mice were shown to contain a greatly reduced number of IL-10-producing CD4+ CD25+ T cells, suggesting that the absence of these cells is related to their autoimmune disease (9). A regulatory cell defect rather than a cell intrinsic abnormality was suspected in IL-2-deﬁcient mice because aspects of this autoimmune syndrome or experimental autoimmune encephalomyelitis (EAE) are contained by wild-type (WT) populations of T cells or IL-2-treated IL-2−/− T cells in various adoptive transfer settings (10–13). The demonstration that highly puriﬁed WT CD4+ CD25+ T cells are sufﬁcient to prevent all the abnormalities associated with the lack of IL-2R signaling provides direct data that the main problem in IL-2/IL-2R-deﬁcient mice is the inability to produce Treg cells (14, 15). The issue concerning the importance for IL-2 during T cell–dependent immune responses in vivo still remains. Early on, IL-2deﬁcient mice were shown to develop protective immunity upon challenge with a variety of infectious agents (16–18). A major caveat with these experiments is that the rapid autoimmunity might bypass a requirement for IL-2. This complication, however, does not account for these ﬁndings because more recent studies show that IL-2- or IL-2R-deﬁcient mice develop effective immunity in settings where

Autoimmune disease in C57BL/6 IL-2Rβ (CD122)-deficient mice

Birth

No symptoms

2 weeks old

Enlarged lymph nodes; increased proportion of activated T cells

3–8 weeks old

Lymphadenopathy; splenomegaly; considerable proportion of activated T cells, especially CD4 T cells; increased granulocytes in the bone marrow and spleen; hemolytic anemia; anti-DNA auto-antibodies; multi-organ inﬂammatory inﬁltrates; wasting disease

8–12 weeks old

Many symptoms above continue; thymic aplasia; sometimes decreased lymph node and spleen cellularity; impaired B cell development and cellularity; death

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the associated autoimmunity is greatly abated or completely prevented (19). Thus, although IL-2 is essential for many T cell responses in vitro, it is dispensable for induction of T cell–dependent immunity in vivo. Nevertheless, there remains one aspect of long-lasting T cell immunity that may strictly require IL2, i.e., recall responses by CD8+ memory T cells (20, 21). There are several past reviews that provide detailed information concerning the role of IL-2 in vitro and its signaling pathways, as well as detailed discussion concerning initial studies implicating IL-2 in Treg cell production (19, 22–24). This review primarily deals with our current understanding of IL-2 in the production of Treg cells and in T cell– dependent immune responses in vivo where autoimmunity is not a confounding factor. To start, I introduce the basics of the IL-2/IL-2R system.

IL-2 IL-2 is a 15,000-kDa α-helical cytokine produced predominately by activated CD4+ and CD8+ T cells, the latter to a lesser extent. Activated dendritic cells (DCs), natural killer (NK) cells, and NKT cells also produce IL-2 (25–29), but the biological relevance of IL2 from these cells remains unclear. IL-2 is rapidly and transiently produced upon engaging the TCR and costimulatory molecules such as CD28 on naive T cells. The transient nature of IL-2 secretion depends on transcriptional induction by TCR signals and stabilization of IL-2 mRNA by costimulatory signals, followed by transcriptional silencing of the IL-2 gene and rapid degradation of IL-2 mRNA (30–34). As described in more detail elsewhere (see 30–32), much is known concerning the stringent transcriptional induction of the IL-2 gene. A minimal proximal promoter/enhancer region extends approximately 500 base pairs upstream of the transcriptional start site of the IL-2 gene. The importance of this region is highlighted by

the high DNA sequence conservation in the human and the mouse IL-2 genes. TCR signaling induces AP-1, increases the levels of active NF-κB p65/rel, and causes calcineurinmediated dephosphorylation of NFAT (nuclear factor of activated T cells), promoting its translocation into the nucleus. These transcription factors, in conjunction with constitutive factors such as OCT-1, bind to speciﬁc sites in a cooperative fashion within the ﬁrst 300 base pairs of the minimal promoter, contributing to IL-2 gene transcription. HMGI(Y) and Ets factors bind to regulatory sites just upstream. AML1/Runx1 is another positive regulator that binds to sites approximately 1.4 kb upstream of the transcriptional start site (35). Although the minimal IL-2 promoter is very active in vitro, this region or a larger 2.8 kb upstream IL-2 gene fragment does not give tissuespeciﬁc and copy number–dependent activation in vivo that recapitulates the endogenous IL-2 gene (36–38). Thus, other regulatory elements must lie further 5 downstream, controlling IL-2 gene expression in vivo. Consistent with this notion, the expression of green ﬂuorescent protein (GFP) under the control of 8.4 kb of the IL-2 5 upstream sequence in mice more closely resembles the expression of the endogenous IL-2 gene (27, 28). The mechanism that dictates IL-2 gene silencing after T cell activation is less well understood, but it represents another critical aspect controlling IL-2’s transient expression. Zfxla (ZEB, TCF8) and CREM (Icer) bind to the IL-2 minimal promoter and inhibit reporter gene transcription, but their mechanism of action is undeﬁned (39–43). More recently, T-bet (Tbx21) has been implicated in repressing IL-2 production through interaction with Rel-A within the proximal IL-2 promoter (44). A classical auto-regulatory feedback loop has recently been described in which IL-2 inhibits its own production (45, 46). This autoregulatory loop depends on activation of signal transducer and activator of transcription-5 (Stat5) and IL-2-dependent induction of the www.annualreviews.org • The Function of IL-2

GFP: green ﬂuorescent protein Auto-regulatory loop: a gene product that promotes or inhibits its own production Stat: signal transducer and activator of transcription

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Activated T cell APC

Naive T cell IL-2R

TCR MHC

pSTAT5 Il2

B7

IL-2R

Blimp-1

Il2

Il2

CD28

1 pim Bl Il2

IL-2

TCR signaling IL-2 transcription

IL-2 secretion IL-2R expression

IL-2R signaling

IL-2 repression IL-2R upregulation

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Figure 1 The autocrine IL-2 auto-inhibitory loop.

Blimp-1: B lymphocyte maturation protein-1 High-affinity IL-2R: the functional IL-2R, composed of IL-2Rα (CD25), IL-2Rβ (CD122), and γc (CD132)

transcriptional repressor B lymphocyte maturation protein-1 (Blimp-1) (Figure 1). Thus, after antigen-activation of a naive T cell, IL2 is produced; the high-afﬁnity IL-2R is expressed; secreted IL-2 binds to the IL-2R, leading to Stat5 activation and Blimp-1 induction; and ultimately the IL-2 gene is repressed. Blimp-1 is a key downstream mediator of IL-2 repression because ectopic expression of Blimp-1 in activated T cells inhibits IL-2 production and the 8.4 kb IL2/GFP reporter (46), and Blimp-1-deﬁcient T cells produce increased IL-2 (47, 48). Nevertheless, it remains to be determined whether IL-2-dependent Stat5 activation directly targets Blimp-1 induction and whether Blimp1 directly represses IL-2. Interestingly, mice with T cell conditional knockout of Blimp1 exhibit severe inﬂammatory bowel disease leading to early death (47, 48). The basis for this autoimmunity, including whether it is due to improper production of IL-2, is not known.

IL-2R

Jak: Janus kinase MAPK: mitogen-activated protein kinase PI3K: phosphatidylinositol 3-kinase

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The IL-2R consists of three subunits, IL-2Rα (CD25), IL-2Rβ (CD122), and the common gamma chain or γc (CD132). CD122 and γc are primarily folded into β-sheet structures and are related to members of the class I cytokine receptor superfamily. In contrast, CD25 and IL-15Rα contain “sushi” modules in their extracellular region (22). All three Malek

subunits are required for formation of the high-afﬁnity IL-2R (Kd ∼10−11 M). The IL2R does not exist as a preformed heterotrimer, but rather CD25 on its own initially binds IL2 (Kd ∼ 10−8 M), which promotes association with CD122 and γc. The crystal structure of IL-2 bound to the IL-2R reveals that each receptor subunit contacts IL-2, with most contacts at the IL-2/CD25 interface, and that signiﬁcant interactions occur between CD122 and γc, leading to a stable quaternary complex of IL-2, CD25, CD122, and γc (49, 50). This quaternary complex induces IL-2 signaling that depends on the cytoplasmic tails of CD122 and γc. When in close proximity, Jak-3 via γc and Jak-1 via CD122 phosphorylate key tyrosine residues on CD122, leading to association of the adapter Shc and either Stat5 or Stat3, the latter to a lesser extent. Shc provides a platform to activate the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) pathways, important for cell growth and survival (22, 24). The associated Stats are further phosphorylated, allowing their dimerization and translocation into the nucleus, where Stat5 is the main IL-2-induced Stat in activated T cells that regulates genes important for effector function and T cell growth. Although activation of the MAPK, PI3K, and Stat5 pathways are important for conventional activated T cells, Stat5 is the main pathway by which IL-2R contributes to Treg cell production and maintenance.

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IL-2 bound to the high-afﬁnity IL-2R is short-lived on the cell surface as this complex is rapidly internalized (t1/2 10–20 min). IL-2, CD122, and γc are targeted for lysosomal degradation that is regulated by the proteasome and that depends on the cytoplasmic tail of γc, whereas CD25 recycles to the cell surface (51–54). Importantly, driving IL-2-dependent T cells into S-phase of the cell cycle requires sustained IL-2R signaling for several hours (55). Thus, IL-2-dependent T cell clonal expansion requires a somewhat prolonged source of IL-2 and a continued expression of all subunits of the IL-2R. However, some key downstream targets of the IL2R may be properly activated by the initial IL-2/IL-2R interaction. The γc subunit is expressed on virtually all hematopoeitic cells and is shared by the receptors for IL-4, IL-7, IL-9, IL-15, and IL-21 (22, 56). CD122 is a subunit of both the IL2R and the IL-15R. CD122 is constitutively expressed on a subset of early lymphoid progenitors, NK cells, NKT cells, memory phenotypic CD8 T cells, and Foxp3+ Treg cells, whereas its expression is induced on virtually all antigen-activated T cells. IL-2 weakly binds to cells expressing only CD122 and γc, but this is unlikely to be biologically relevant, as the phenotype of IL-2- and CD25-deﬁcient mice is very similar (6, 7). Besides binding soluble IL-15, CD122 and γc also readily bind IL-15 through a process of trans-presentation in which IL-15 bound to IL-15Rα on one cell presents IL-15 and engages CD122 and γc on another cell (57). Accordingly, IL-15- or IL15Rα-deﬁcient mice show defects in production of NK and NKT cells and in the homeostasis of CD8 memory cells (57). Most lymphoid cells do not express CD25 when directly examined ex vivo. However, CD25 is readily found on subsets of developing pre-T and pre-B cells, but these cells lack CD122 and cannot respond to IL-2. Thus, mainstream T and B cell development is normal in young CD25-deﬁcient mice before their accompanying severe autoimmunity disrupts lymphocyte development (6). Activated

effector cells readily, but transiently, express CD25. The activation of naive T cells in vitro leads to very high levels of CD25 expression through a two-step process (Figure 1). First, moderate levels of CD25 are rapidly induced by TCR and costimulatory signals, in part by activation of NF-κB, NFAT, AP-1, and CREB/AFT (58). Subsequently, IL-2 binds to the IL-2R and increases the initial level of CD25 through a Stat5-dependent positive feedback loop. Such a mechanism increases IL-2 binding and hence signaling by activated T cells through enhanced capture of IL-2 by CD25. CD25 is also found on natural CD4+ Foxp3+ Treg cells. Like activated effector T cells, CD25 expression by Treg cells is inﬂuenced by TCR activation and IL-2 upregulation. Another means for CD25 expression is based on TCR activation and transforming growth factor (TGF)-β1 signaling (59). Treg cells may also express CD25 in part based on this mechanism through their production of TGF-β1.

EXPRESSION OF IL-2 AND IL-2R IN VIVO Although individual IL-2R subunits are widely distributed on many different cell types in the lymphoid compartment when directly examined ex vivo, only two major cell subsets readily coexpress CD25, CD122, and γc, which are required for the high-afﬁnity IL2R, i.e., CD4+ Foxp3+ Treg cells and activated conventional CD4+ and CD8+ T cells. Thus, Treg cells and antigen-activated T cells represent the main population of cells poised to respond to IL-2 in vivo. CD25 is very transiently expressed at a high level by activated T cells in vivo (60). Treg cells, rather than conventional activated T cells, consistently express the highest level of CD25 in vivo (61). This is somewhat surprising given the ability to drive very high levels of CD25 on activated T cells in vitro. CD25 has proven to be a useful marker, albeit not a perfect one, for isolating natural Treg cells, especially in the mouse, www.annualreviews.org • The Function of IL-2

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where most of the CD25+ CD4+ T cells are also Foxp3+ Treg cells (61). In human, conventional activated CD25+ T cells are more prevalent and the isolation of Treg cells based on CD25 is more problematic. An important issue with respect to IL-2dependent control of Treg cells and activated T cells in vivo is an understanding of how IL2 is made available to these cell populations. One issue is what proportion of cells produce IL-2 spontaneously or in response to endogenous immune stimuli. Without direct immunological challenge, IL-2-producing cells, all of T cell origin, are readily detected by in situ hybridization and immunohistochemistry within the thymus from day 14 of gestation until at least 5 weeks of age, in the epidermis of the skin in neonates with a clusters of IL-2+ cells near hair follicles from 3–5 weeks of age, and from day 16 of gestation through adult life within the intestine (62). The signiﬁcance of the IL-2-producing cells in the skin and intestine is not clear, but their embryonic and early neonatal appearance suggests some developmental role. Along this line, production of Vγ3Vδ1+ T cells in CD122−/− mice and CD8αα intraepithelial lymphocytes in IL-2or CD122-deﬁcient mice is impaired, consistent with a requirement for IL-2 or IL-15 for the development and/or maintenance of these T cells, respectively (63–66). IL-2-producing T cells in the thymus may reﬂect T cells undergoing positive and negative selection as T cell activation accompanies these processes, including IL-2 production. IL-2-secreting thymocytes are readily found at the cortical medullary junction and within the medulla and are surrounded by a halo of IL-2 protein, suggesting that IL-2 is available only to T cells within close proximity (62). This thymic IL-2 may promote the development of Treg cells, which are also found within the inner thymus (67, 68). The use of GFP transgenic reporter mice under control of 8.4 kb of 5 IL-2 regulatory sequence reveals approximately 1% GFP+ cells in the thymus (27). These GPF+ cells are distributed between conventional TCRαβ+ , TCRγδ+ , and

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NKT cells, and direct enumeration of IL-2producing thymic T cells supports a contribution by both TCRαβ+ and TCRγδ+ T cells. One limitation with this reporter is that it may overstate IL-2 expression by TCRγδ+ and NKT cells, as GFP mRNA is substantially proportionally higher than IL-2 mRNA in these T cell subsets (28). Peripheral immune tissues also contain a resident population of IL-2-producing cells. Using the same IL-2/GFP reporter, the spleen contains approximately 2% GFP+ T cells, the majority of which were TCRαβ+ cells (27, 28). For CD4+ GFP+ T cells, most express an effector/memory phenotype, suggesting that these cells are recently activated. Real-time PCR revealed that the CD4+ CD25low splenic T cell subset, which primarily corresponds to activated conventional T cells, expresses the highest levels of IL-2 mRNA. NK and NKT cells also express IL-2 mRNA but at strikingly lower levels, whereas DCs lack this mRNA (26). As IL-2 dominantly controls the homeostasis of Treg cells in peripheral immune tissue, one expects a source of IL-2 for these cells in the spleen and lymph nodes, and these ﬁndings are consistent with activated T cells as a source of IL-2. Whether endogenous antigens or autoantigens provide the immune stimulus for this IL-2 production remains to be determined. With respect to antigen-speciﬁc induction of IL-2 by naive CD4+ T cells in vivo, early studies indicate that IL-2 mRNA and protein peak 6–12 h after antigen challenge, with less than 50% of the antigen-activated cells IL-2+ , and are then undetectable 24 h post-challenge (60, 69). The use of a more sensitive IL-2 secretion assay reﬁned this analysis and reveals that IL-2 protein is secreted by 1 h after antigen challenge of naive T cells, and by 5–6 h later nearly 80% of antigen-speciﬁc T cells produce maximal IL-2 (70). After this time, IL-2 secretion declines and is undetectable by 16 h after the initial stimulation. IL-2 production by memory T cells is similarly transient, with a peak response as rapid as 1–2 h after antigen challenge. A similar high frequency of

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IL-2-producing CD8+ T cells is detected after viral infection of mice (71). IL-2 is secreted directionally at the immune synapse favoring use by IL-2R-bearing T cells in close proximity to antigen-presenting cells in either an autocrine or paracrine manner (72). In vivo IL-2 production is somewhat more rapid and transient than seen in vitro and is consistent with the recently described IL-2 auto-inhibitory loop (45, 46). Importantly, this time course indicates that IL-2 is available primarily during the initial programming phase of antigenactivated T cells that precedes T cell proliferation. This implies a role for IL-2 during an immune response independent of driving extensive clonal expansion. The considerable T cell expansion after immune challenge of IL-2- or IL-2R-deﬁcient mice is consistent with an IL-2-independent mechanism for T cell growth.

IL-2 AND THE GENETIC BASIS OF AUTOIMMUNITY Mice deﬁcient in IL-2, CD25, or CD122 exhibit lethal autoimmunity (4–7) and are impaired in Treg cell production (14, 15, 61). These ﬁndings and the related phenotype of Foxp3-deﬁcient mice, which lack Treg cells, are consistent with the genetic basis for IL2/IL-2R in immune tolerance at the level of Treg cells. Even though IL-2 and IL-15 share common receptor signaling subunits, i.e., CD122 and γc, IL-15- and IL-15Rαdeﬁcient mice are autoimmune-free and contain a normal number of Foxp3+ Treg cells (57, 73), ﬁrmly establishing that IL-2 is the critical cytokine for Treg cells. Foxp3 is a transcriptional regulator that is essential for Treg cell production (74, 75) and is currently the best marker for Treg cells. In WT mice, Foxp3 is uniformly expressed at a high level in only natural Treg cells and is not induced in activated effector T cells (61). In humans, high levels of Foxp3 are also found in Treg cells, but some activated effector T cells also transiently express Foxp3 at a low level (76). Foxp3-deﬁcient or scurfy mice that con-

tain mutations within the Foxp3 gene exhibit a very severe systemic autoimmune disease that parallels the autoimmunity seen in IL2/IL-2R-deﬁcient mice, although the tempo of the disease in Foxp3-deﬁcient mice is quicker. One difference between Foxp3−/− animals and IL-2-, CD25-, and CD122-deﬁcient mice is that only the former completely lacks Foxp3+ T cells. In comparison with WT mice, the Foxp3+ CD4+ T cells within IL2/IL-2R-deﬁcient mice contain an approximately twofold reduced level of Foxp3, essentially lack expression of CD25, and are found in markedly reduced proportions, especially within peripheral immune tissues (75, 77, 78). Investigators have argued that the CD4+ Foxp3low CD25neg cells in IL-2/IL-2Rdeﬁcient mice are functional Treg cells, but they cannot keep up with the autoreactive T cells, accounting for the slower pace of autoimmunity (61). In support of this idea, the Foxp3+ T cells within IL-2-deﬁcient mice show some capacity to inhibit T cell proliferation in a Treg cell–dependent suppression assay in vitro (61, 79). The overall relevance of this in vitro suppression has been questioned because, in tumor-bearing vaccinetreated CD25−/− mice, the resident Foxp3+ T cells do not suppress antitumor immunity (78). Alternatively, less rapid autoimmunity in IL-2/IL-2R-deﬁcient mice may be due to impaired autoimmune effector T cells that delay disease. In support of this idea and discussed more fully below, IL-2/IL2R-deﬁcient mice are immunocompetent but show variable abnormalities in clonal expansion and effector function. Similarly, acute disease by autoreactive effector CD4+ T cells was shown to be milder when induced by IL2−/− effector cells (80). Furthermore, when autoimmunity in CD122-deﬁcient mice is prevented by the adoptive transfer of WT Treg cells, a normal proportion and number of donor WT CD4+ CD25+ Foxp3+ Treg cells are found in the peripheral immune compartment while the number of endogenous Foxp3low CD25neg CD122−/− T cells remains www.annualreviews.org • The Function of IL-2

Autocrine: production and use of a cytokine by the same cell Paracrine: a cell that responds to a cytokine produced by a neighboring cell

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very low (81). This ﬁnding implies that these Foxp3low cells are largely nonfunctional and perhaps analogous to the Foxp3low phenotype associated with effector rather than regulatory human T cells (82–84). Thus, the problem with Treg cells in IL-2/IL-2R-deﬁcient mice is more complex than simply a failure to keep up with autoreactive cells. In autoimmune-prone NOD mice, one of the autoimmune susceptibility genes, Idd3, spans 650 kb of genomic DNA and contains the IL-2 gene (85, 86). Molecular and functional studies indicate that Idd3 regulates IL-2 transcription such that mice containing the NOD Idd3 gene segment express twofold lower IL-2 (86). Importantly, by speciﬁcally targeting the IL-2 gene within the Idd3 locus, autoimmune diabetes is accelerated by this lower IL-2 production. Thus, a mouse IL-2 gene polymorphism contributes to autoimmune susceptibility owing to fewer Treg cells with less suppressive activity. With respect to IL-2R and human disease, several patients have been reported with mutations in the CD25 gene (87–90). Analogous to CD25-deﬁcient mice, such patients exhibit a wide spectrum of abnormalities, including severe autoimmunity, lymphadenopathy, and persistent viral infections. Problems with Treg cells are suspected but not deﬁnitively established. In one case, Foxp3+ CD4+ T cells were detected, analogous to IL-2/IL2R-deﬁcient mice (88). Activation of their T cells shows impaired production of IL-10. In another case, myeloablative conditioning followed by an allogeneic bone marrow transplant completely resolved the symptoms (89), consistent with reconstitution of Treg cells. In addition, genome-wide association studies that evaluated approximately 2000 patients, each with rheumatoid arthritis or type 1 diabetes, associate SNPs mapping to CD25 or CD122 genes, further relating possible polymorphisms of the IL-2R with autoimmune diseases (91). Stat5 activation is a main outcome of IL2R signaling, and Stat5 is known to be critical for production of Treg cells on the ba-

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sis of the phenotype of mice deﬁcient in this transcriptional activator (92–95). A patient with a homozygous missense mutation in the Stat5b gene has also been identiﬁed with immune dysregulation and impaired expression of Foxp3 and Treg cell function (96), implicating Stat5 activation with human Treg cell production.

IL-2 IN Treg CELL DEVELOPMENT Natural CD4+ CD25+ Foxp3+ T cells develop within the thymus and require TCRmediated selection on self-antigens (97). Foxp3 expression is primarily restricted to CD4+ “single positive” thymocytes. However, a few CD4+ CD8low thymocytes are also Foxp3+ (61). This expression pattern is consistent with commitment to a Treg lineage at a somewhat late phase of T cell development. Besides TCR engagement, Treg development also requires costimulation through CD28 (98, 99). One role of CD28 signaling is cell intrinsic for Treg cell development, i.e., induction of Foxp3 expression (100), and this may explain the detection of Foxp3+ T cells in the absence of IL-2/IL-2R. A second role is to induce IL-2 production by T cells undergoing selection to provide a potential source of paracrine IL-2 to developing Treg cells (100). Some have argued that IL-2 is dispensable for thymic Treg cell development because Foxp3+ thymocytes are detected in IL2- and IL-2R-deﬁcient mice (61, 79, 101). This is most striking for the 6.5 TCR transgene selected upon its cognate antigen, viral hemagglutinin that is widely expressed within the thymus, where the frequency of Foxp3+ thymocytes is similar in IL-2+/+ and IL-2−/− mice (79). However, the development of Foxp3+ thymocytes is nearly ﬁvefold lower in IL-2−/− mice that expressed the 3.9 TCR transgene, speciﬁc for hen egg lysozyme (HEL), when selected upon HEL under control of the insulin promoter (101). A major difference in this setting is that HEL expression is not ubiquitous but dependent on expression

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by Aire. Thus, under more physiological expression of a model self-antigen, development of Foxp3+ thymocytes is highly dependent on IL-2. Similarly, when compared with WT mice, there is a consistent twofold reduction in Foxp3+ thymocytes in IL-2- or IL-2Rdeﬁcient mice containing a polyclonal TCR repertoire (61, 73, 77). The reduction is even greater when WT and CD25−/− thymocytes develop in competition with each other (61). In these settings, the level of Foxp3 protein is also twofold lower (61, 77). These ﬁndings also support a role for IL-2 in thymic Treg cell development. In addition to the observations mentioned above, three additional lines of evidence directly implicate IL-2 for thymic Treg cells. First, thymic-directed expression of WT CD122 in CD122−/− mice restores Treg cell production and prevents the associated autoimmunity (14, 102). More recent studies show that this transgenic IL2R is functionally active within the thymus by increasing to normal levels the number of CD4+ Foxp3+ thymocytes and the expression of Foxp3 and CD25 (77). The increase in Foxp3 expression may be particularly important because lower expression of Foxp3 is directly related to development of autoimmunity (103). Second, treatment of normal mice with anti-IL-2 reduces the number of thymic CD4+ CD25+ GITR+ Treg cells (104). Third, thymic development of CD4+ Foxp3+ T cells is restored in Rag2−/− mice reconstituted with CD122−/− bone marrow transduced with WT CD122 or a variant of CD122 designed to selectively activate Stat5 (73). This latter study shows an important role for IL-2-induced Stat5 in thymic Treg development. This regulation may be direct, as IL-2 activation of human Treg cells leads to Stat5 and Stat3 binding to a conserved Stat-binding site in the ﬁrst exon of the Foxp3 gene (105). Interestingly, although T cell development is severely blocked in γc−/− mice, the few CD4 T cells that are present contain a negligible proportion of Foxp3-bearing T cells (61). Analysis of Stat5- and Jak-3-deﬁcient mice re-

veals a similar lack of CD4+ Foxp3+ T cells (106). Collectively, these data indicate a requirement for one or more γc-dependent cytokines that activate Stat5 for Foxp3 expression and Treg cell development. IL-2, IL7, and IL-15 are the most likely possibilities as they readily activate Stat5. IL-7−/− , IL7Rα (CD127)−/− , IL-15−/− , and IL-15Rα−/− mice, however, contain a normal proportion of CD4+ CD25+ Foxp3+ T cells (73, 95), and IL-2/IL-2R-deﬁcient mice contain CD4+ Foxp3low CD25neg T cells, demonstrating that these cytokines do not solely control the development of Foxp3+ T cells. Alternatively, another γc-dependent cytokine contributes to the Foxp3low phenotype associated with IL-2/IL-2R-deﬁcient mice. In one study, the thymus of CD122−/− mice was shown to contain fewer CD4+ Foxp3+ thymocytes than do IL-2−/− mice (73), implying a contribution by IL-15. The decrease reported, however, is not as severe as in γc-deﬁcient mice, and this impairment was not observed in another study (61), indicating that IL-15 is not an essential cytokine for Treg cells. IL-7 and IL-2 appear to be the relevant cytokines because, in the absence of both IL-7R and IL2R, CD4+ Foxp3+ T cells are not detected (A.L. Bayer & T.R. Malek, unpublished data). The TSLPR (thymic stromal-derived lymphopoietin receptor) also utilizes IL-7Rα, but not γc, as subunits. As Treg development depends on multiple γc-dependent cytokines, as mentioned above, IL-7 is favored over TSLP as the key cytokine that functions in tandem with IL-2. An important question is whether the action of the IL-7R and IL-2R largely overlap at the same stage of Treg cell development. One possibility is that the Foxp3low phenotype in IL-2/IL-2R-deﬁcient mice is due to minimal signaling by the IL-7R at the same developmental step. This scenario seems unlikely because when IL-7R is increased on Treg cells by expressing CD127 as a transgene throughout most thymocytes in CD122−/− mice, Treg cell development is not rescued, and the mice exhibit autoimmunity indistinguishable www.annualreviews.org • The Function of IL-2

Model self-antigen: germ-line expression of a deﬁned foreign antigen, usually as a transgene, resulting in its immune tolerance γc-dependent cytokines: cytokines whose receptor contains the γc subunit, i.e., the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21

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from nontransgenic CD122−/− mice (107). However, it was informative to observe the activity of chimeric CD127/CD122, linking IL-7 binding to IL-2R signaling, or reciprocally chimeric CD122/CD127, linking IL-2 binding to IL-7R signaling, when expressed as transgenes in all T lineage cells in CD122-deﬁcient mice (107). Only chimeric CD122/CD127 increases the number of Foxp3+ thymocytes, upregulates Foxp3 and CD25, and provides robust protection from autoimmunity. These results indicate that IL7R signaling promotes Treg cell development in the absence of a functional IL-2R, but IL2 must trigger this signaling. The complete failure of the chimeric CD127/CD122 or the WT transgenic CD127 to rescue Treg cell development strongly suggests that IL-7 is unavailable in the niche that is normally dependent on IL-2. These data are consistent with a model in which signaling by CD127 and CD122 occurs at distinct stages of Treg development and indicate that there is not a strict requirement for signal transduction through the cytoplasmic tail of CD122.

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IL-2 IN Treg CELL HOMEOSTASIS In addition to contributing to thymic Treg development, considerable data indicate that IL-2 also functions to maintain Treg cells in the peripheral immune compartment. Peripheral immune tissues of IL-2- and IL-2R-deﬁcient mice contain an approximate tenfold reduction in the proportion of CD4+ Foxp3+ T cells when enumerated as a fraction in all CD4+ T lymphocytes (61, 77). These cells are also Foxp3low and largely CD25neg , analogous to their thymic counterparts. This reduction in peripheral CD4+ Foxp3+ CD122−/− T cells remains at least tenfold in absolute number upon correcting autoimmunity by adoptive transfer of WT Treg cells into lL-2Rβ−/− mice (81). This ﬁnding illustrates the striking inability of these Foxp3low CD122−/− T cells, which are continually produced by the thymus, to 462

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compete with the IL-2-responsive WT donor Treg cells. A key observation implicating IL-2 in peripheral Treg cell production is the demonstration that adoptively transferred CD4+ T cells from IL-2−/− , but not CD25−/− , mice protect IL-2-sufﬁcient recipient mice from spontaneous EAE (13). This leads to an increase in donor CD4+ CD25+ T cells that depends on host-derived IL-2. Besides providing data consistent with the rescue of IL-2−/− Treg cells by paracrine IL-2 in the periphery, this ﬁnding suggests that the requirement for IL-2 can also be delivered extrathymically. Subsequently, anti-IL-2 treatment of normal mice was shown to block Treg cells in the periphery of neonatal and adult mice and to cause autoimmunity in several disease-prone strains (26, 104). Gene expression proﬁling of peripheral CD4+ Foxp3+ T cells derived from IL-2−/− mice after treatment with IL-2 revealed upregulation of growth-related mRNAs rather than mRNAs important for immune function (61). Furthermore, peripheral Treg cells with impaired expression of IL-2R exhibit markedly lower homeostatic proliferation as assessed by BrdU uptake (14, 77). Thus, these data are consistent with IL-2 providing key signals in shaping the numbers of peripheral Treg cells. Nevertheless, homeostasis of Treg cells also depends on other signals, including TCR recognition of selfantigen in the context of MHC class II and costimulation through CD28 (98, 99, 108). In competitive environments when enumerating CD4+ Foxp3+ T cells in peripheral immune tissue, WT Treg cells strongly outcompete the Foxp3+ cells of IL-2R nonresponding origin, even after correcting IL-2R signaling within the thymus (61, 77, 79, 81). These data further support a critical role for IL-2 in the periphery. A potentially important issue, however, is the extent that IL-2 shapes the pool of peripheral Treg cells after exiting the thymus, particularly in neonatal or irradiated adult mice, as these settings readily support proliferation owing to their lymphopenic environment (109). In this

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regard, when WT Treg cells are transferred to 1- to 2-day-old CD122-deﬁcient mice, they undergo extensive IL-2-dependent proliferation, expanding six- to eightfold in one week (104). Furthermore, 10- to 20-day-old mice were administered anti-IL-2 to induce autoimmunity that was observed later in life (26). These experiments indicate that the Treg cells present in the neonatal peripheral immune compartment are especially dependent on IL-2. Thus, one caveat concerning conclusions about the requirement for IL-2 for peripheral homeostasis of Treg cells is that it is difﬁcult to distinguish this early developmental requirement versus subsequent maintenance in adult peripheral immune tissue. In any case, the available data indicate that, under physiological conditions, IL-2 begins to shape the number of Treg cells and levels of Foxp3 during thymic development and continues to function importantly in the periphery during neonatal and adult life. Treg cells with defective IL-2R restricted to peripheral immune tissues are of some interest because they suppress autoimmunity and are maintained at normal numbers even after adult thymectomy, probably because of slower death rate (14, 77). IL-2 induces very weak and transient Stat5 activation in these IL-2R-defective Treg cells. Correspondingly, downstream IL-2R-dependent responses are markedly impaired in vitro and in vivo. Thus, this very minimal IL-2 signal is sufﬁcient for peripheral Treg cell homeostasis, or there are IL-2-independent mechanisms in the periphery once Treg cells receive a productive IL-2 signal in the thymus. In either case, this result has an important implication, i.e., that peripheral Treg cells may be relatively resistant to anti-IL-2 or IL-2R blockade. Indeed, peripheral Foxp3+ T cells were transiently reduced by <50% after anti-IL-2 treatment of adult mice (61, 110). Another report showed a greater reduction of peripheral adult Treg cells by anti-IL-2, but investigators measured CD25, not Foxp3 (26). As anti-IL-2 also inhibits CD25 expression on Treg cells in vivo (61, 110), this latter study likely overstated

the effect on Treg cell numbers. Furthermore, anti-CD25 monoclonal antibody (mAb) or daclizumab immunotherapy is used in a variety of clinical settings without obvious autoimmune complications or dysregulation of the Treg cell compartment (111–113). Although Treg cells are one of the most highly proliferating lymphocyte populations in vivo (14, 114), these cells are characterized as anergic and hyporesponsive to TCR and IL-2 signaling in vitro. Nevertheless, Treg cells efﬁciently suppress TCR-induced proliferation when mixed with conventional T cells in vitro by targeting inhibition of IL2 production (70, 115, 116). IL-2 initially produced from activated conventional T cells acts on Treg cells to promote their survival, growth, and suppressor activity that in turn cause inhibition of IL-2 production and subsequent proliferation by conventional T cells (117–119). Treg cells may not have a strict requirement for IL-2 for suppressor function in vitro because Foxp3+ T cells from IL-2nonresponsive mice are suppressive in vitro (14, 61, 79, 120). Thus, there is some redundancy for Treg cell inhibition in this assay, and other factors, such as IL-4, produced by activated T cells may substitute for IL-2 (121). This in vitro assay, therefore, may model some aspects of IL-2-dependent Treg cell homeostasis in vivo where autoreactive or antigenactivated T cells likely provide IL-2 in close proximity to Treg cells. This IL-2 is expected to promote Treg cell homeostasis and may enhance their functional activity. The ﬁnding that the number of IL-2-producing effector cells is indexed to the number of Treg cells in vivo is consistent with this view (122).

Peripheral homeostasis: constant number of a lymphocyte subset in peripheral immune tissues through regulated cell growth and death

IL-2 AND INDUCED Treg CELLS Besides thymic-derived natural Treg cells, accumulating data indicate that conventional T cells can develop in the periphery to become Foxp3+ -induced Treg (iTreg) cells (123). Naive T cells become iTreg cells in vitro upon activation through the TCR when www.annualreviews.org • The Function of IL-2

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Adoptive immunotherapy: the transfer of in vitro–derived effector or regulatory cells into diseased individuals to enhance or suppress immunity Regulatory program: expression of a key set of genes required for functional Treg cells

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cultured with TGF-β and IL-2 (124–126). These cells exhibit potent suppressive activity in vitro and are easily grown, representing another possible source of cells for adoptive immunotherapy. The role of IL-2 for these cells is twofold: induction of Foxp3 and T cell growth (127, 128). TGF-β-induced expression of Foxp3 is strictly dependent on IL-2, whereas once Foxp3 is induced, other cytokines, including γc-dependent cytokines, maintain this expression. These ﬁndings are consistent with a role for IL-2 in Treg lineage commitment and are similar to IL-2 signaling in vivo, where high levels of Foxp3 depend on IL-2 signaling (61, 77). Recent evidence suggests that development into auto-aggressive pathogenic Th17 cells and development into Treg cells are reciprocally regulated cell fate choices (Figure 2) (129–131). Another interesting aspect of IL-2 signaling is that it prevents generation of Th17 cells in vitro and within the tumor microenvironment in vivo (132, 133). Thus, IL-17-producing T cells develop after stimulation through TCR and CD28 when TGF-β and IL-6 are present. This devel-

Tnaive Anti-CD3 Anti-CD28

Tact TGF-β

Th17

iTreg

IL-2

IL-6

Relative cytokine levels Figure 2 IL-2 and the interrelationship between Th17 and induced Treg (iTreg) cells. IL-6 and IL-2 refer to their levels during T cell priming in the presence of TGF-β 464

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opment is inhibited by IL-2 through activation of Stat5. Blocking IL-2R signaling, on the other hand, increased production of Th17 cells (132). These investigators propose two mechanisms by which IL-2 limits antigen- or auto-antigen-induced inﬂammation: production of Treg cells and inhibition of effector cell differentiation into Th17 cells. Thus, altering IL-2 levels, perhaps even in a narrow range, may have important pathogenic or therapeutic consequences. For example, a seemingly modest twofold reduction of IL-2 results in a genetic predisposition to type 1 diabetes in NOD mice (86). In addition, the ready detection of IL-2-producing cells in the intestine (62) may function in part to prevent development of Th17 cells.

IL-2 AND THE Treg CELL PROGRAM Most T cells have the potential to secrete IL2. Treg cells, however, do not produce IL2, and this represents one of their deﬁning features. In contrast to naive T cells, the IL2 proximal promoter in Treg cells does not undergo chromatin remodeling upon TCR activation (134). Moreover, Foxp3 directly binds to the minimal IL-2 promoter in association with NFAT and further downstream in association with AML1/Runx1, contributing to active transcriptional repression of IL2 (35, 135). Blimp-1 is another candidate to silence IL-2 (46), as its mRNA (47, 48) and protein (D. Gong & T.R. Malek, unpublished data) are also highly expressed in Treg cells. Foxp3 cooperation with NFAT and AML1/Runx1 has a broader role in Treg cells than just repressing IL-2. Rather, these also act to establish aspects of the regulatory program of Treg cells. Thus, they positively regulate expression of CD25, cytotoxic T lymphocyte-associated antigen-4 (CTLA4), and glucocorticoid-induced tumor necrosis factor (TNF) receptor (GITR) by binding to their promoters, and they thereby contribute to other important characteristics of the Treg cell phenotype (35, 135).

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Recent studies indicate that Treg lineage commitment ensues without expression of Foxp3 (130, 136). An important function of Foxp3 is to amplify and ﬁx these pre-existing features required for the suppressive program. Moreover, paracrine IL-2 was suggested as essential for establishing this program (130). As the Treg cell suppressive program depends on consistent high levels of Foxp3 (103, 130, 137), a fundamental role of IL-2 likely lies in its ability to increase Foxp3. Another link between IL-2R signaling and expression of important molecules for Treg function is the fact that enforced expression of Foxp3, TGF-β, or CTLA-4 into IL-2- or CD122deﬁcient T cells controls many aspects of autoimmunity when such cells are present in IL2- or CD122-deﬁcient mice (120, 138, 139). Collectively, these ﬁndings, especially the increase in Foxp3 after IL-2R signaling (77), raise the possibility of a more fundamental role for this cytokine in Treg lineage commitment, along with a role in maintenance and homeostasis. Thus, an important question that remains to be fully deﬁned is the precise role for IL-2 in the production of Treg cells.

IL-2 IN PRIMARY IMMUNE RESPONSES Depending on how and when IL-2R signaling is enhanced during viral infections, the initial response, the number of persistent memory T cells, and recall proliferation have all been reported to increase (140–142). Such studies reveal the potential of cells to respond to IL-2 but do not establish whether IL-2 is required. Nevertheless, given these results and the dominant role of IL-2 for T cell expansion and effector cell development in vitro, surprisingly, effective immune responses occur in most studies in which IL2-, CD25-, or CD122-deﬁcient mice were directly challenged (Table 2), although immune deﬁcits are sometimes noted. For example, the magnitude of the response to lymphocytic choriomeningitis virus (LCMV) in IL-2−/− mice is lower with impaired cytotoxic

Table 2

IL-2/IL-2R-deficient mice develop effective immunity

Deficiency IL-2

IL-2 and IL-4

Immune response to

References

Vaccinia virus, LCMV, VSV

16

Islet allografts

18

Superantigen (SEA and SEB)

182

Cardiac allografts

183

Vaccinia virus, LCMV

17

Islet allografts

184

IL-2Rα (CD25)

HSV-2

185

IL-2Rβ (CD122)

Superantigen (SEB)

186

Salmonella entericia

187

HSV, herpes simplex virus; LCMV, lymphocytic choriomeningitis virus; SEA, SEB, staphylococcal enterotoxin A or B; VSV, vesicular stomatitis virus.

T lymphocyte (CTL) activity and interferon (IFN)-γ production (143, 144). Nevertheless, the simplest interpretation of these ﬁndings is that immune responses are largely IL2-independent. An important consideration with these studies, however, is that autoimmunity associated with these mice may have bypassed normal physiological mechanisms. A number of more recent studies use a variety of approaches either to inhibit or to prevent completely this autoimmune disease. One approach is breeding a TCR transgene onto IL-2−/− , CD25−/− , or CD122−/− genetic backgrounds. This tactic substantially delays autoimmune disease, or, if these mice are further crossed to Rag1−/− or Rag2−/− mice, T cells bearing autoreactive TCRs do not develop, preventing autoimmunity. The use of T cells from such TCR transgenic mice also permits quantiﬁcation of proliferative and functional responses. One common result from all such studies is that there is substantial T cell proliferation by CD4 (145, 146) or CD8 (147–151) T cells without IL2/IL-2R. Sometimes, these proliferative responses are comparable to that developed by WT T cells, and at other times expansion by the IL-2/IL-2R-deﬁcient cells is reduced twoto threefold in lymphoid or nonlymphoid tissues. Alternatively, CD25−/− or CD122−/− mice are rendered autoimmune-free by correcting the Treg cell defect through preparing mixed bone marrow chimeras, by adoptively www.annualreviews.org • The Function of IL-2

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transferring Treg cells during neonatal life, or by selectively reconstituting the IL-2R in the thymus of CD122−/− mice. Again, substantial and generally normal proliferative responses are noted by the IL2R-deﬁcient T cells after challenging with LCMV, Listeria monocytogenes, vaccinia virus, or superantigen (20, 21, 152, 153). Notably, in these settings, which depend on the endogenous TCR repertoire, effective primary immune responses are elicited to the infectious agent. Besides functioning as a T cell growth factor, IL-2 also sensitizes activated T cells to undergo apoptosis upon restimulation through the TCR (154) and promotes development of effector function in vitro (155). IL-2, therefore, might contribute to the contraction phase of an immune response and/or promote effector cell differentiation. In this regard, CD25−/− and IL-2−/− TCR transgenic CD4 T cells readily contract after proliferation to antigenic peptide (145, 146). For CD8 T cells without IL-2 or IL-2R signaling, their contraction and subsequent development into memory cells is relatively normal (20, 21, 148, 149, 152, 153). Functional responses (i.e., CTL activity or IFN-γ or TNFα secretion) by the activated CD8 T cells are also often readily detected (20, 21, 151, 152). Sometimes, a particular effector activity is impaired without IL-2R signaling, e.g., direct ex vivo CTL activity during rejection of allogeneic skin grafts or IFN-γ secretion during vaccinia virus infection (152). These studies demonstrate considerable IL-2-independent T cell proliferation and effector cell development leading to effective primary immune responses. However, in some model systems, speciﬁc responses are lower and not comparable to those in WT mice, indicating that IL-2 contributes to immune responses. IL-2-independent immunity likely depends on inﬂammatory or other γcdependent cytokines produced during the immune response. In this regard, the near strict dependency on IL-2 for in vitro effector responses is bypassed by including IL-12 or IL-

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4 in the cultures (153, 155). There are two observations, however, that suggest that γcdependent cytokines may not readily account for IL-2-independent expansion in vivo. First, TCR transgenic CD4+ T cells from γc−/− mice undergo normal antigen-driven expansion when transferred into immunodeﬁcient hosts (156). Second, superantigen-mediated expansion and contraction by autoimmunefree CD122-deﬁcient CD4+ and CD8+ T cells are not impaired by blocking mAbs to γc (153). Co-stimulation by CD27 has emerged as a possible candidate for IL-2-independent clonal expansion by CD8+ T cells (157).

IL-2 AND T CELL MEMORY Several recent studies raise the possibility that IL-2 is essential to develop efﬁcient memory responses. CD4+ IL-2−/− TCR transgenic T cells readily expand and contract after in vivo activation with peptide-pulsed DCs, but these IL-2−/− T cells poorly survive and yield a low number of memory cells (146, 158). This is due to failed expression of the IL-7R, which is known to be critical for CD4 T cell memory (159–161). Particularly informative are studies of immune responses by donor T cells after preparing mixed bone chimeras that receive a mixture of WT and CD25-deﬁcient bone marrow. This approach prevents autoimmunity by Treg cells that develop from WT precursor cells and permits an assessment of the requirement for IL-2 in a competitive environment. Although some preference for WT T cells is noted, these experiments conﬁrm that the primary CD8 T cell response and memory cell generation after infection by LCMV or Listeria monocytogenes is largely IL2-independent (20, 21). However, the recall response to these agents is severely impaired. Interestingly, the requirement for IL-2 is not for expansion of the primed T cells in the secondary response, but rather for IL-2 early during the primary response. Two mechanistic issues emerge from these latter experiments. First, past studies by several laboratories indicate that CD4 T cell help

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during the primary response is essential for CD8 memory expansion (162). There is much debate about this helper requirement, and the CD40/CD40L interaction between the antigen-presenting DC and antigen-speciﬁc CD4+ T cells is considered one necessary signal for a CD8 T cell response. However, IL-2 production by CD4+ T cells during the very early activation of an immune response might also represent a form of T cell help. Second, the ability to temporally dissociate the requirement for IL-2 from when recall responses are elicited suggests a programming function for IL-2. This notion is in line with the role for IL-2 programming autonomous antigen-independent expansion and effector cell development of CD8 T cells, although these activities are also provided by redundant signals in vivo (155, 163, 164). Collectively, all this work with IL-2/IL-2R-deﬁcient mice raises the possibility that memory recall responses, rather than primary responses, may be a more important target when immune responses are inhibited after anti-IL-2 or antiIL-2R immunotherapy.

CONSIDERATIONS FOR IL-2-TARGETED IMMUNOTHERAPY Based on the foregoing discussion, the requirements for IL-2 are quite distinctive for the T regulatory and T effector arms of the immune system (Table 3). Much data now support a dominant role for IL-2 for Treg cell production and homeostasis. Emerging ﬁndings also suggest an essential function for IL-2 during immune responses at the level of memory responses. These ideas are much different from the early paradigm in which IL-2 is cenTable 3

In vivo dependency on IL-2

Treg cell development

High

Treg cell homeostasis

High

T effector cell expansion

Low

T effector cell function

Low/Intermediate

T effector contraction

Low

T memory recall responses

High

tral for immune responses by promoting T cell growth and effector differentiation that has driven most therapeutic interventions targeting IL-2 or its receptor. Accordingly, clinical outcomes not predicted by this model are expected and have been reported. For example, IL-2 is used in immunotherapy with the intention to boost immunity in HIV/AIDS and cancer patients. In both cases, there are reports of expansion of Treg cells (165–168). For HIV/AIDS, increasing Treg cells might have some beneﬁt in restoring T cell homeostasis and controlling T cell activation associated with this disease. For cancer patients, increasing Treg cells is likely to be detrimental by preventing induction of tumor-speciﬁc T cell responses and may explain the limited efﬁcacy of IL-2 in antitumor therapy. Anti-CD25 immunotherapy is widely used with some success to inhibit unwanted immune responses in patients with autoimmune disease or recipients of allogeneic transplants. On face value, this success ﬁts with the model that blocking IL-2 prevents immune responses. However, for patients with multiple sclerosis and autoimmune uveitis, the mechanism of immune inhibition is the induction of a regulatory CD56bright NK cell population, rather than inhibition of effector cells (169, 170). Newly revealed functions of IL-2 need to be carefully considered in future use of IL-2-directed therapies. Besides promoting Treg cells, two other important issues are the auto-inhibitory loop where IL-2 inhibits its own production (Figure 1) and the capacity of IL-2 to antagonize development of autoaggressive Th17 cells (Figure 2). Whether these mechanisms are functional within the human system remains to be determined, as they have just been recently described for the mouse. Given this caveat, one likely consequence not previously considered when IL-2 is blocked in vivo may be increased IL-2 production through impaired auto-inhibition. As low-dose IL-2 in humans selectively increases CD56bright NK cells (171), it is tempting to speculate that the increase in regulatory NKT cells in autoimmune patients undergoing www.annualreviews.org • The Function of IL-2

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anti-CD25 therapy may be the result of preventing the IL-2 auto-inhibitory loop, leading to increased IL-2. Another consequence of blocking IL-2 may be increased development of auto-aggressive Th17 cells. However, increased IL-2, through failed autorepression, may counteract this greater potential for Th17 cells by preventing Th17 development and/or by promoting Treg cells. There remains much hope that adoptive immunotherapy with Treg cells will provide therapeutic beneﬁt to patients with severe autoimmunity or that receive allogeneic transplants. Another very important property of IL-2, therefore, is its ability to support the growth of Treg cells in vitro. Indeed, in the most successful reports of Treg cell expansion, IL-2 is a key ingredient in the growth protocol in which activation through the TCR and CD28 is also required (172, 173). Somewhat paradoxically and in stark contrast to antigen-activated effector cells, Treg cells do not directly proliferate to exogenous IL-2 even though they constitutively express the high-afﬁnity IL-2R (174, 175). One reason for the poor direct response by IL-2 is that Treg cells contain increased levels of SOCS2 (suppressor of cytokine signaling 2), which generally dampens IL-2 signaling (175, 176). Another reason is that IL-2 signal transduction differs in Treg cells versus activated effector cells. IL-2 readily activates MAPK, PI3K, and Stat5 pathways in conventionally activated T cells, whereas IL-2 activates Stat5, but not downstream targets of the PI3K pathway, in Treg cells (175). The poor activation of the PI3K pathway in Treg cells is due to increased activity of phosphatase and tensin homolog (PTEN) (174, 175). This may be particularly relevant, as efﬁcient IL-2-driven T cell growth normally requires prolonged activation of the PI3K pathway (177). Accordingly, Treg cells directly proliferate to IL-2 after deletion of PTEN (174). Moreover, TCR activation of Treg cells in vitro downregulates PTEN, which may be permissive for their subsequent IL-2-dependent growth.

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When targeting IL-2 in immunotherapy, new strategies are needed to selectively enhance or inhibit the regulatory versus the effector components of the immune response. Blocking particular pathways of IL-2R signal transduction holds promise because IL2R signaling appears more diverse in effector than in Treg cells, the latter of which dominantly require Stat5. In this regard, inhibiting downstream targets of the PI3K pathway with rapamycin aids selective expansion of Treg cells in vitro and in type 1 diabetes patients by preventing IL-2-dependent growth and promoting depletion of T effector cells, respectively (178, 179). Another approach is to independently block the effector arm while enhancing Treg cells. Indeed, the combined application of the immunosuppressant dexamethasone with IL-2 is more effective than IL-2 alone to expand Treg cells in peripheral tissues in normal mice and to inhibit EAE (180). Agonists that selectively target the IL-2R on distinct cell types may also be of value. The ﬁnding that anti-IL-2/IL-2 preformed complexes function as IL-2R agonists in vivo in the mouse is an unanticipated example (181). One mAb/IL-2 complex activates cells bearing the high-afﬁnity IL-2R (CD25, CD122, and γc), whereas another activates cells bearing only CD122 and γc, and hence does not activate Treg cells. These agonists are used at much lower concentrations than is direct IL-2 infusion, with less expected toxicity, but persist longer, leading to prolonged IL-2R signaling. Although it may not be feasible to develop an IL-2R agonist that distinguishes the high-afﬁnity IL-2R on regulatory versus effector T cells, these types of agonists alone or in conjunction with IL-2R pathway antagonists may represent an approach to improved IL-2-based immunotherapy. Ultimately, further deﬁning quantitative and qualitative differences in IL-2R signaling between Treg and T effector cells and downstream targets represents a key step in devising more selective therapeutics.

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DISCLOSURE STATEMENT The author is not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS My research is supported by the National Institutes of Health and by the Juvenile Diabetes Research Foundation.

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93. Burchill MA, Goetz CA, Prlic M, O’Neil JJ, Harmon IR, et al. 2003. Distinct effects of STAT5 activation on CD4+ and CD8+ T cell homeostasis: development of CD4+ CD25+ regulatory T cells vs CD8+ memory T cells. J. Immunol. 171:5853–64 94. Antov A, Yang L, Vig M, Baltimore D, Van Parijs L. 2003. Essential role for STAT5 signaling in CD25+ CD4+ regulatory T cell homeostasis and the maintenance of selftolerance. J. Immunol. 171:3435–41 95. Peffault de Latour R, Dujardin HC, Mishellany F, Burlen-Defranoux O, Zuber J, et al. 2006. Ontogeny, function, and peripheral homeostasis of regulatory T cells in the absence of interleukin-7. Blood 108:2300–6 96. Cohen AC, Nadeau KC, Tu W, Hwa V, Dionis K, et al. 2006. Cutting edge: decreased accumulation and regulatory function of CD4+ CD25high T cells in human STAT5b deﬁciency. J. Immunol. 177:2770–74 97. Liston A, Rudensky AY. 2007. Thymic development and peripheral homeostasis of regulatory T cells. Curr. Opin. Immunol. 19:176–85 98. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, et al. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+ CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12:431–40 99. Tang Q, Henriksen KJ, Boden EK, Tooley AJ, Ye J, et al. 2003. Cutting edge: CD28 controls peripheral homeostasis of CD4+ CD25+ regulatory T cells. J. Immunol. 171:3348–52 100. Tai X, Cowan M, Feigenbaum L, Singer A. 2005. CD28 costimulation of developing thymocytes induces Foxp3 expression and regulatory T cell differentiation independently of interleukin 2. Nat. Immunol. 6:152–62 101. Liston A, Siggs OM, Goodnow CC. 2007. Tracing the action of IL-2 in tolerance to islet-speciﬁc antigen. Immunol. Cell Biol. 85:338–42 102. Malek TR, Porter BO, Codias EK, Scibelli P, Yu A. 2000. Normal lymphoid homeostasis and lack of lethal autoimmunity in mice containing mature T cells with severely impaired IL-2 receptors. J. Immunol. 164:2905–14 103. Wan YY, Flavell RA. 2007. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 445:766–70 104. Bayer AL, Yu A, Adeegbe D, Malek TR. 2005. Essential role for interleukin-2 for CD4+ CD25+ T regulatory cell development during the neonatal period. J. Exp. Med. 201:769–77 105. Zorn E, Nelson EA, Mohseni M, Porcheray F, Kim H, et al. 2006. IL-2 regulates FOXP3 expression in human CD4+ CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood 108:1571–79 106. Yao Z, Kanno Y, Kerenyi M, Stephens G, Durant L, et al. 2007. Nonredundant roles for Stat5a/b in directly regulating Foxp3. Blood 109:4368–75 107. Yu A, Malek TR. 2006. Selective availability of IL-2 is a major determinant controlling the production of CD4+ CD25+ Foxp3+ T regulatory cells. J. Immunol. 177:5115–21 108. Gavin MA, Clarke SR, Negrou E, Gallegos A, Rudensky A. 2002. Homeostasis and anergy of CD4+ CD25+ suppressor T cells in vivo. Nat. Immunol. 3:33–41 109. Min B, McHugh R, Sempowski GD, Mackall C, Foucras G, Paul WE. 2003. Neonates support lymphopenia-induced proliferation. Immunity 18:131–40 110. Bayer AL, Malek TR. 2007. The role of IL-2 in the development and peripheral homeostasis of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells. In Regulatory T Cells and Clinical Applications, ed. S Jiang, RI Lechler. New York: Springer. In press 111. Vlad G, Ho EK, Vasilescu ER, Fan J, Liu Z, et al. 2007. Anti-CD25 treatment and FOXP3-positive regulatory T cells in heart transplantation. Transpl. Immunol. 18:13–21

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:453-479. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

vi

Contents

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The Biochemistry of Somatic Hypermutation Annu. Rev. Immunol. 2008.26:481-511. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

∗

∗

Jonathan U. Peled,1, Fei Li Kuang,1, Maria D. Iglesias-Ussel,1 Sergio Roa,1 Susan L. Kalis,1 Myron F. Goodman,2 and Matthew D. Scharff1 1

Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461

2

Biological Sciences and Chemistry, University of Southern California, Los Angeles, California 90089; email: [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:481–511

Key Words

First published online as a Review in Advance on December 12, 2007

activation-induced cytidine deaminase (AID, aicda), mismatch repair, antibody diversity, base excision repair, error-prone repair, germinal center

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090236 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0481$20.00 ∗

These authors contributed equally to this review.

Abstract Afﬁnity maturation of the humoral response is mediated by somatic hypermutation of the immunoglobulin (Ig) genes and selection of higher-afﬁnity B cell clones. Activation-induced cytidine deaminase (AID) is the ﬁrst of a complex series of proteins that introduce these point mutations into variable regions of the Ig genes. AID deaminates deoxycytidine residues in single-stranded DNA to deoxyuridines, which are then processed by DNA replication, base excision repair (BER), or mismatch repair (MMR). In germinal center B cells, MMR, BER, and other factors are diverted from their normal roles in preserving genomic integrity to increase diversity within the Ig locus. Both AID and these components of an emerging error-prone mutasome are regulated on many levels by complex mechanisms that are only beginning to be elucidated.

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INTRODUCTION H, or IgH: immunoglobulin heavy chain GC: germinal center AID: activation-induced cytidine deaminase (gene symbol: aicda)

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SHM: somatic hypermutation CSR: class switch recombination V, or V region: variable region of the Ig gene C, or C region: constant region of the Ig gene Transitions: mutations that change a pyrimidine into another pyrimidine (e.g., C to T) or a purine to the other purine (e.g., G to A) Transversions: mutations that change a pyrimidine (C or T) into a purine (G or A) or vice versa

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Vertebrates have evolved a complex set of mechanisms to protect themselves from infections and foreign substances. The adaptive humoral response plays an important role in this process by providing antibodies that circulate throughout the body and into secretions, where they bind strongly and speciﬁcally to invading organisms and other foreign substances and dispose of them through a variety of effector functions (1). Although all vertebrates can make antibodies, species differ in the details of how they use a small amount of genetic material to generate sufﬁcient antibody diversity to deal with all possible antigens (2). Prior to antigen exposure, mice and humans constantly recreate a highly diverse repertoire of antigen-binding sites in pro- and pre-B cells in the bone marrow through the rearrangement of germ line immunoglobulin (Ig) variable (V), diversity (D), and joining ( J) elements to form the heavy (H) and light (L) chain V regions in the Ig genes (1, 3–5). These germ line–encoded IgM antibodies are of low afﬁnity and are usually not effective in inactivating pathogenic organisms and their products. Once an antigen appears, however, cognate mature B cells are stimulated to proliferate, differentiate, and migrate to the dark zone of the germinal centers (GC) in secondary lymphoid organs, where they become centroblasts (6, 7). In the GC microenvironment, centroblast B cells begin to express large amounts of activation-induced cytidine deaminase (AID), which initiates somatic hypermutation (SHM) of the antibody V regions that encode the antigen-binding sites (8, 9) (Figure 1). These point mutations result in the amino acid replacements in the H and L chain V regions that are responsible for the afﬁnity maturation and changes in ﬁne speciﬁcity that are required to produce effective neutralizing antibodies (1, 3–5, 10, 11). Centroblast B cells also carry out class switch recombination (CSR), which requires AID and utilizes many of the same mechanisms of mutation and repair that

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are involved in SHM (12). In this review, we discuss only SHM of the V region genes in mice and humans, but we also draw on some of the studies that have been done with the DT40 chicken B cell line that provide insights into the mechanisms of SHM in mice and humans (13). We focus on the more recent biochemical studies of AID and the characterization of additional genetically defective mice that provided convincing support for and extend the biochemical basis of this model. In this discussion, we draw heavily on and refer to the ideas and data summarized in a number of excellent recent reviews (1, 3–5, 10, 11) as we try to understand how the different enzymatic systems that are involved in SHM are organized and regulated. We ﬁrst describe general features of SHM, followed by each of the mutation and repair systems involved in V region hypermutation, and then address the issue of the overall regulation of SHM. CSR is discussed in detail in the review by Stavnezer et al. (14) in this volume.

FEATURES OF SOMATIC HYPERMUTATION If we consider the characteristics of SHM of the H chain V region (Figure 1), most of the mutations are single base changes that accumulate starting 100–200 bp from the transcription initiation site and end 1.5–2.0 kb downstream (15–19). The frequency of mutations is highest in the V(D)J coding region and the J region introns, sparing the important regulatory elements in the promoter, the intronic enhancer, and the constant (C) region genes that are responsible for effector functions (17, 19) (Figure 1). The frequency of V region mutation is approximately 10−5 – 10−3 /base pair/generation, which can be compared to the basal level of mutation in the genome of ∼10−9 . There are more transitions (e.g., C to T, G to A) than transversions (e.g., C to A or G; G to C or T), and many of the mutations are preferentially targeted to the deoxycytidines (dC) within WRC

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Antibody structure Antigenbinding sites

Constant region (C)

Light (L) chain Heavy (H) chain

b

Somatic hypermutation in Ig genes

Mutation frequency

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Variable region (VDJ, VJ)

P

L

V(D)J

Core MAR MAR

hs3a hs1,2 hs3b,4 hs5, 6, 7

IgH L

V DJ

L

V J

iEµ

SR

CH

3' RR

IgL iE

C

3' RR

Figure 1 (a) Antibody structure. An antibody molecule is a dimer of heterodimers (H+L)2 , connected by disulﬁde bonds. The heavy (H) and light (L) chain V regions (orange) form the antigen-binding site, while the C regions (blue) form the effector arm. (b) Somatic hypermutation in Ig genes. Mutation frequency along the IgH and IgL locus is depicted. Representative IgH and Igκ genes are depicted below the graph that include the following elements: Leader (L), V region [V(D)J], intronic enhancer (iEμ and iEκ), C region (CH or Cκ), switch region (SR), and the 3 regulatory region (3 RR). The arrow indicates the transcription start site. The V region (orange) experiences signiﬁcant SHM, whereas the C region (blue) does not. SHM is sharply delimited by the V promoter (P) at the 5 end and starts approximately 100–200 bp from the transcription start site. Mutation frequency is maximal over the V(D)J coding exon and exponentially decays at the 3 end at 1.5–2 kb downstream from the transcription start site. (Mutation data adapted from Reference 15; boundaries described in References 18, 19.)

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C G

AID U G UNG

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Replication

MMR

G Short-patch BER

G

Long-patch BER

A PCNA recruits error-prone polymerases (Pol η)

C T T A

G

Phase 1a

Phase 1b

Mutations at G:C basepairs ~40% of total mutations

G Phase 2 Mutations at A:T basepairs ~60% of total mutations

Figure 2 Model of somatic hypermutation. AID deaminates a cytidine residue, creating a uridine:guanosine (U:G) mismatch that is resolved by several pathways that may compete with one another. AID deaminates single-stranded DNA formed during transcription of both strands of the DNA (not shown). The subsequent steps, however, might not occur equally on both strands. (Left) The general replication machinery can interpret the U as if it were a deoxythymidine (T). One of the daughter cells will acquire a C-to-T transition mutation. (Center) UNG can remove the uracil, leaving behind an abasic site. Short-patch base excision repair (BER) can ﬁll the gap with error-prone polymerases, which can insert any nucleotide in place of the U, leading to transitions and transversions at G:C bases. (Right) Mismatch repair (MMR) can recognize the U:G mismatch. The U-bearing strand is excised and, at loci that undergo SHM, monoubiquitylated PCNA (proliferating cell nuclear antigen) recruits error-prone polymerases to ﬁll the gap, leading to transition and transversion mutations at A:T bases as well as at neighboring G:C bases. (Dashed line) Long-patch BER can also be a source of mutations at A:T bases and may compete with MMR.

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motifs that are hot-spots for SHM (20–22). High rates of transcription are required for SHM to occur, and the frequency of mutation is roughly proportional to the rate of transcription (23–25). Both the transcribed (template, bottom) and the nontranscribed (nontemplate, top) strands undergo AID-induced deamination of dC to deoxyuridine (dU) to produce C-to-T mutations at the same frequency (26). Transcription alone is not sufﬁcient, as many transcribed genes in GC B cells are not targeted for SHM (27, 28). A general model for the enzyme systems involved in SHM includes a ﬁrst phase that depends on the mutagenic activity of AID and a second phase that depends on the errorprone repair of the AID-induced mutations (Figure 2). Even before the discovery of AID, Rada et al. (29) proposed two distinct phases of SHM because mice deﬁcient in DNA mismatch repair (MMR) exhibited a selective loss of mutations at A:T bases but retained the ability to mutate G:C bases. Mutations at G:C bases were termed phase 1 mutations, with MMR-dependent mutations at A:T bases occurring in phase 2. Further studies (30) demonstrated that mutations detected in the antibody V regions require the propagation of the C-to-U mutation through replication, generating a C-to-T transition, or, in the opposite strand, a G-to-A transition (phase 1a). Phase 1b accounts for transversions at G:C bases, which depend on one of the uracil DNA glycosylase enzymes, UNG, to trigger base excision repair (BER), remove uracil from DNA, and create an abasic site that can be ﬁlled in with any of the four bases. Long-patch BER and MMR are responsible for the large number of mutations at A and T bases (3) generated in phase 2. Although the BER and MMR enzymes normally maintain genomic stability, they become error prone in the GC centroblast B cells when they are acting on the Ig genes and thus greatly increase the number of mutations that accumulate in antibody V regions. A critical conclusion from this model is that in mice and humans ∼60% of the mutations

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that arise as the result of SHM are in A:T bases, and about half of those are transversions rather than transitions (5). Thus, more than half of the mutations in the V region are not the result of the direct biochemical action of AID, but rather depend on the error-prone BER and MMR of the AID-induced mutation (Figure 2). This error-prone repair is contributed by members of a family of low-ﬁdelity translesional DNA polymerases (31). These translesional polymerases, including Pol η, θ, ι, ζ, λ, and REV1, are ubiquitously expressed and appear to have a shallower and less constrained binding pocket than the high-ﬁdelity polymerases. This allows them to recognize DNA lesions and bypass them in newly replicated or mutated DNA by inserting bases opposite the lesion (32). As a consequence of their more permissive binding sites, they are also error prone. The discovery that AID introduces dU mutations at a high frequency in the V regions (8, 9) led us to propose that these translesional polymerases were responsible for the error-prone repair of the dU introduced by AID (33). The discovery of an important role for BER and MMR led to an expansion and reﬁnement of this DNA deamination model by Neuberger and his colleagues (3, 30, 34) (Figure 2) that serves as the working model for most investigators in the ﬁeld. BER and MMR (and perhaps AID itself) participate in large complexes of proteins, the nature of which are under intense investigation. The extent to which these proteins interact with each other and other factors is important because this is one way in which SHM may be regulated. The Supplemental Table lists the proteins reported to be involved in SHM and selected interactions among them (follow the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org).

BIOCHEMICAL BASIS OF AID ACTIVITY When AID was ﬁrst discovered, investigators proposed that it worked as an RNA-editing

enzyme on an mRNA that encodes a yet to be discovered endonuclease that may initiate SHM (8, 9, 35). Many subsequent studies, however, have revealed that AID initiates the process of V region hypermutation by deaminating dC to dU on single-stranded DNA (ssDNA) (1, 3–5, 10, 11). The role of AID as the initiating factor in SHM was conﬁrmed by studies showing that genetically engineered mice that lack AID and patients with type II hyper-IgM immunodeﬁciency syndrome (HIGM-2) who have inactivating mutations in AID were unable to carry out SHM (36, 37). Although there is still much to be learned about the biochemistry and targeting of AID, a critical observation came from the ﬁrst biochemical studies: In the absence of cofactors, AID deaminates dC to dU on ssDNA, but not on double-stranded DNA (dsDNA), DNA:RNA hybrids, or RNA in any form (38–40). Furthermore, in this cell-free system, semipuriﬁed AID preferentially deaminates dC in WRC hot-spot motifs, while exhibiting much lower activity for dC in SYC cold-spot motifs (39, 40). As has been observed in vivo, not every dC in a hot-spot is deaminated, and some non-hot-spot motifs undergo frequent mutation in vitro. AID will only deaminate dC in dsDNA in vitro if there is ongoing transcription at the site or if single-stranded bubbles are otherwise introduced (40–46). Thus, the requirement for active transcription to initiate SHM (and CSR) is explained by the generation of the ssDNA substrate for AID within a moving transcription bubble. These observations provided direct evidence that the substrate of AID was DNA and suggested that the AID protein itself has the inherent information to preferentially deaminate dC in the context of certain sequences such as WRC and to reduce deamination in other sequences such as SYC (38–42, 44–48). A second important ﬁnding from the studies with AID puriﬁed from insect cells was that AID has high processivity on ssDNA substrates. After binding ssDNA in vitro, www.annualreviews.org • Somatic Hypermutation

BER: base excision repair MMR: mismatch repair Translesional polymerase: a DNA polymerase that can bypass a bulky DNA lesion, sometimes with the introduction of non-Watson-Crick base pairings HIGM-2: type II hyper-IgM immunodeﬁciency syndrome Hot-spot: a short sequence of DNA where AID-induced point mutations are preferentially found: WRC, where W is either of the weakly hydrogen-bound bases A or T, and R is either of the purines G or A Cold-spot: a short sequence of DNA where AID-induced point mutations are rare: SYC, where S is either of the strongly hydrogen-bound bases G or C, and Y is either of the pyrimidines C or T

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AID causes multiple deaminations prior to dissociation (39, 40, 49). Although it appears that the processive behavior of AID would allow it to translocate along with a transcription bubble in vitro, generating dC deaminations principally in hot-spot motifs (38–42, 44, 47), evidence is lacking that AID catalyzes multiple deaminations during transcription in vivo because only a few V-gene mutations appear to occur per cell division. An alternative possibility, however, is that AID induces many mutations per cell division but that the majority are rapidly and discreetly repaired in an error-free fashion (see Conclusions). Another perhaps more compelling teleological rationale for AID processivity is that the enzyme is globally targeted to regions of ssDNA, but not to speciﬁc target dC motifs. AID binds with roughly equal afﬁnity to ssDNA that contains either WRC

5' WRC

AID

Jumping

Active site WRC

Sliding

Sli din g

ssDNA

3'

WRC

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Processive: a processive enzyme catalyzes multiple reactions on a single substrate prior to acting on a different substrate

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WRC Figure 3 Model describing processive C-to-U deaminations. AID is depicted as a dimer on ssDNA. Current biochemical data suggest that AID binding occurs randomly, and enzyme motion, for example sliding and jumping, occurs in either direction along the ssDNA substrate. Deamination by AID occurs processively (making multiple deaminations per substrate molecule) and equally in 5 and 3 directions, with preferential targeting to WRC motifs. Notably, there is no external energy source present, for example ATP or GTP hydrolysis.

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motifs or SYC motifs and even to DNA with no C residues or with only U residues (M.F. Goodman, unpublished data). Therefore, AID would have to remain bound to the DNA to catalyze deamination when it does encounter a dC, perhaps while tracking along with a moving transcription bubble (5, 10, 50). Once bound to ssDNA, AID catalyzes deamination in what appears to be a “hit-andmiss” process of the sort described for restriction enzymes rather than for polymerases (39, 48, 51). For example, deamination patterns on individual DNA clones exposed to AID in vitro contain small clusters of deaminations, often separated by lengthy regions where dC is left intact even though there are numerous WRC hot-spot motifs. Each DNA clone exhibits a different deamination pattern (39, 40, 51), and, similarly, clonal patterns have been reported for different B cell clones bearing the same Ig transgene (52). When deaminations do occur, WRC hot-spot motifs are favored over SYC cold-spots by about 6:1. Multiple deaminations in vitro tend to congregate near WRC sites, suggesting that after a hotspot deamination, AID can slide to and attack a proximal C residue and then jump to another region on the same ssDNA strand. The biochemical data suggest that AID binds randomly to ssDNA and performs a bidirectional random search for C residues by jumping and sliding along the DNA backbone (Figure 3). This behavior of AID reﬂects special properties of the enzyme because it differs signiﬁcantly in this cell-free system from one of its homologs, APOBEC-3G (Apo3G), which favors deamination toward the 5 region of ssDNA (51). It is unclear how the processive properties of AID are used in vivo, where directional deamination might be imposed upon it by the 5 to 3 motion of RNA polymerase II (RNA Pol II), which has been reported to associate with AID (53) (see the Supplemental Table). An important biochemical challenge will be to recapitulate the salient features of transcription-dependent deamination with puriﬁed mammalian enzymes.

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Strand-Specific Targeting On the basis of the biochemical studies described above, we might expect that only the nontranscribed strand would be attacked by AID. In fact, a single cytosine in the nontranscribed strand is sufﬁcient to recruit AIDinduced deamination and allow the mutation of upstream and downstream A and T residues, whereas in one experimental system this does not seem to happen when the cytosine is in the transcribed strand (54). The analysis of this strand asymmetry in vivo has revealed that error-prone MMR preferentially targets dU in the nontranscribed strand, suggesting different post-AID repair of the two strands. However, mutations at dC sites in cells lacking both BER and MMR occur with roughly equal frequencies on both DNA strands (3, 55, 56). Assuming that the deamination events that trigger mutation occur during V-gene transcription, one possibility is that both strands can undergo transcription to provide ssDNA on which AID can act, and this has now been observed in Ramos cells, a human centroblast-like B cell line that constitutively undergoes SHM (57). As far 1aa

3aa

AID Protein Structure A sketch of the AID domain structure is shown in Figure 4. HIGM-2 disease-causing mutations are found throughout the protein

53aa

Exon 1

143aa

Exon 2

R24W Select HIGM-2 patients Site-directed mutagenesis R19E R24E

P

NH2

back as 1992, antisense promoters were identiﬁed in another part of the Ig locus, the Ig μ-switch regions that are associated with c-myc in a Burkitt’s lymphoma cell line (58), and antisense transcription has been reported in other regions of the Ig gene (59, 60). However, if bidirectional transcription is not occurring in vivo, then how C-to-T mutation occurs on both strands is unclear. One proposal is that DNA supercoiling may generate transient single-stranded regions on the transcribed strand (5, 47, 50, 61). Although the use of T7 polymerase has revealed only occasional deamination of the nontranscribed strand (48), deamination of both strands has been reported using Escherichia coli polymerase in vitro (44) and in the E. coli chromosomal rpoB locus when AID is overexpressed (30). The addition of AID to a mammalian transcription system may provide a clearer answer to how both strands are targeted in vivo.

Charged

Exon 3

182aa Exon 4

R112H

H56Y

198aa

Exon 5 R190X

R35E/ R36D

P

Active site

Zn coordination

P

H56 E58

C87 C90

Y184

+++ ++ T27

NLS

COOH S38

Cytidine deaminase motif

Linker

Apobec-like domain

SHM specific (13–23)

NES

CSR specific (172 –198)

Figure 4 Inactivating mutations and functional domains in AID. The exon domains of AID are depicted (aa, amino acids). Select inactivating mutations found in HIGM-2 patients (red ) or generated by site-directed mutagenesis (blue) that result in changes in amino acids are indicated with arrows off of the exon domain diagram. The AID protein is depicted in green, with critical residues indicated and functional domains drawn below. Known phosphorylation sites are represented by yellow or orange sunbursts. The model is not to scale. www.annualreviews.org • Somatic Hypermutation

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(http://bioinf.uta.fi/AICDAbase), and most result in loss of both SHM and CSR (62). Some of these mutations (shown in Figure 4) and complementary studies using site-directed mutagenesis have provided clues to the functional domains of AID (39, 63, 64). The C-terminal portion of AID is required for CSR but not for SHM (63–65). This region is not required for deamination: Biochemical analysis of the mutation spectrum generated by a C-terminal deletion mutant of AID revealed close resemblance to that generated by wild-type AID (39). Thus, some aspect of AID targeting to switch regions versus V regions resides within these last few amino acids, perhaps via the presence of CSR-speciﬁc cofactors (63, 64). The N-terminal region of AID contains a remarkably high concentration of basic amino acid residues, resulting in a +11 net positive charge (39). An AID double mutant (R35E/R36D, Figure 4), in which the Nterminal charge has been reduced to +7, exhibits reduced processivity in vitro (39), which is not surprising if it binds with lower afﬁnity to the negatively charged ssDNA backbone. Yet this double mutant also shows a change in speciﬁcity in that the highest deamination rate occurs in a non-WRC motif (39). This modiﬁed deamination speciﬁcity is surprising because the R35E/R36D N-terminal mutations are far removed from the catalytic region. Our understanding of the functional properties of AID would be helped signiﬁcantly by structural data. Although there is as yet no crystal structure for AID, a member of the APOBEC family of enzymes, APOBEC2 (APO2), has been crystallized and shown to form a rod-shaped tetramer (66). Owing to a signiﬁcant degree of sequence similarity, APO2 can be used as a surrogate for AID to make structure-based predictions. First, AID complexed with Zn is necessary for deamination activity. The APO2 structure suggests that Zn hydroxylation may regulate substrate access and enzyme activity. A site-directed mutation at the equivalent site in AID (R19E, Figure 4) was predicted to alter Zn hydrox-

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ylation and, when tested in vitro, resulted in a ∼ﬁvefold reduction in AID deamination activity (66). Second, AID has been identiﬁed as a dimer (63, 71, 162), and mutations predicted to disrupt a putative dimer interface (F46A/Y48A) resulted in a fourfold loss of activity (66). Finally, a residue in APO2 is predicted to help stabilize a β1 -hairpin conformation and thus allow access to nucleic acid substrate. When the corresponding residue is mutated in AID (R24E), deamination activity is completely abrogated. The residues of AID that are mutated in HIGM-2 syndrome, causing impaired production of high-afﬁnity antibodies, are well conserved in APO2. Notably, the aforementioned R24 residue is mutated in some HIGM-2 patients, whereas additional HIGM-2 mutations occur on the predicted surface of an AID monomer (66). Despite biochemical and physical (66) evidence that AID likely works principally as a dimer in vivo, we cannot rule out that an AID monomer or even higher polymers may also be active (41).

Posttranslational Modifications AID isolated from stimulated primary B cell nuclei is phosphorylated at multiple sites, including Ser38 (68–70) (Figure 4). Only about 10% of the protein contains phosphate at this residue, and, interestingly, the phosphorylated form is enriched in the chromatin fraction (69). AID expressed in E. coli, which is not expected to be phosphorylated, deaminates transcribed dsDNA in vivo (43, 46), and protein partially puriﬁed from E. coli deaminates dC residues on ssDNA in vitro (43–45). Thus, phosphorylation per se is not necessary for AID activity. It has been reported that AID enriched from B cells must be phosphorylated at Ser38 in order to deaminate linear dsDNA undergoing transcription with T7 RNA polymerase in a reaction that also requires replication protein A (RPA), a factor that binds ssDNA (71). Human AID obtained from insect cells is also phosphorylated at Ser38, yet it is able

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to deaminate ssDNA in a T7-based transcription assay in the absence of added RPA (39, 40). Unphosphorylated AID expressed in E. coli also appears to be active in the setting of in vitro transcription assays without added RPA (43, 44). Therefore, uncertainties concerning the role of phosphorylation at Ser38 and perhaps at other sites remain an outstanding issue. At present, phosphorylation at Ser38 appears to play a role in the efﬁciency of SHM. In activated B cells deﬁcient for AID, a Ser38Ala mutant exhibits signiﬁcantly delayed appearance of SHM (69). Other residues in AID may be phosphorylated as well (Figure 4). A thorough biochemical comparison using phosphorylated and nonphosphorylated AID could shed light on how AID interacts with ssDNA, RPA, and perhaps transcription factors that may serve to target AID to DNA undergoing transcription.

AID Splice Variants Another aspect of AID that needs to be examined in more detail is the role, if any, of the various isoforms that have been observed. Alternative transcripts of AID have been reported in asthmatic patients (72), human B cell non-Hodgkin’s lymphomas (B-NHL) (73), chronic lymphocytic leukemia (74–77), and normal B cells stimulated with CD40L and IL-4 (74, 75). The constitutive expression of AID and its splice variants may contribute to B-NHL formation (73). Five different transcript variants of AID have been detected: (a) the full-length AID transcript (36), which is the most prevalent AID transcript in healthy and neoplastic B cells (73); (b) a variant that lacks the ﬁrst 30 bp of exon 4 (72, 74, 75); (c) a variant that lacks all of exon 4 (72–75); (d ) a variant in which intron 3 is retained (73, 76, 77); and (e) a variant that includes a short neo-exon located in intron 3, but lacks exons 3 and 4 (73). The biochemical examination of these isoforms would provide useful structural and functional information and may also pro-

vide some insights into the role of AID in B cell malignancies.

BASE EXCISION REPAIR BER is a DNA repair pathway in which altered bases are removed by a DNA glycosylase, followed by subsequent steps to repair the lesion. Approximately 60% of the mutations that accumulate in vivo in mice and humans are in A:T bases and are not caused by the direct action of AID. As illustrated in Figure 2, once AID has mutated dC to dU in DNA, the uracil may be either replicated or excised by UNG to create an abasic site. This intermediate can be converted into a single-stranded break by apurinic/apyrimidic endonucleases (APEs) (not shown in Figure 2) that can in turn be repaired by error-prone polymerases. This UNG-dependent pathway can generate both transition and transversion mutations, whereas replication yields only transitions (78, 79). UNG is primarily responsible for initiating BER of the Ig genes in centroblast B cells, and its genetic inactivation causes a profound defect in both SHM and CSR in mice and humans (34, 80), whereas the genetic inactivation of other uracil DNA glycosylases has little or no effect (3, 81, 82). The dominance of UNG in SHM (and CSR), compared with the many other uracil DNA glycosylases that are available (83, 84), illustrates a common theme in the SHM of the Ig genes: Even in the presence of considerable redundancy, often one particular enzyme and its downstream protein partners are hijacked by the centroblast B cell to repair the antibody V regions in an error-prone manner. A great deal has been learned in recent years about high-ﬁdelity BER that may be relevant to the error-prone BER of Ig genes. For example, it is now clear that the abasic sites generated by UNG and the single-stranded breaks created by the action of APE1 can be repaired by either of two alternative pathways: short-patch repair of one base or long-patch repair that involves the excision of 2–8 or www.annualreviews.org • Somatic Hypermutation

Short-patch repair: BER subpathway where the length of the repair patch is exactly one nucleotide Long-patch repair: BER subpathway where the length of the repair patch is 2–8 nucleotides or possibly more

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possibly more bases and their replacement (84, 85) (Figure 2). The repair of AIDinduced mutations by BER can in principle be limited to mutations at single dC bases after the dU has been removed by UNG, in which case it is called short-patch repair. Because replication over the uracil that arises from the direct action of AID results only in transition mutations, transversion mutations at C or G could arise as a result of such short-patch BER. In animal model systems, the absence of transversion mutations at C and G residues is used to identify defects in this aspect of BER in SHM. Because C-to-G transversions are lost in mice that are genetically defective in REV1, this translesional enzyme is thought to be primarily responsible for the short-patch BER of abasic sites in SHM (78). As with UNG, REV1 is yet another example of one of the many potential enzymes that is preferentially recruited to carry out error-prone repair of AID-induced mutations in the Ig V regions. Recent studies suggest that the switch from high-ﬁdelity polymerases to error-prone polymerases is mediated by the monoubiquitylation of proliferating cell nuclear antigen (PCNA) (86–89) (see below) so that modiﬁed PCNA may be required to recruit REV1 to repair the abasic sites or the ssDNA breaks that arise in short-patch BER. Barring the existence of an A:T deaminase, mutations at A:T bases must be brought about by excision of bases surrounding the initially targeted dC. One important pathway that carries this out is MMR (discussed below). However, MMR-deﬁcient mice still accumulate some A:T mutations. Interestingly, all A:T mutations disappear in MSH2-UNG (90) and MSH6-UNG double-deﬁcient mice (56), suggesting that a UNG-dependent, BER pathway also contributes to A:T mutagenesis. Since short-patch BER only acts at the initially targeted dC (Figure 2), it cannot be responsible for those residual A:T mutations seen in MMR-deﬁcient mice. Therefore, it is likely that long-patch BER contributes to A:T mutagenesis, although the exact enzymatic players are not as well understood as they are

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in MMR. In addition, there is some evidence that the MRN (MRE11-RAD50-NBS1) complex, normally involved in double-stranded break repair, can also participate in this process, perhaps in concert with or downstream from BER (84, 92, 93). Once UNG has removed the uracil, it is also unclear what regulates the recruitment of short-patch and long-patch BER during SHM. Although uracils that arise during replication more often undergo long-patch repair at least in some cell lines (91), this may not be true of AID-induced mutations in centroblast B cells. In general, the presence of PCNA at a DNA lesion seems to favor longpatch over short-patch repair, whereas Pol β tends to favor short-patch repair (85). We now know that monoubiquitylated PCNA (see below) is involved in SHM and likely facilitates A:T mutagenesis in the Ig gene via both MMR and long-patch BER. (86–89; S. Roa & M.D. Scharff, unpublished data). Another emerging principle is that the relative abundance of the various players makes a critical difference in determining error-prone versus error-free repair of AID-induced mutations. This is nicely illustrated in the case of the BER of V region mutations where Pol β, which usually mediates the high-ﬁdelity postreplicative short-patch repair of abasic sites, is downregulated in SHM-proﬁcient BL2 cells, a centroblast-like human B cell line (94). This appears to allow the error-prone polymerases, especially REV1 and perhaps Pol η or other translesional polymerases, to replace Pol β and allow for error-prone repair in SHM. In Pol β–deﬁcient B cells under certain experimental conditions, CSR can be more proﬁcient, although the levels of Pol β are not modulated during CSR in wild-type mice (78, 95). Taken together, all these studies suggest that both short-patch and long-patch BER play a critical role in the SHM of mouse and human V regions. Because high-ﬁdelity postreplicative BER is also important in maintaining genome stability, it is unclear how the centroblast B cell targets error-prone

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BER to the Ig. One possibility, which is discussed below, is that AID mutations and their error-prone repair may be restricted to the nonreplicative G1 and G2 phases of the cell cycle. It is probably also important to understand how BER and MMR compete to repair the dU mutations produced by AID. That competition could be between UNG and MSH2-MSH6 or for factors such as RPA, PCNA, and the error-prone translesional polymerases that are used in both BER and MMR. Because monoubiquitylated PCNA can recruit error-prone polymerases, the state of modiﬁcation of PCNA could determine whether there is long- or short-patch BER of V region mutations and whether it is error free or error prone. Most of the translesional error-prone polymerases are not processive and add only one, or a few, bases. The repair of longer patches of excised DNA either in BER or MMR (see below) requires that a more processive DNA polymerase like Pol ζ and/or high-ﬁdelity polymerases like Pol δ or ε assist the translesional enzyme in replacing the excised DNA (32). One report, for instance, has suggested that Pol η may replace the ﬁrst nucleotide, followed by extension of the new strand by Pol θ (79). There could also be competition for these translesional enzymes between long-patch BER and MMR. Because these different BER processes are mediated by different complexes (96), it would be interesting to compare the protein complexes that are present in centroblast B cells at different stages of the cell cycle to resolve some of these questions.

MISMATCH REPAIR As with BER, the mutagenic role that MMR plays in SHM is surprising, considering its critical role in maintaining the integrity of the genome. MMR is a complex process requiring the sequential action of many proteins to increase the overall ﬁdelity of DNA replication and the repair of genotoxic damage (97, 98). Postreplicative MMR is conserved from prokaryotes to mammals, and in

eukaryotic organisms it is mediated by the following proteins: (1) the MSH2-MSH6 heterodimer (called MutSα) that recognizes single mismatched base pairs, or MSH2-MSH3 (MutSβ) that recognizes larger mismatches or loops resulting from deletions or insertions; (2) following the initial binding of MSH2MSH6 or MSH2-MSH3 to the mismatch, there are a series of ATP-dependent events that lead to the recruitment of MLH1 and PMS2 (or MLH1-MLH3) that recruit other downstream elements and introduce a singlestranded nick near the mismatch (99); (3) exonuclease 1 (EXO1), which excises the mismatch and a yet to be determined stretch of the surrounding DNA strand; (4) Pol δ and ε, high-ﬁdelity polymerases that resynthesize the excised DNA stand; and (5) DNA ligase I, which ligates the ends. Other proteins that are also involved in long-patch BER, such as RPA, replication factor chaperone-like complex (RFC), and PCNA, play an important role in MMR (97, 98) (see Supplemental Table). Like BER, the centroblast B cell has hijacked the MMR process and made it error prone when it encounters AID-induced G:U mismatches in the Ig V regions (Figure 5). Error-prone MMR accounts for more than 50% of all the mutations and for most of the transversion mutations at A:T bases (3, 100). The genetic inactivation of both Ung and either Msh2 or Msh6 leads to a loss of virtually all mutations at A:T bases in V regions and a profound loss of CSR (55, 56). In conﬁrmation of a direct role for MMR in SHM and CSR, chromatin immunoprecipitation showed that EXO1 and MLH1 are associated with mutating V regions in the BL2 cell line and that MSH6 is associated with the switch regions involved in CSR in primary B cells (101, 102). As described by Stavnezer et al. (14), MMR is also important in CSR. Because the mutations introduced by AID create single-base G:U mismatches and because short-patch BER generates G:abasic site mismatches, it is not surprising that the absence of Msh3, which recognizes larger www.annualreviews.org • Somatic Hypermutation

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e Figure 5 Mismatch repair in SHM. A model of phase II SHM mediated by the mismatch repair pathway and error-prone polymerases. (a) A uridine (U) created by AID deamination of a cytidine creates a mismatch. (b) The Msh2-Msh6 complex recognizes the mismatch. Msh3 competes with Msh6 for Msh2. (c) Other components of the mismatch repair pathway are recruited, including Pms2, Mlh1, and Exo1. Each subunit of the homotrimeric PCNA can be monoubiquitylated (Ub). Mlh3 competes with Pms2 for Mlh1. The exact nature of the MMR complex is unknown. (d ) Exo1 excises the U-bearing strand, and monoubiquitylated PCNA recruits error-prone polymerases, especially Pol η, to resynthesize the gap. (e) The region near the deaminated residue acquires point mutations (red stars), including transitions and transversions at A:T bases. The newly transcribed strand is ligated in place (not shown).

mismatches, has little effect on SHM (103– 105). Although both Msh2 and Msh6 bind DNA, only Msh6 bears a conserved Phe-XGlu motif that directly interacts with mismatched nucleotides and is inserted into the 492

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minor groove of the helix at the mismatch site, bending the DNA some 60◦ (98, 106). In addition to triggering the recruitment of the other proteins required to excise and repair the mismatch (Figure 5), the binding of the

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MSH2-MSH6 heterodimer also informs the cell of the presence of damaged DNA and can either directly or indirectly activate the DNA damage response pathway, including the ATR kinase and the CHK1/CHK2 G2 /M checkpoint proteins, causing cell cycle arrest and signaling for apoptosis. Since error-prone MMR is responsible for so many of the mutations that accumulate in the V region, it is important to understand how this process is regulated. How does errorprone MMR continue to maintain the integrity of most of the genome of the rapidly dividing centroblast B cell, while simultaneously contributing many of the mutations in the V region? Not surprisingly, the proteins that are involved in MMR are highly expressed in rapidly dividing centroblast B cells that are undergoing SHM (107). There is probably tight control of the relative abundance of each of the MMR proteins because the inactivation of MLH3, which can also form a dimer with PMS2 (Figure 5), results in a change in the spectrum of mutations in vivo (108), suggesting that in wild-type mice it is competing with PMS2 to form complexes that are acting on the Ig gene. Studies with genetically defective mice and patients with the variant form of Xeroderma Pigmentosum, in whom Pol η is mutated, clearly demonstrate a critical role for Pol η in the introduction of transversion mutations at A:T bases that are the hallmark of the MMR-dependent second phase of SHM (109, 110) (Figures 2 and 5). Moreover, the complete absence of mutations at A:T bases in MSH2-Pol η double-deﬁcient mice indicates that the residual A:T mutagenesis in the single MMR-deﬁcient mice is contributed by Pol η (110). Additional studies in Pol θ–defective mice suggest that it works in concert with Pol η and other more processive, high-ﬁdelity DNA polymerases to ﬁll in the gap created by EXO1 (11, 79, 111–113). The question then becomes how these error-prone polymerases are recruited to the repair of the AID-induced mutations in the Ig V region and not to the postreplicative repair of uracils that are introduced during

the normal course of replication. The beginning of an answer comes from studies in yeast showing that the recruitment of these errorprone polymerases is mediated by PCNA that has been monoubiquitylated at residue 164 (32, 114–116). Although polyubiquitylation is a well-known mechanism for targeting proteins for degradation in the proteasome, the addition of a single ubiquitin monomer is a posttranslational modiﬁcation that recruits and activates various repair pathways. PCNA is a homotrimer that is central to all forms of DNA replication and serves as a sliding platform that recruits polymerases and other factors. These interactions are competitive and allow PCNA to serve as a central organizing factor, or trafﬁc cop, for DNA repair and replication (117). In eukaryotes, the monoubiquitylation of PCNA at residue 164 is mediated primarily by the Rad6-Rad18 ubiquitin ligase pathway, and the levels of monoubiquitylated PCNA are adjusted by deubiquitylation, primarily by USP1 (118). During normal DNA replication, if replication forks stall because of bulky lesions, PCNA becomes monoubiquitylated so that it can recruit translesional enzymes that bypass those lesions. In addition, PCNA plays a critical role in the resynthesis of the excised strand in MMR and seems to be involved at some level in the recognition of MMR enzymes; it physically interacts with MSH6, MSH3, MLH1, and EXO1 as well as with the high-ﬁdelity and low-ﬁdelity polymerases (Supplemental Table). Very recently, a few reports addressed the role of PCNA in recruitment of error-prone polymerases by using modiﬁed DT40 chicken B cell lines that have been rendered defective in the Rad6-Rad18 pathway or that express PCNA that cannot be ubiquitylated because of a lysine-to-arginine mutation at 164 (86– 88). Although DT40 cells do not seem to target A:T bases for mutation, their inability to monoubiquitylate PCNA is associated with a decrease in SHM (87, 88). These studies have now been extended to mice, in which the expression of PCNA with a lysine-to-arginine www.annualreviews.org • Somatic Hypermutation

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mutation at 164 has resulted in a signiﬁcant loss of mutations at A:T bases, which is comparable to that seen in mice that are genetically defective in Pol η (89; S. Roa & M.D. Scharff, unpublished data). Although many details of this modiﬁcation in mammalian B cells must still be worked out, the recruitment of monoubiquitylated PCNA to V region repair provides a possible mechanism for how error-prone polymerases can be recruited to Ig V regions (see Figure 5). This may be another example of the hijacking of a normal process by the centroblast B cell to generate SHM. Nevertheless, it remains unclear how PCNA, or any other protein, could distinguish AID-induced mutations from mutations that arise during normal DNA replication and repair. This is discussed below in the context of the targeting of SHM.

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TARGETING OF SHM Historically, most of the studies on the targeting of SHM have been conceptually organized around how AID itself is targeted to the V regions of Ig genes in centroblast B cells. However, because more than half of the mutations are dependent on error-prone BER and MMR, it is also important to determine whether the G:U created by AID is sufﬁcient to recruit those processes and mediate their switch from error-free to error-prone repair or if other factors are involved. The targeting of SHM probably occurs at multiple levels and in multiple layers. The critical and recurring question now is how the mutagenic properties of AID combine with error-prone BER and MMR to target parts of the Ig V region for very high rates of mutation, whereas some other genes accumulate fewer mutations, and C regions and most of the genome are spared or protected.

Expression of AID As AID is the initiating factor in SHM, its expression is, not surprisingly, an important regulatory step. In mice and humans, AID is 494

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highly expressed in centroblast B cells in the GC microenvironment (9). This ensures that high levels of SHM of Ig V regions will occur during a brief stage of B cell development and will then be turned off in the memory and plasma cell stages of B cell differentiation. However, AID expression and lower rates of SHM have been observed in T-independent responses and in immature B cells (119, 120), and AID protein has been detected in vivo in extrafollicular B cells (121). Recent studies with AID indicator mice (122) conﬁrm the original observations that AID is primarily restricted to the GC stage of B cell development. When AID is artiﬁcially expressed in non-GC B cells (such as hybridomas) or in non-B cells (such as CHO cells), the mutation rate is at least tenfold lower (123, 124). Surprisingly, mice engineered to constitutively express AID in all cells succumb to T cell but not B cell malignancies (125). These observations suggest that other factors must synergize with AID to produce effective SHM in the B cell, and there must be protective measures in place in B cells to prevent their malignant transformation. At a cellular level, most of the AID protein in centroblast B cells is cytoplasmic, and it shuttles back and forth from the cytoplasm to the nucleus, with only 10%–15% located in the nucleus (68, 126, 127). Although there are both nuclear localization and nuclear export motifs in AID (Figure 4), the subcellular localization of AID is likely regulated by posttranslational modiﬁcations such as phosphorylation (see above) and associated proteins. Once modiﬁed AID and its presumed associated proteins have entered the nucleus, they must be recruited to the Ig V regions and largely restricted from acting on other sites. In normal individuals several genes outside of the Ig locus, including Bcl6, CD79, and CD95, undergo SHM, albeit at much lower levels than in the Ig V region (128–130). In malignant B cells, several other loci, including Bcl6, Pim1, Myc, RhoH/TTF, and Pax5, can undergo aberrant SHM (131, 132). AID will even mutate many different types of reporter genes if they are highly expressed in cultured

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B and non-B cells and in vivo (124, 125, 133). Yet not all highly transcribed genes accumulate AID-induced mutations in centroblast B cells. These ﬁndings and the AID-inactivating mutations in patients (Figure 4) indicate that there must be other cis or trans factors that are responsible for the accumulation of mutations in the Ig V region and for the lower rates of mutation in genes like Bcl6 and c-myc and in reporter genes that are distributed throughout the genome (134, 135). The importance of these other putative factors is also consistent with the surprising ﬁnding that when naive splenic B cells are stimulated in shortterm culture to express high levels of AID and undergo high rates of CSR, the V regions of those same activated B cells are not mutated (136). Another surprising ﬁnding that suggested other cellular factors is that AID conditionally overexpressed in B cells leads to fewer mutations, indicating mechanisms that speciﬁcally negatively regulate AID (137). The many detailed studies addressing the mechanism of AID targeting in SHM are the subject of recent reviews (5, 50, 138, 139). Several explanations have been explored, including (a) that the V region is made more accessible to AID than is the C region or other genes in centroblast B cells; (b) that there are cis-acting sequences in or around the V region, and perhaps a few other genes, that either recruit DNA-binding proteins which in turn recruit AID or form macromolecular DNA structures that serve as a nidus for the recruitment of AID; (c) that there are proteins that associate with AID especially in B cells that both target it to the V region and perhaps restrict it from acting on most other parts of the genome; and (d ) that AID-induced mutations and/or error-prone repair occur only during the nonreplicative stages of the cell cycle, temporally segregating SHM from replication and error-free repair, and there are subnuclear domains in which the targeted Ig genes and SHM enzymes are compartmentalized. Importantly, many of these mechanisms could also be used to regulate or target errorprone BER and MMR. We offer a brief synop-

sis of evidence supporting each of these models; however, these hypotheses are not mutually exclusive.

Accessibility Regulation of transcription, repair, and replication of particular genes is often associated with epigenetic changes such as changes in DNA methylation and modiﬁcations of histones. These changes regulate the accessibility of those genes to the protein complexes responsible for these DNA transactions. Investigators have surmised that a similar paradigm applies to SHM: The selective targeting of AID and error-prone BER and MMR could be facilitated by increased accessibility of the V region in SHM (and of the switch regions in CSR). Recent studies in transgenic mice have suggested that DNA demethylation of cytosines early in B cell development may play a role in targeting the active kappa light chain allele for SHM (140). These studies showed that SHM occurs with a tenfold preference on the unmethylated allele compared with an identical methylated allele of the Igκ gene. Earlier studies in the IgH gene and the Igλ gene found no difference between V regions versus C regions with respect to DNA methylation (141), but those studies did not address speciﬁc alleles. How DNA methylation can affect targeting of AID is unclear, as both methylated and demethylated alleles have similar rates of transcription in vivo, and the presence of a methylated CpG does not have a negative effect on AID deamination activity on nearby cytosine residues (142). As described in the review on CSR by Stavnezer et al. in this volume (14), a number of studies in primary B cells have shown that the histones associated with AID-targeted switch regions that undergo CSR are hyperacetylated compared with switch regions that do not undergo CSR (53, 143, 144). As occurs in the switch regions, the acetylation of the H3 and H4 histones associated with the H chain V region increases when compared with www.annualreviews.org • Somatic Hypermutation

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Chromatin modifications: posttranslational modiﬁcations of histone residues, such as acetylation, that can change the local accessibility of genes to various protein complexes cis-acting elements: sequences that recruit protein factors in a sequence-speciﬁc manner or that form higher-order structures (e.g., stem-loops) that recruit protein factors

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the C region in BL2 cells (101). When BL2 cells were treated with trichostatin A (TSA), a drug that globally inhibits histone deacetylases, mutations were observed to accumulate in the C region and within the ﬁrst 100 bases downstream from the transcription start site. Because mutations are not usually observed in these two regions of the Ig gene (Figure 1), this effect of TSA suggests that changes in histone acetylation can affect the targeting of AID. The global hyperacetylation effects of TSA, however, make it difﬁcult to demonstrate that histone modiﬁcations are required for proper targeting of SHM. Hyperacetylation of H3 and H4 histones in the H chain V region compared with the C region has also been observed in primary B cells in vivo (141). These differences in chromatin modiﬁcations preceded the expression of AID, suggesting that if they play a role in the targeting of SHM, they may be part of a broader program of regulation that prepares the V region so it can be targeted by AID. In this study, there was no difference in histone acetylation between the V and C region of the λ light chains from these same mice, but this particular C region is only 1.6 kb away from the promoter and still within the domain that can be targeted for SHM. Because histone H3 and H4 hyperacetylation are also marks of transcribed genes, it is difﬁcult to know whether this chromatin modiﬁcation pattern is a reﬂection of transcription per se or important for SHM targeting in this context. In the same study, phosphorylation of histone H2B-Ser14 (H2B-pSer14) was associated with V regions of both L chain and H chain genes, but not with C regions. On the one hand, this chromatin modiﬁcation pattern was dependent on AID, as AID-deﬁcient mice did not exhibit these changes, which suggests that H2B-pSer14 is unlikely to be responsible for the initial recruitment of AID. On the other hand, this chromatin modiﬁcation could play a role in the recruitment of error-prone BER or MMR, a possibility that remains to be explored. Thus, there is circumstantial but not deﬁnitive evidence that speciﬁc epigenetic Peled et al.

changes in V regions translate into increased accessibility to SHM enzymes and play a role in targeting SHM. In addition, it is likely that a combination of chromatin modiﬁcations work in parallel to target the action of SHM.

Cis-Acting Sequences Analogous to transcription, in which particular DNA motifs recruit speciﬁc transcription factors, cis-acting sequences may recruit AID, BER, and MMR to the Ig V regions in centroblast B cells. The Ig genes are rich in promoters and enhancers that have been well studied over the years for their roles in transcription and activation of the locus (Figure 1). Because deletion or mutation of these elements often compromises transcription rates, it has been difﬁcult to distinguish between the loss of speciﬁc SHM targeting that leads to decreases in mutation levels and loss of SHM owing to decreases in transcription. Nevertheless, many studies of the endogenous Ig locus or of ectopically located transgenes in cell lines and mice have attempted to identify cis-acting sequences that might recruit SHM enzymes to the Ig V region (reviewed in 138, 145). Although the results of these studies are conﬂicting, likely owing to variability within transgenic mice and within or between B cell lines, in general the studies of L chain genes reveal that the SHM machinery could be activated at the correct time during B cell differentiation and could be recruited to almost any ectopically located reporter gene, as long as it is ﬂanked by a strong transcriptional promoter and carries both the intronic and 3 L chain enhancers. These observations suggest that the V region coding exon itself does not contain sequences that are necessary for the targeting of AID but that important motifs must reside in the promoter and/or enhancer regions. However, any strong promoter seems able to target SHM to the V region and, at least in cell lines, even viral enhancers are sufﬁcient (146, 147). Recent studies in the chicken B cell line, DT40, in which it is easier to examine different

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modiﬁcations of promoters or enhancers, have suggested that the situation may be more complicated. In DT40 B cells, the human EF1-α promoter, which induces as much transcription as the beta-actin or the endogenous L chain promoters, did not support high levels of SHM in the endogenous L chain (148). With respect to the H chain gene, a variety of promoters were able to target SHM to the V region or to other genes that were inserted in place of the V region. Studies in which the core Eμ intronic H chain enhancer was deleted resulted in mice that had a small but not signiﬁcant effect on SHM, suggesting that it does not play an important role in the targeting of SHM (149). Similarly, deletion of various known parts of the endogenous 3 regulatory regions of the H chain did not affect SHM in mice (150), but the roles of the recently described elements hs5, 6, and 7 have yet to be determined (151). However, some recent studies with cell lines suggest that the situation may be more complicated. To circumvent the problem of maintaining high levels of transcription in the absence of the H chain enhancer, we have used Sp6 hybridoma cell lines in which the endogenous intronic enhancer is in fact deleted but the H chain continues to be stably transcribed at high levels through variegated expression (152). In addition, the 3 regulatory region is insulated by the insertion of a gpt gene, which allowed us to examine whether the intronic enhancer is required for SHM without any compensating assistance from the 3 regulatory region. Because the Sp6 hybridoma does not express AID, we stably transfected the cells with AID. These studies revealed that the intronic core Eμ enhancer was required for SHM of the endogenous H chain V region if the matrix attachment regions (MARs) were still present but that if both the MARs and core Eμ were deleted, then SHM was restored to wild-type levels. We concluded that at least in this hybridoma system, cis-acting sequences associated with these transcriptional regulatory regions were affecting the targeting of SHM, even if they were no longer required to con-

trol transcription. In addition, these results suggested to us that the MARs and core Eμ elements can singly or in combination impose positive and negative regulation of SHM. This general idea is supported by studies in which the intronic enhancer has been manipulated in other ways (153). For example, in Ramos cells, when the core Eμ enhancer is deleted in an ectopic H chain gene, there is a twofold reduction in SHM. If part of the 3 regulatory region is added, mutations in the V region of the transgene occur primarily in A:T bases rather than G:C bases. In the same cells, the mutations in the endogenous H chain V region continue to be primarily in G:C. This suggests that cis-acting sequences could also regulate the recruitment or activity of error-prone BER or MMR because the spectrum of mutations is altered in a cis-acting manner. In vivo, the presence of trans factor– binding sites, such as E-box motifs for E2A, PU.1, and NF-EM5, alters the rate of SHM in H and/or L chain V regions (52, 154). Because these motifs are not found in all H and L chain genes and are present in many nonmutating genes, they are unlikely to be the sole regulators of targeting of SHM. In addition, these trans factors alter the rate of transcription in Ig genes, so it is difﬁcult to know if the changes in SHM are primary or secondary to changes in rates of transcription. However, these binding sites and presumably other cis-acting sequences and DNA-binding proteins are likely to play an as yet to be deﬁned role in the targeting process. Another type of cis-acting sequence that might recruit the SHM enzymes to Ig regions undergoing high rates of mutation is ssDNA structures. For example, G-loops have been observed in transcribed switch regions, and they might offer AID a stable ssDNA target in the nontranscribed strand (155), whereas collapsed R-loops may provide the same in the transcribed strand (156). To probe for ssDNA associated with mutating V regions, we have crosslinked the nucleic acids and proteins in intact B cell nuclei and used sodium bisulﬁte www.annualreviews.org • Somatic Hypermutation

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to identify regions of ssDNA. We found more patches of ssDNA in crosslinked chromatin in genes that are undergoing SHM than in highly transcribed genes that are not targeted by AID (57). In addition, a number of investigators have used computer programs to identify potential stem-loop structures in the V and switch regions that may stabilize ssDNA in regions that are frequently mutated (157, 158).

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Trans-Acting Protein (Co-)Factors As discussed above in the AID section, the location of some of the inactivating mutations in AID (Figure 4) and the importance of its phosphorylation sites suggest that there are interacting proteins that could inﬂuence AID targeting to either the V or switch regions (64, 65). One of the ﬁrst AID cofactors identiﬁed was RNA Pol II (53). Additionally, the 5 boundaries of SHM may reﬂect the transition between the initiating and elongating forms of RNA Pol II, with AID activity or binding requiring the elongating form (50). If true, this requirement for elongation might explain the relatively protected 100–200 bp from the transcription start site. Similarly, stochastic dissociation of AID from RNA Pol II has been invoked to explain the exponential decay of SHM after the ﬁrst 1.5–2 kb (15, 19). Direct evidence for this model is lacking, and it does not explain how other highly transcribed genes, which also use RNA Pol II, would be protected from AID. Other identiﬁed protein cofactors include RPA; MDM2, which is a ubiquitin ligase 3 that modiﬁes P53; protein kinase A (PKA), which presumably phosphorylates serine 38 and/or perhaps other residues in AID; and DNA PKcs. All these cofactors have been reported to associate with AID (70, 71, 159, 160) (Supplemental Table). Considering that these proteins are ubiquitously expressed and perform general cellular functions, it seems unlikely that they are responsible for mediating the preference of AID for the Ig genes or for speciﬁc targeting to V and switch regions.

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Cell Cycle and Subnuclear Restriction In the discussion of BER and MMR, we emphasized the important role of error-prone repair in SHM and CSR, whereas error-free repair is an essential characteristic of DNA replication during the S phase of the cell cycle. The recent crystal structures of human Msh2-Msh6 bound to various types of mismatches (106) suggest that the conformation of the heterodimer is identical, whether it is bound to a typical postreplicative G:T mismatch, the G:U mismatch that is the result of AID deamination, or even bulkier adducts caused by genotoxic agents. Therefore, the nature of the DNA lesion itself is unlikely to explain the recruitment of error-prone repair to AID-induced lesions. By restricting SHM and CSR to the nonreplicative phases of the cell cycle, the centroblast B cell could potentially separate the error-free repair needed during DNA replication from the error-prone repair of antibody V and switch regions that have undergone AID-induced mutations. This seemingly straightforward mechanism might work because error-free repair is an essential characteristic of DNA replication during S phase. Only a few studies have been done that are relevant to this possibility. In BL2 cells in which SHM was induced by incubation with antiIgM, anti-CD19, and anti-CD21, V region mutations were detected during G1 and G2 but not during S phase (161). Similarly, during CSR the colocalization of the IgH gene with proteins involved in nonhomologous end joining (NHEJ) was observed in G1 (136). Immunohistochemical studies on the subcellular location of AID also suggest that there may be differences in localization at various stages of the cell cycle (121, 162). Clearly, a great deal is unknown in this area, including when during the cell cycle AID is itself expressed and has access to the Ig genes. Two other ways that SHM may be targeted and restricted are through subnuclear localization of those genes that will undergo

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SHM and through the restriction of AID and the various other enzymes that are involved to that region of the nucleus. A number of studies have reported changes in the subnuclear localization of the H chain gene around the time of V(D)J rearrangement (163), as well as colocalization of the locus with proteins involved in NHEJ during isotype switching (164). However, neither colocalization of the Ig genes with AID nor the proteins involved in BER and MMR has been reported. Particularly intriguing is the recent ﬁnding that the IgH and IgL genes colocalize with c-myc in the same “transcription factories” in primary resting B cells (165), but it remains to be determined if this colocalization is also present during SHM or CSR and whether AID and its associated cofactors are in such transcription factories.

CONCLUSIONS Although a great deal of genetic and biochemical evidence supports the model of SHM depicted in Figure 2, the discussions of BER and MMR in the preceding sections reveal many unresolved issues and raise a number of provocative questions. Even if one focuses on just V region mutation, it is unclear how the B cell manages the ordered recruitment of the many different components of each system to carry out the SHM of antibody V regions (110, 166). Although AID may initiate this process by converting dC to dU, we know that a whole program of gene expression changes occurs as B cells enter the GC and become centroblasts (167, 168). These changes include the transcriptional activation of AID, the increased expression of enzymes involved in MMR and BER, changes in the chromatin to make the Ig V regions accessible to AID (101, 141), and suppression of the DNA damage response (169, 170). Although AID may deaminate dC on both strands at about the same frequency, a strand bias for mutations in A:T bases has been recognized (26), which suggests that the targeting of AID and of the repair processes that it recruits may be differ-

ent. A very recent paper suggests that errorprone MMR preferentially targets dU in the nontranscribed strand (54). Dividing SHM into two phases has been convenient, but whether they are really distinct is unclear. We have assumed that the presence of dU is responsible for recruiting BER, whereas the G:U and probably the G:abasic sites create mismatched bases that recruit MMR (1, 3–5, 10). However, the AID protein, the molecules that are associated with it, and/or some as yet unidentiﬁed protein or transcription complex may also play a role in recruiting and managing the competition between BER and MMR. For example, a dominant-negative mutant of MSH6 affects the targeting of AID to particular hot-spots, suggesting a connection between the ﬁrst and second phase (102). This may merely reﬂect that the accumulation of 10–15 mutations, which is often seen in a single V region, requires multiple cycles of mutation and repair and that mutations introduced by BER and MMR introduce new mismatches that must be repaired. But phase 1 and phase 2 may also be connected by an organizing molecule such as PCNA, or, perhaps, B cell–speciﬁc complexes or mutasomes contain both phase 1 and phase 2 enzymes and associated proteins (171), which coordinately regulate SHM. Even more perplexing is how a high rate of mutation can be selectively targeted to small regions of the antibody genes, whereas other regions of the Ig genes and non-Ig genes that are highly expressed in centroblast B cells do not undergo such high rates of mutation. Although the number of mutations that accumulate in antibody V regions is clearly much higher than in other genes in AID-expressing B cells, mutations have been observed in other genes. Many of the genes that appear to be mistargeted by AID are proto-oncogenes, and the mutations in them are observed in B cell tumors (131). However, high mutation rates have been reported in normal primary cells in genes that do not contribute to malignancies (128). In addition, reporter genes that are integrated throughout the genome also www.annualreviews.org • Somatic Hypermutation

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accumulate mutations at frequencies that are lower than the V region but are still very high when compared with the genome-wide mutation rate (134, 135). This suggests that AID-induced mutations are not as restricted as previously believed and raises the question of how much DNA damage is actually being produced throughout the genome in B cells undergoing SHM and CSR. There is accumulating evidence that the amount of damage is substantial because centroblast B cells appear to have developed mechanisms to ignore or deal with such damage (137, 169). There is already evidence that many of the mutations in the V region are repaired and not scored as mutagenic events (161). This raises the possibility that AID is causing mutations in many genes but that the mutations in non-Ig genes are effectively repaired in an error-free way. A third and related question is how the centroblast B cell organizes the mutation of the antibody V regions so that the BER and MMR of the V region is error prone, whereas the repair of other genes is error free. Approximately 70–250 dUs are normally introduced during each cycle of DNA replication, and all or most of these are repaired by BER, MMR, and other mechanisms with high ﬁdelity (172). How are these postreplicative lesions distinguished from the AID-induced lesions, and, if AID is not restricted to the antibody genes, how are the AID-induced lesions

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in non-Ig genes distinguished from those in the V regions, or are they? These are important issues because the B cell needs to create genomic instability in the V region while still maintaining sufﬁcient stability in the rest of its genome so that it will survive to produce clonal progeny that can be positively selected for high-afﬁnity antibodies. These afﬁnitymatured B cells must then either differentiate into plasma cells that secrete useful antibodies or into memory B cells that can meet future challenges against the same antigen. Notably, the loss of control of this process may be responsible for the many B cell lymphomas that arise from GC B cells. To address these issues, it will be necessary to identify the proteins that interact with AID and contribute to its differential targeting to Ig genes and to the V region and different switch regions, to learn more about when in the cell cycle SHM is occurring, and to identify mutation and repair complexes that participate in error-prone and error-free repair. We will also have to gain a better understanding of how the levels of these SHMparticipating proteins and their high-ﬁdelity, error-free competitors are regulated in centroblast B cells and identify the signal transduction pathways that are responsible for controlling these events. Such studies not only will reveal how SHM is regulated and targeted but also will lead to new insights into the basic mechanisms of mutation and repair.

SUMMARY POINTS 1. Afﬁnity maturation of the humoral response occurs through diversiﬁcation of Ig genes by AID-induced somatic hypermutation (SHM) of the Ig V regions, followed by selection of high-afﬁnity B cells. 2. AID initiates SHM by deaminating cytidine residues in ssDNA during transcription of the Ig locus. 3. AID is recruited to speciﬁc regions by a variety of targeting mechanisms that may include increased accessibility, cis-acting elements, proteins that associate with AID, and regions of stabilized ssDNA.

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4. The resulting uridine is either replicated over or processed by a complex series of enzymes, each of which paradoxically functions outside the Ig locus in high-ﬁdelity DNA repair and maintains genome stability.

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5. Mismatch repair, base excision repair, and DNA polymerases are diverted from their typical cellular roles of preserving genomic integrity to process uridines and surrounding sequences in an error-prone fashion, which leads to signiﬁcant diversiﬁcation of the Ig locus. 6. The regulation of this process occurs at many levels that likely include posttranslational modiﬁcation of AID and PCNA, downregulation of components of high-ﬁdelity repair, subcellular localization and trafﬁcking of AID, chromatin changes in the targeted loci, differences in protein interactions and complex formation at different phases of the cell cycle, and global changes that occur in GC B cells in transcription programs and the DNA damage response.

FUTURE ISSUES 1. How is AID targeted to speciﬁc genomic loci? 2. How are mismatch repair and base excision repair induced to recruit error-prone repair to sites of AID action while performing high-ﬁdelity repair elsewhere in the genome? 3. What is the crystal structure of AID and what is its mechanism of deamination and targeting? 4. Are the chromatin modiﬁcations that are observed in the areas of AID activity required for AID targeting? What other chromatin modiﬁcations are involved? 5. What are the cis-acting elements that recruit AID and the error-prone mutasome? 6. What are the other components of the error-prone mutasome, which protein-protein interactions are critical for its regulation, and which are the proteins that interact with AID? 7. How do posttranslational modiﬁcations of AID regulate its activity and targeting, and what role, if any, do alternative isoforms of AID play in SHM? 8. Are there AID-induced mutations within or outside of the Ig locus that are repaired in an error-free fashion, and if so how does the centroblast B cell target error-free or error-prone repair to the sites of AID activity?

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We thank Phuong Pham and Janet Stavnezer for thoughtful comments on the manuscript. F.L.K. and J.U.P. are supported by the Medical Scientist Training Program T32 GM 007288 www.annualreviews.org • Somatic Hypermutation

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at Albert Einstein College of Medicine. S.R. is supported by a fellowship from the Spanish Ministry of Education and Science EX-2006-0732. S.L.K. is supported by the Immunology and Immuno-oncology Training Program T32 CA 09173 at Albert Einstein College of Medicine. M.F.G. acknowledges funding from NIH ES013192 and NIH R37GM21422. M.D.S. is supported by RO1CA72649 and R01CA102705.

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RELATED RESOURCES Stavnezer, J, Guikema JEJ, Schrader CE, 2008. Mechanism and regulation of class switch recombination. Annu. Rev. Immunol. 26:261–92 Database of HIGM Type II causing mutations: http://bioinf.uta.fi/AICDAbase

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Annual Review of Immunology

Annu. Rev. Immunol. 2008.26:481-511. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:481-511. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Contents

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al.

Supplemental Table Interactions that occur amongst the proteins involved in somatic hypermutation

Protein

Select Functions

Effect in SHM

AID

Cytidine deaminase: deaminates dC into dU on ssDNA (1, 2).

Abolished, both in patients with mutations in AID (3) and in knock-out mice (4).

PKA

RPA

MDM2

Phosphorylates AID at serine 38 and tyrosine 184 (8) -DNA replication, recombination and repair. -ssDNA binding protein. -NER: hRPA binds to the undamaged strand and directs incisions to the damaged strand. DNA damage: is a ring finger E3 ubiquitin ligase that promotes ubiquitination and proteasomal degradation of p53.

Select protein–protein interactions -RNA pol II: by co-IP (5). -RPA (32 kDa subunit): by cloning a complementing factor required for AID activity in vitro (6). -MDM2: through C-terminus of AID (7). -PKA: by coIP and co-purification (8). -DNA-PKcs: (through C-terminal AID domain): by IP followed by MALDITOF (9).

AIDS38A has diminished SHM activity on artificial and physiological DNA targets (10)

-AID: by coIP and co-purification (8) in the cytoplasm of B cells (11).

AID–RPA complexes may preferentially bind to ssDNA of small transcription bubbles at SHM hotspots (6).

-Phosphorylated AID: (8) unphosphorylated AID from 293 cells does not interact with RPA (6). -PCNA: by affinity chromatography and mass spectrometry (12). -hUNG2: interacts with RPA2 (34KDa subunit) by two hybrid (13). -XPG: by gel retardation assays (14). -ATR: ATR and RPA colocalize to nuclear foci after DNA damage and RPA is required for the recruitment of ATR to sites of DNA damage (15).

_

-AID: through C-terminus of AID (7). -MRE11, RAD50 and NBS1: by co-IP in HeLa cell lysates (16).

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al.

MSH2

MSH3

MMR: -Recognition of point mutations or short mismatches (in complex with MSH6) and large mismatches, insertions or deletions (with MSH3). -Scaffolding: recruits accessory proteins involved in processing of the mismatch. MMR: mediates repair of large mismatches, insertions and deletions (in complex with MSH2). MMR: recognition of point mutations and short mismatches (in complex with MSH2).

MSH6 In complex with MSH2 physically recognizes dU:dG mismatches (36). PMS2

MMR

In Msh2 -/- mice: *5-fold reduction in mutation frequency. *Reduced mutations at A:T. *Increase in hotspot targeting. (17-19)

-MSH3: by co-IP with human proteins (20). Contact regions have been mapped (21). -MSH6: by co-IP with human proteins (20). Contact regions have been mapped (21). -MLH1 and PMS1: Msh2-Msh6 forms a ternary complex with MLH1-PMS1 on DNA substrate containing a mismatch. (22). -EXO1: by GST fusion pull-down (23). -PMS2: interacts with MSH2-MSH6 (24). -Controversial interaction with PCNA: not detected by co-IP but found to interact in yeast two-hybrid assay (25). PCNA binds to MSH2 when MSH2 is in a heterodimer with MSH6 (26) (27) or with MSH3 (28). -RAD50: in a complex (29). -POL η: in cell extracts (30). -EXO I: by GST pull-down analysis (23). -ATR: by co-IP in HeLa and sf9 cells (31).

In Msh3 -/-: No effect (32, 33) or slight decrease in mutations at G/C basepairs and slight decrease in mutations at A:T bases in JH2-JH4 regions (34)

-MSH2: by co-IP with human proteins (20). Contact regions have been mapped (21). -PCNA: by GST pull-down (27). -EXO I: by GST/IVTT bait-prey interaction assay (35).

In Msh6 -/- mice: *Decrease in mutation frequency. *Reduced mutations at A/T. *Increase in Ts mutations at G/C. *Increase in hotspot targeting. (32-34)

-MSH2: by co-IP with human proteins (20). Contact regions have been mapped (21). -MLH1: by co-IP (29). -PCNA by GST pull-down (27). -EXO I: weak interaction by GST/IVTT bait-prey interaction assay (35). -ATR: by co-IP in sf9 cells (31).

In Pms2 -/- mice: *2-4 fold reduction in mutation frequency (37-39) *6-22-fold reduction in mutation frequency (40). *High frequency of dinucleotide mutations (39). *Slight increase in mutations at G:C (37).

-MSH2 in complex with MSH6: (24). -MLH1: by coIP (41) (42) (24). -PCNA: by co-IP (43). -EXO I: weak interaction by GST/IVTT bait-prey interaction system (35).

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al.

MLH1

MLH3

MMR

In Mlh1 -/- mice: normal to ∼2-fold reduction in frequency of mutation and altered patterns of mutation in a nonselectable Ig-κ gene with an artificial insert in the V region with a preference for targeting G and C nucleotides (37).

-MSH2: in the presence of DNA (homoduplex or heteroduplex), Mg2+ and ATP (24, 43). -MLH3: by yeast two-hybrid assay (44). -MSH6: by co-IP (29). -PMS2: by co-IP (41-43). -PCNA: MLH1 interacts with PCNA (26). Human MLH1 and PCNA coimmunoprecipitate (43). Yeast PCNA interacts with yeast MLH1 in a two hybrid system (25). -MRN complex: by IP anti RAD50 and mass spectrometry (29). -EXO I: by GST/IVTT bait-prey interaction system (35).

MMR

In Mlh3 -/-: Slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged JH4 gene (45) or increase in the frequency of mutations in the intronic DNA downstream of the rearranged JH4 gene (46).

-MLH1: by yeast two-hybrid assay (44).

Since PCNA -/- is embryonic lethal PCNA K164R (that prevents ubiquitination, but does not interfere with its function in replication) has been used to study the effect of PCNA ubiquitylation in SHM: *PCNA K164R knock-in in DT40 cells shown reduction frequency of mutations at the lightchain VJ segment. Although all types of mutations are reduced the most pronounced decreases seen are C-to-G and G-to-C Tv (48). *PCNA K164R knock-in mice: altered mutation spectrum of at variable Ig region (JH4 intronic region), strong reduction of mutations at A/T basepairs associated with a compensatory increase at G/C (49). *Transgenic mice expressing a PCNA-/- + Tg K164R: decrease in mutations per sequence and at A/T basepairs in the VH 186.2 region (S. Roa et al. unpublished results).

-RPA: by affinity chromatography and mass spectrometry (12) -Controversial interaction with MSH2: not detected by co-IP but found to interact in yeast two-hybrid assay (25). PCNA binds to MSH2 when MSH2 is in a heterodimer with MSH6 (26) (27) or with MSH3 (28). - MSH3 and MSH6: from human and yeast, by GST pull-down (27). -MSH2-MSH6 heterodimer: by glycerol gradient (26) and by GST pull-down (27). -MSH2-MSH3 heterodimer (28). -PMS2: by co-IP (43). -MLH1: interaction between human proteins by co-IP (43) and yeast proteins by two-hybrid assay (25). -RAD18: by two-hybrid (50). -RFC: in complex with CHL12 (12). -hUNG2: physical and fuctional interaction (51, 52). -APE1: by co-IP (53). -POL μ: detected both with yeast and human proteins, in vitro (by gel filtration) and in vivo by yeast two-hybrid analysis (54). -POL η: physical and fuctional interaction and mutations in the PCNA binding motif of hPol η inactivate this interaction (55). -POL ι: physical interaction (56). -REV1: binds PCNA (through the BRCT domain located near the N terminus of REV1) and monoubiquitylation of PCNA enhances this interaction (57). -XPG: interaction between human proteins by co-IP (58).

-MMR: DNA replication and DNA re-synthesis. -Long-patch BER.

PCNA

Monoubiquitination of PCNA at Lys 164 triggers switching from a damage sensitive, high fidelity DNA polymerase (δ, ε) to one of the damage tolerant, low fidelity TLS polymerases (47). High fidelity replication over U favors Ts (C to T and G to A), whereas low fidelity replication over abasic sites favors Tv (C to G/A or G to C/T).

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al.

RAD18

RFC

Ubiquitination of PCNA

Long-patch BER: loads PCNA onto DNA

Inactivation of Rad18 in DT-40 cell line: *In a hypermutation reporter construct: Impaired SHM (59). *In the endogenous Ig locus: decrease in the mutation frequency and decrease in all types of mutations at dG/dC residues, as well as an increase in deletions (59) _

Transcription factors that regulates immunoglobulin gene expression.

Base substitutions in the 3’ enhancer in the PU.1 binding motif cause reduction in the mutation rate and in hotspot mutations in an Igκ transgene (60).

BER: Removal of uracil bases by deglycosylation: hydrolizes the Nglycosidic bond between the nucleotide base and the deoxyribose phosphate creating an AP site.

-In Ung -/- mice: mutations at G/C are shifted toward Ts (95%), indicating inhibition in the generation of abasic sites (that introduce Tv at C/G) and the pattern of mutations at A/T remains unaffected (61). -In patients: recessive mutations in ung (hyper-IgM syndrome type 5) cause a partial disturbance in the SHM pattern in the a VH3-23 region of IgM+ B (CD19+) memory (CD27+) cell populations, such that mutations at G/C baispairs are biased toward Ts, whereas at A/T residues the ratio of Ts/Tv remains unaffected (62).

APE (1 and 2 )

Short-patch BER: excises AP sites at 5’, generating a ssDNA break.

Not determined in SHM. Involved in CSR (Stavnezer et al, this volume)

MRE11/ RAD50/ NBS1 (MRN) complex

Long-patch BER: DNA break repair.

PU1

UNG

Overexpression of NBS1 (nibrin) in a hypermutating B cell line increases mutation (65)

-PCNA: physical interaction by two-hybrid (50).

-PCNA: in complex with CHL12 (12).

-RPA2 (34KDa subunit): by two-hybrid (13). -PCNA: physical and fuctional interaction (51, 52).

-PCNA: by co-IP (53). -POL β: by two-hybrid assay (63). Fail to detect this interaction using affinity chromatography (64) might indicate that this interaction occurs in the context of DNA. -MSH2: interacts with RAD50 in a complex (29). -MLH1: by IP antiRAD50 and mass spectrometry (29). -MDM2 interacts with MRE11, RAD50 and NBS1: by co-IP in HeLa cell lysates (16).

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al.

POL β (beta, B)

POL η (eta, H)

REV1

POL ι (iota, I):

Short-patch BER: processes ssDNA breaks with high fidelity.

TLS

TLS: -Possible role in shortpatch BER

TLS:

-Correlation between pol β expression and SHM in various hypermutating BL2 cell line subclones: pol β expression is downregulated in SHM-proficient clones and upregulated in SHM-deficient subclones (66) -No direct effect in SHM (67). -Pol η is downregulated in mutating human B cells (68). -Pol η -/- mice show an 80% reduction of mutations at A/T base pairs and increase of mutations at G/C base pairs in the JH4 intron (69, 70) (71) -In patients with xeroderma pigmentosum variant disease (with a defect in pol η) the frequency of SHM is normal but mutations at A/T basepairs is decreased (72) in WA (A/T, A) motifs (73). -In Rev1 -/- mice: C to G Tv are absent (and A to T, C to A, and T to C increased) in the nontranscribed (coding) strand and reduced in the transcribed strand. In addition, loss of REV1 causes compensatory increase in mutagenesis by other TLS polymerases (77). -In REV1-deficient DT40: reduced level of immunoglobulin gene mutation, indicating a defect in translesion bypass (78). Controversial: -When the Pol ι gene is knocked out in BL2 cell line: no detectable increase in mutation frequency after induction for hypermutation (80). -Mice from the 129 strain (which have an inactivating nonsense mutation in exon 2 that abrogates production of POL ι) have normal SHM (81).

-APE1: by two-hybrid assay (63). Fail to detect this interaction using affinity chromatography (64) might indicate that this interaction occurs in the context of DNA.

-MSH2: in cell extracts (30). -PCNA: physical and fuctional (55). -REV1: (through C-terminal region of Rev1): by yeast two-hybrid and co-IP in mouse (74) and human (75). Interaction between human proteins is strong (76) .

-PCNA: REV1 binds PCNA (through the BRCT domain located near the N terminus of REV1) and monoubiquitylation of PCNA enhances this interaction (57). -POL ζ: Interaction between hREV1 and hREV7 (subunit of DNA polymerase ζ) proteins by yeast two-hybrid, GST pull-down assays, and co-IP (79). -POL ι: (through C-terminal region of Rev1): by yeast two-hybrid system and co-IP in mouse (74). -POL η: (through C-terminal region of Rev1): by yeast two-hybrid co-IP using in mouse (74).

-PCNA: physical interaction (56). -REV1: (through C-terminal region of Rev1): by yeast two-hybrid system and co-IP in mouse (74). Interaction between human proteins is interaction (76).

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al.

POL θ (theta, Q)

TLS

POL ζ (zeta, Z):

TLS

POL μ (mu, M)

NHEJ. Is an error prone polymerase.

EXOI

MMR: exonuclease that creates a ssDNA break.

Somewhat controversial: -In mice carrying a gene disruption that completely eliminates the POLQ enzyme: 60-80% reduction in overall frequency of SHM, pattern of mutations moderately shifted towards more Ts at both A/T and C/G basepairs, but no overall change in the proportion of events at A/T and C/G positions (82). -In mice carrying an in-frame deletion of part of the DNA polymerase domain, so that a catalytically inactive POLQ protein is still produced: slight reduction in the overall mutation frequency, unaffected % of mutations at A/T, significant decrease in % of mutations at C/G particularly at hotspots and both Ts and Tv are similarly reduced at JH4 intronic sequences. Partial impairment in the production of high-affinity specific antibodies. (83). -POL ζ is upregulated in mutating human B cells (68). -Antisense RNA (in mice)/oligos (in cell line) to a portion of mouse REV3: *In mice: decreased frequency of mutation but no effect in pattern of mutations (84). *In the mutating CL-01 cell line: moderate reduction of both A:T and G:C mutations (68). Overexpression of hPOL μ in Ramos cells increases SHM in the IgVH4–34 gene, mutations occur preferably at C and G residues in hotspots rather than in A/T residues. More C G Tv (34%) than C T Ts (23%) (85)

ExoI -/- mice have: increase in Ts at G/C, reduced mutations at A/T and increase in hotspot targeting (86).

-REV1: Interaction between hREV1 and hREV7 (subunit of DNA polymerase ζ) proteins was detected by yeast two-hybrid assay, GST pull-down and co-IP (79). Interaction also reported between mouse proteins (74). Interaction between human proteins is strong (76).

-PCNA: detected both with yeast and human proteins, in vitro (by gel filtration) and in vivo by yeast two-hybrid analysis (54). Mutations in the PCNA binding motif of hPol μ inactivate this interaction (55). -MSH2: by yeast two-hybrid (87) and between human proteins (23). -hExo1 binds to hMLH1: by the GST/IVTT bait-prey interaction assay (35). -hExo1 binds to hMSH3: by the GST/IVTT bait-prey interaction assay (35). -hExo1 binds weakly to MSH6: by the GST/IVTT bait-prey interaction assay (35). -hExo1 binds weakly to PMS2: by the GST/IVTT bait-prey interaction assay (35). -hExoI forms an immunoprecipitable complex with hMLH1/hPMS2 in vivo (35).

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al.

DNA-PKcs

DSB repair by NHEJ.

XPG

NER endonuclease

ATR

-DNA damage signaling during Sphase (90). -DNA replication (S/M) checkpoint (91).

GANP/ MCM3AP

Inhibitor of DNA replication

BACH2

THO complex

B-cell-specific transcription repressor highly expressed before the plasma cell stage Co-transcriptional recruitment of export factors to package mRNA into an exportable mRNA ribonucleoprotein.

Small to no effect (88). xpg deficient mice have 3-fold increase in mutation frequency and increase of mutations at A/T pairs, particularly A/T to C/G Tv in a supF shuttle vector (89). Patients with mutations in the ATR gene have a normal-to-slightly-elevated frequency of SHM and an altered pattern of mutations at VH3-23-C transcripts, with fewer mutations at A and more mutations at T residues and more Ts at T residues (92). Ganp–/– mice have decreased affinity maturation: decrease in overall mutation in VH186.2 region after NP-CG immunization and severe decrease in highaffinity type W33L, mutations biased to known SHM hotspots (93) Bach2-/- mice have impaired SHM (94)

THO mutants (in yeast) that generate suboptimal mRNP formation strongly increase hypermutation in a transcription-dependent manner and preferentially at the non transcribed strand (95).

AID: activation-induced cytidine deaminase AP sites: apurinic/apyrimidinic or abasic sites, where a nucleotide base has been removed APE: apurinic/apyrimidinic endonuclease ATR: ataxia telangiectasia and Rad3-related protein BER: Base excision repair Co-IP: coimmunoprecipitation DNA-PKcs: DNA-dependent protein kinase catalytic subunit EXO1: exonuclease 1 GANP: Germinal center-associated nuclear protein MLH1: MutL homologue 1 MMR: mismatch repair MSH: MutS homologue NER: Nucleotide excision repair NHEJ: non homologous end joining

-AID (C-terminal domain): Showed by transient expression of mouse AID in HeLa doing AID IP followed by MALDI-TOF (9). -RPA: on 3'-protruding substrates the 5'- oriented side of hRPA is positioned toward the DNA junction allows specific interaction with XPG, which cleaves the strand opposite the one that is bound by hRPA (14). -PCNA: interaction detected between human proteins by co-IP (58). -RPA: ATR and RPA colocalize to nuclear foci after DNA damage and RPA is required for the recruitment of ATR to sites of DNA damage (15). -MSH2: by co-IP in HeLa and sf9 cells (31). -MSH6: by co-IP in sf9 cells (31).

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al. PCNA: Proliferating cell nuclear antigen PKA: protein kinase A PMS2: postmeiotic segregation 2 POL: polymerase RFC: Replication Factor Chaperone like complex RPA: replication protein A ssDNA: single stranded DNA TLS: translesion DNA synthesis Ts: transition mutations Tv: transversion mutations UNG: uracil DNA glycosylase XPG: xeroderma pigmentosum complementation group G

Supplementary Table Legend Interactions among proteins implicated in somatic hypermutation are summarized. A brief description of their known cellular functions and the effect on SHM when those proteins are deficient, overexpressed, or otherwise altered are provided. Early references and evidence that support those interactions are listed. Supplementary Table Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Bransteitter R, Pham P, Scharff MD, Goodman MF. 2003. Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc Natl Acad Sci U S A 100: 4102-7 Pham P, Bransteitter R, Petruska J, Goodman MF. 2003. Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424: 103-7 Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, Catalan N, Forveille M, Dufourcq-Labelouse R, Gennery A, Tezcan I, Ersoy F, Kayserili H, Ugazio AG, Brousse N, Muramatsu M, Notarangelo LD, Kinoshita K, Honjo T, Fischer A, Durandy A. 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102: 565-75 Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102: 553-63 Nambu Y, Sugai M, Gonda H, Lee CG, Katakai T, Agata Y, Yokota Y, Shimizu A. 2003. Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 302: 2137-40 Chaudhuri J, Khuong C, Alt FW. 2004. Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature 430: 992-8 MacDuff DA, Neuberger MS, Harris RS. 2006. MDM2 can interact with the C-terminus of AID but it is inessential for antibody diversification in DT40 B cells. Mol Immunol 43: 1099-108 Basu U, Chaudhuri J, Alpert C, Dutt S, Ranganath S, Li G, Schrum JP, Manis JP, Alt FW. 2005. The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation. Nature 438: 508-11 Wu X, Geraldes P, Platt JL, Cascalho M. 2005. The double-edged sword of activation-induced cytidine deaminase. J Immunol 174: 934-41 McBride KM, Gazumyan A, Woo EM, Barreto VM, Robbiani DF, Chait BT, Nussenzweig MC. 2006. Regulation of hypermutation by activation-induced cytidine deaminase phosphorylation. Proc Natl Acad Sci U S A 103: 8798-803 Pasqualucci L, Kitaura Y, Gu H, Dalla-Favera R. 2006. PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells. Proc Natl Acad Sci U S A 103: 395-400 Ohta S, Shiomi Y, Sugimoto K, Obuse C, Tsurimoto T. 2002. A proteomics approach to identify proliferating cell nuclear antigen (PCNA)-binding proteins in human cell lysates. Identification of the human CHL12/RFCs2-5 complex as a novel PCNA-binding protein. J Biol Chem 277: 40362-7 Nagelhus TA, Haug T, Singh KK, Keshav KF, Skorpen F, Otterlei M, Bharati S, Lindmo T, Benichou S, Benarous R, Krokan HE. 1997. A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A. J Biol Chem 272: 6561-6 de Laat WL, Appeldoorn E, Sugasawa K, Weterings E, Jaspers NG, Hoeijmakers JH. 1998. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair. Genes Dev 12: 2598-609 Zou L, Elledge SJ. 2003. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300: 1542-8 Alt JR, Bouska A, Fernandez MR, Cerny RL, Xiao H, Eischen CM. 2005. Mdm2 binds to Nbs1 at sites of DNA damage and regulates double strand break repair. J Biol Chem 280: 18771-81

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Examination of Msh6- and Msh3-deficient mice in class switching reveals overlapping and distinct roles of MutS homologues in antibody diversification. J Exp Med 200: 47-59 35. Schmutte C, Sadoff MM, Shim KS, Acharya S, Fishel R. 2001. The interaction of DNA mismatch repair proteins with human exonuclease I. J Biol Chem 276: 33011-8 36. Warren JJ, Pohlhaus TJ, Changela A, Iyer RR, Modrich PL, Beese LS. 2007. Structure of the human MutSalpha DNA lesion recognition complex. Mol Cell 26: 579-92 37. Kim N, Bozek G, Lo JC, Storb U. 1999. Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications. J Exp Med 190: 21-30 38. Kong Q, Maizels N. 1999. PMS2-deficiency diminishes hypermutation of a lambda1 transgene in young but not older mice. Mol Immunol 36: 83-91 39. Winter DB, Phung QH, Umar A, Baker SM, Tarone RE, Tanaka K, Liskay RM, Kunkel TA, Bohr VA, Gearhart PJ. 1998. Altered Spectra of Hypermutation in Antibodies from Mice Deficient for the Mismatch Repair Protein PMS2. Proceedings of the National Academy of Sciences USA 95: 6953-8 40. Cascalho M, Wong J, Steinberg C, Wabl M. 1998. Mismatch repair co-opted by hypermutation. Science 279: 1207-10 41. Guerrette S, Acharya S, Fishel R. 1999. The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer. J Biol Chem 274: 6336-41 42. Brieger A, Trojan J, Raedle J, Plotz G, Zeuzem S. 2002. Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants. Gut 51: 677-84 43. Gu L, Hong Y, McCulloch S, Watanabe H, Li GM. 1998. ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair. Nucleic Acids Res 26: 1173-8 44. Kondo E, Horii A, Fukushige S. 2001. The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2. Nucleic Acids Res 29: 1695-702 45. Wu X, Tsai CY, Patam MB, Zan H, Chen JP, Lipkin SM, Casali P. 2006. A role for the MutL mismatch repair Mlh3 protein in immunoglobulin class switch DNA recombination and somatic hypermutation. J Immunol 176: 5426-37 46. Li Z, Peled JU, Zhao C, Svetlanov A, Ronai D, Cohen PE, Scharff MD. 2006. A role for Mlh3 in somatic hypermutation. DNA Repair (Amst) 5: 675-82 47. Plosky BS, Woodgate R. 2004. Switching from high-fidelity replicases to low-fidelity lesion-bypass polymerases. Curr Opin Genet Dev 14: 113-9 48. Arakawa H, Moldovan GL, Saribasak H, Saribasak NN, Jentsch S, Buerstedde JM. 2006. A role for PCNA ubiquitination in immunoglobulin hypermutation. PLoS Biol 4: e366 49. Langerak P, Nygren AO, Krijger P, van den Berk P, Jacobs H. 2007. A/T mutagenesis in hypermutated immunoglobulin genes strongly depends on PCNA K164 modification. J Exp Med 204 50. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. 2002. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419: 135-41 51. Ko R, Bennett SE. 2005. Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen. DNA Repair (Amst) 4: 1421-31

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Haracska L, Johnson RE, Unk I, Phillips BB, Hurwitz J, Prakash L, Prakash S. 2001. Targeting of human DNA polymerase iota to the replication machinery via interaction with PCNA. Proc Natl Acad Sci U S A 98: 14256-61 57. Guo C, Sonoda E, Tang TS, Parker JL, Bielen AB, Takeda S, Ulrich HD, Friedberg EC. 2006. REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo. Mol Cell 23: 265-71 58. Gary R, Ludwig DL, Cornelius HL, MacInnes MA, Park MS. 1997. The DNA repair endonuclease XPG binds to proliferating cell nuclear antigen (PCNA) and shares sequence elements with the PCNAbinding regions of FEN-1 and cyclin-dependent kinase inhibitor p21. J Biol Chem 272: 24522-9 59. Bachl J, Ertongur I, Jungnickel B. 2006. Involvement of Rad18 in somatic hypermutation. Proc Natl Acad Sci U S A 103: 12081-6 60. Kodama M, Hayashi R, Nishizumi H, Nagawa F, Takemori T, Sakano H. 2001. The PU.1 and NF-EM5 binding motifs in the Igkappa 3' enhancer are responsible for directing somatic hypermutations to the intrinsic hotspots in the transgenic Vkappa gene. Int Immunol 13: 1415-22 61. Rada C, Williams GT, Nilsen H, Barnes DE, Lindahl T, Neuberger MS. 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr Biol 12: 1748-55 62. Imai K, Slupphaug G, Lee WI, Revy P, Nonoyama S, Catalan N, Yel L, Forveille M, Kavli B, Krokan HE, Ochs HD, Fischer A, Durandy A. 2003. Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nat Immunol 4: 1023-8 63. Bennett RA, Wilson DM, 3rd, Wong D, Demple B. 1997. Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway. Proc Natl Acad Sci U S A 94: 71669 64. Prasad R, Singhal RK, Srivastava DK, Molina JT, Tomkinson AE, Wilson SH. 1996. Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis. J Biol Chem 271: 16000-7 65. Yabuki M, Fujii MM, Maizels N. 2005. The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions. Nat Immunol 6: 730-6 66. Poltoratsky V, Prasad R, Horton JK, Wilson SH. 2007. Down-regulation of DNA polymerase beta accompanies somatic hypermutation in human BL2 cell lines. DNA Repair (Amst) 6: 244-53 67. Esposito G, Texido G, Betz UA, Gu H, Muller W, Klein U, Rajewsky K. 2000. Mice reconstituted with DNA polymerase beta-deficient fetal liver cells are able to mount a T cell-dependent immune response and mutate their Ig genes normally. Proc Natl Acad Sci U S A 97: 1166-71 68. Zan H, Komori A, Li Z, Cerrutti M, Flajnik MF, Diaz M, Casali P. 2001. The translesional polymerase zeta plays a major role in Ig and Bcl-6 somatic mutation. Immunity 14: 643-53 69. Delbos F, De Smet A, Faili A, Aoufouchi S, Weill JC, Reynaud CA. 2005. Contribution of DNA polymerase eta to immunoglobulin gene hypermutation in the mouse. J Exp Med 201: 1191-6 70. Masuda K, Ouchida R, Hikida M, Kurosaki T, Yokoi M, Masutani C, Seki M, Wood RD, Hanaoka F, J OW. 2007. DNA polymerases eta and theta function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes. J Biol Chem 282: 17387-94 71. Martomo SA, Yang WW, Wersto RP, Ohkumo T, Kondo Y, Yokoi M, Masutani C, Hanaoka F, Gearhart PJ. 2005. Different mutation signatures in DNA polymerase eta- and MSH6-deficient mice suggest separate roles in antibody diversification. Proc Natl Acad Sci U S A 102: 8656-61 72. Zeng X, Winter DB, Kasmer C, Kraemer KH, Lehmann AR, Gearhart PJ. 2001. DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes. Nat Immunol 2: 53741 73. Yavuz S, Yavuz AS, Kraemer KH, Lipsky PE. 2002. The role of polymerase eta in somatic hypermutation determined by analysis of mutations in a patient with xeroderma pigmentosum variant. J Immunol 169: 3825-30 74. Guo C, Fischhaber PL, Luk-Paszyc MJ, Masuda Y, Zhou J, Kamiya K, Kisker C, Friedberg EC. 2003. Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis. Embo J 22: 6621-30 75. Ohashi E, Murakumo Y, Kanjo N, Akagi J, Masutani C, Hanaoka F, Ohmori H. 2004. Interaction of hREV1 with three human Y-family DNA polymerases. Genes Cells 9: 523-31 76. Tissier A, Kannouche P, Reck MP, Lehmann AR, Fuchs RP, Cordonnier A. 2004. Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein. DNA Repair (Amst) 3: 1503-14 77. Jansen JG, Langerak P, Tsaalbi-Shtylik A, van den Berk P, Jacobs H, de Wind N. 2006. Strand-biased defect in C/G transversions in hypermutating immunoglobulin genes in Rev1-deficient mice. J Exp Med 203: 319-23 78. Simpson LJ, Sale JE. 2003. Rev1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line. Embo J 22: 1654-64 79. Murakumo Y, Ogura Y, Ishii H, Numata S, Ichihara M, Croce CM, Fishel R, Takahashi M. 2001. Interactions in the error-prone postreplication repair proteins hREV1, hREV3, and hREV7. J Biol Chem 276: 35644-51 80. Faili A, Aoufouchi S, Flatter E, Gueranger Q, Reynaud CA, Weill JC. 2002. Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota. Nature 419: 944-7 81. McDonald JP, Frank EG, Plosky BS, Rogozin IB, Masutani C, Hanaoka F, Woodgate R, Gearhart PJ. 2003. 129-derived strains of mice are deficient in DNA polymerase iota and have normal immunoglobulin hypermutation. J Exp Med 198: 635-43

Supplemental Material: Annu. Rev. Immunol. 2008. 26: 481-511 doi: 10.1146/annurev.immunol.26.021607.090236 The Biochemistry of Somatic Hypermutation Peled et al. 82. Zan H, Shima N, Xu Z, Al-Qahtani A, Evinger Iii AJ, Zhong Y, Schimenti JC, Casali P. 2005. The translesion DNA polymerase theta plays a dominant role in immunoglobulin gene somatic hypermutation. Embo J 24: 3757-69 83. Masuda K, Ouchida R, Takeuchi A, Saito T, Koseki H, Kawamura K, Tagawa M, Tokuhisa T, Azuma T, J OW. 2005. DNA polymerase theta contributes to the generation of C/G mutations during somatic hypermutation of Ig genes. Proc Natl Acad Sci U S A 102: 13986-91 84. Diaz M, Verkoczy LK, Flajnik MF, R. KN. 2001. Decreased Frequency of Somatic Hypermutation and Impaired Affinity Maturation but Intact Germinal Center Formation in Mice Expressing Antisense RNA to DNA Polymerase zeta. J Immunol 167: 327-35. 85. Ruiz JF, Lucas D, Garcia-Palomero E, Saez AI, Gonzalez MA, Piris MA, Bernad A, Blanco L. 2004. Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate. Nucleic Acids Res 32: 5861-73 86. Bardwell PD, Woo CJ, Wei K, Li Z, Martin A, Sack SZ, Parris T, Edelmann W, Scharff MD. 2004. Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice. Nat Immunol 5: 224-9 87. Tishkoff DX, Boerger AL, Bertrand P, Filosi N, Gaida GM, Kane MF, Kolodner RD. 1997. Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2. Proc Natl Acad Sci U S A 94: 7487-92 88. Bemark M, Sale JE, Kim H-J, Berek C, Cosgrove RA, Neuberger MS. 2000. Somatic hypermutation in the absence of DNA-PK or Rag1 activity. J. Exp. Med. 192: 1509-14 89. Shiomi N, Hayashi E, Sasanuma S, Mita K, Shiomi T. 2001. Disruption of Xpg increases spontaneous mutation frequency, particularly A:T to C:G transversion. Mutat Res 487: 127-35 90. Dart DA, Adams KE, Akerman I, Lakin ND. 2004. Recruitment of the cell cycle checkpoint kinase ATR to chromatin during S-phase. J Biol Chem 279: 16433-40 91. Cliby WA, Roberts CJ, Cimprich KA, Stringer CM, Lamb JR, Schreiber SL, Friend SH. 1998. Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. Embo J 17: 159-69 92. Pan-Hammarstrom Q, Lahdesmaki A, Zhao Y, Du L, Zhao Z, Wen S, Ruiz-Perez VL, Dunn-Walters DK, Goodship JA, Hammarstrom L. 2006. Disparate roles of ATR and ATM in immunoglobulin class switch recombination and somatic hypermutation. J Exp Med 203: 99-110 93. Kuwahara K, Fujimura S, Takahashi Y, Nakagata N, Takemori T, Aizawa S, Sakaguchi N. 2004. Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses. Proc Natl Acad Sci U S A 101: 1010-5 94. Muto A, Tashiro S, Nakajima O, Hoshino H, Takahashi S, Sakoda E, Ikebe D, Yamamoto M, Igarashi K. 2004. The transcriptional programme of antibody class switching involves the repressor Bach2. Nature 429: 566-71 95. Gomez-Gonzalez B, Aguilera A. 2007. Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex. Proc Natl Acad Sci U S A 104: 8409-14

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Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn1 and Jeffrey V. Ravetch2 1

Laboratory of Experimental Immunology and Immunotherapy, Nikolaus-Fiebiger-Center for Molecular Medicine, University of Erlangen-Nuremberg, 91054 Erlangen, Germany; email: [email protected]

2

Laboratory for Molecular Genetics and Immunology, Rockefeller University, New York, New York 10021; email: [email protected]

Annu. Rev. Immunol. 2008. 26:513–33

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

Fc receptor, inﬂammation, glycosylation, autoimmunity, autoantibody, sialic acid, IVIG

This article’s doi: 10.1146/annurev.immunol.26.021607.090232 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0513$20.00

Abstract The remarkable success story of the therapeutic application of pooled immunoglobulin G (IgG) preparations from thousands of donors, the so-called intravenous IgG (IVIG) therapy, to patients with a variety of hematological and immunological disorders began more than half a century ago. Since then, the use of this primary blood product has increased constantly, resulting in the serious danger of shortages in supply. Despite its widespread use and therapeutic success, the mechanisms of action, especially of the antiinﬂammatory activity, are only beginning to be understood. In this review, we summarize the clinical use of IVIG for different diseases and discuss recent data on the molecular mechanisms that might explain how this potent drug mediates its activity in vivo.

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INTRODUCTION IgG: immunoglobulin G IVIG: intravenous IgG therapy

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ITP: idiopathic thrombocytopenic purpura

Table 1

Immunoglobulins together with T cells are the key mediators of adaptive immunity, and deﬁciencies in either of these two arms of the adaptive immune system can result in a heightened susceptibility to bacterial, fungal, or viral infections. A variety of situations can lead to a constant or transient deﬁciency in immunoglobulins, including primary immunodeﬁciencies, such as several X-linked agammaglobulinemias and common variable immunodeﬁciencies (CVID) (1–3). In addition, hypogammaglobulinemic phenotypes can be caused by viral infections (for example with HIV), in the course of B cell malignancies, or after bone marrow transplantation (4–9). Thus, replacement of immunoglobulin levels, especially of the immunoglobulin G (IgG) isotype, by administration of pooled serum from healthy donors—IVIG (intravenous IgG) as a therapeutic agent—occurred more than 50 years ago. Since then, IVIG use has increased exponentially, although its therapeutic effectiveness in most of these different diseases has not been rigorously addressed, in part because of the variety of diseases it is used to treat and

in part because extant studies include low patient numbers. The popularity of IVIG in the clinic and in research is exempliﬁed by the 360 hits that a PubMed search for IVIG yielded in 2007, when we wrote this review. The two major clinical indications for which IVIG is used are IgG replacement therapy and antiinﬂammation therapy in a variety of acute and chronic autoimmune diseases (Table 1). This latter approach is based on an early observation that a child with idiopathic thrombocytopenic purpura (ITP) showed an attenuated platelet clearance after IVIG administration, which was conﬁrmed in adult patients shortly thereafter (10, 11). Since then, IVIG administration has been included in the therapy of many chronic autoimmune diseases affecting a wide range of tissues and target organs, such as the skin, joints, central nervous system, and hematopoietic system (6). Aside from immunoglobulin replacement therapy, currently licensed applications for IVIG administration include Guillain-Barr`e syndrome, Kawasaki disease, and chronic inﬂammatory demyelinating polyneuropathy

Examples for the clinical use of IVIG (see text for details) Anti-inflammatory (high-dose therapy)

Replacement (low-dose therapy) Primary immunodeﬁciencies (CVID and others) HIV infection Bone marrow transplantation B cell lymphocytic leukemia Multiple myeloma

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Ravetch

Licensed

Off-label

Idiopathic thrombocytopenia purpura Guillain-Barr´e syndrome Kawasaki disease Chronic inﬂammatory demyelinating polyneuropathy (CIDP)

Autoimmune neutropenia Autoimmune hemolytic anemia Anti–Factor VIII autoimmune disease Multiple sclerosis Myesthenia gravis Stiff person syndrome Multifocal neuropathy Systemic vasculitis (ANCA positive) Polymyositis Dermatomyositis Rheumatoid arthritis Systemic lupus erythematosus Antiphospholipid syndrome Toxic epidermal necrolysis Autoimmune skin blistering diseases Steroid-dependent atopic dermatitis Graft-vs-host disease Sepsis syndrome

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(CIDP). Licensed indications, however, only account for approximately 40%–50% of the worldwide IVIG sales, as most IVIG administrations are “off-label” (8, 12). Although in many instances positive results have been reported, well-controlled clinical studies addressing the beneﬁcial effects of IVIG therapy are lacking for most of these diseases. One distinguishing feature between the use of IVIG as a replacement therapy and its use as an anti-inﬂammatory agent is the therapeutically active dose. If used as an IgG replacement, a dose of 300–500 mg/kg body weight of IVIG is recommended to achieve serum levels of approximately 500 mg/dl, which is sufﬁcient to prevent or substantially reduce pulmonary infections, one of the major complications in immunocompromised patients; a dose in this range is considered low-dose therapy. Consistent with the serum half-life of IgG, this treatment must be repeated every 3–4 weeks to maintain a protective serum level (6). To achieve the anti-inﬂammatory effect, however, doses in the range of 1–3 g/kg body weight are required repeatedly (high-dose therapy) (13). Whereas the mechanism of IVIG activity in IgG replacement therapy may be readily explained by the presence of so-called natural antibodies that have an intrinsic capacity to recognize foreign antigens or by the presence of common pathogen-speciﬁc IgG anti-

Table 2

bodies derived from previously immunized or vaccinated serum donors, the explanation of the anti-inﬂammatory activity is more complicated and a matter of much debate. The difﬁculty is to explain how a polyclonal mixture of IgG molecules can suppress the activity of the very same class of molecules, that is, other IgG antibodies recognizing autoantigens. A diverse array of mechanisms, including the blockade of cellular receptors for immunoglobulins (Fc receptors, or FcRs), inhibition of the complement cascade, modulation of cytokine production, neutralization of autoantibodies, and the modulation of inhibitory FcR expression, have been suggested as being responsible for the anti-inﬂammatory activity of IVIG (Table 2) (6, 8, 14, 15). Considering the heterogeneous array of diseases treated with IVIG, one might expect a similarly complex number of mechanisms of action. In this review, we discuss these different possible mechanisms, with an emphasis on results obtained in in vivo model systems. Owing to space limitations, we do not include the immunoglobulin replacement aspect of IVIG therapy, which has been covered in several excellent recent reviews (16, 17). We start with a brief description of the drug IVIG and an introduction into IgG-mediated effector responses, and we follow with a detailed discussion of the different potential mechanisms

Proposed mechanisms for IVIG activity

Replacement therapy

Anti-inflammatory therapy

Preventing infection with pathogenic microorganisms and improving quality of life Reducing risk of graft-vs-host disease after bone marrow transplantation

a b

Autoantibody neutralizationa Modulation of antibody productiona Modulating signaling pathwaysa Modulation of cytokine expression and functiona,b Modulating DC maturation and functiona,b Complement inhibitiona,b Enhancing autoantibody clearance by blocking the FcRnb Functional blockade of activating FcγRsb Upregulation of the inhibitory FcγRIIBb Restoring an anti-inﬂammatory milieub

F(ab)2 -mediated activity. Fc fragment–mediated activity.

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that might explain the therapeutic activity of IVIG in vivo. FcRn: neonatal Fc receptor FcγR: Fcγ receptor

IVIG: PRODUCTION PROCESS, PRODUCT SAFETY, AND SIDE EFFECTS

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IVIG is produced by many different companies and a variety of nonproﬁt organizations such as the American Red Cross (13). With such diverse providers, the compositions of the different IVIG preparations vary in the purity of the IgG preparation, pH, osmolarity, and sodium and sugar content (8, 13). The other most dominant immunoglobulin isotype, IgA, ranges from trace amounts up to 1–2 mg/ml in the different IVIG preparations. IVIG is a primary blood product and must be manufactured according to World Health Organization guidelines. The careful selection and testing of donors for blood transmittable viral diseases, such as HIV, HBV, and HCV, and the incorporation of virus-inactivating steps in the production process are of utmost importance (18). Similarly, the concentration step to obtain a highly enriched IgG preparation has to be selected carefully to prevent loss of biological activity and to minimize unwanted side effects such as the formation of protein aggregates. Most of the currently used techniques were developed in the 1940s and 1950s and only slightly modiﬁed since. Thus, either a cold ethanol precipitation step or the more recently developed caprylate precipitation followed by anion exchange chromatography are used (18). With the optimization of these methods and strict quality control measures, the side effects of IVIG administration are relatively minor today, but they may include a transient headache, nausea, fever, cough, and sore throat (13, 19). In many instances, not the IgG preparation itself but rather product stabilizers such as sugar or salt content or denatured aggregated proteins are responsible for adverse effects (13). Therefore, one quality check is to prevent the presence of signiﬁcant amounts of multimeric IgG aggregates that 516

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may trigger activating FcRs unspeciﬁcally. Ultimately, replacing IVIG with a recombinant product would be advantageous to circumvent these side effects and to prevent supply shortages. For this, however, we must know the molecular mechanism of IVIG activity, which is the focus of the following section.

IgG ACTIVITY IN VIVO As indicated above, both the therapeutically active molecule in the IVIG preparation and the pathology-causing agent in autoimmune diseases are IgG molecules. Thus, we give a brief overview of how IgG antibodies are thought to mediate their activity in vivo. IgG and other immunoglobulin isotypes are composed of an amino acid backbone that contains a sugar moiety attached to an asparagine residue in the antibody constant region (N297) (20, 21). Mice and humans have four different IgG subclasses, denominated IgG1, 2a, 2b, and 3 in mice and IgG1–4 in humans. These are present in the mg/ml range in the serum and have a relatively long halflife, in the range of one week (22). In contrast to other serum proteins, the half-life of the different IgG subclasses is not regulated by the hepatic asialo receptor, which purges the blood of nonfunctional proteins. For IgG molecules, the neonatal Fc receptor, FcRn, plays a crucial role in keeping IgG levels constant. FcRn belongs to the family of major histocompatibility class I (MHC class I) molecules, and mice deﬁcient in this protein or its associated β2-microglobulin (β2M) chain have a dramatically reduced IgG serum concentration and half-life (23, 24). IgG molecules’ proinﬂammatory activity requires recruiting secondary effector functions via their Fc fragment. Cytotoxic or proinﬂammatory pathways that can be activated via the Fc fragment include the complement pathway and crosslinking of cellular FcRs for IgG (Fcγ receptors, FcγR) on innate immune effector cells (25, 26). The family of FcγRs consists of several activating members and one inhibitory member that are

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Figure 1 Activating and inhibitory Fcγ receptors set a threshold for immune effector cell activation. (a) Immune complex (IC) binding to activating FcγRs results in the ITAM phosphorylation of the receptor-associated γc -chain, creating docking sites for Syk kinases. Syk activates several downstream kinases such as the phosphatidylinositol-3 kinase (PI3K), leading to the recruitment of Bruton’s tyrosine kinase (BTK) and phospholipase Cγ (PLCγ) to the plasma membrane, and ultimately leading to calcium inﬂux into the cytosol from intracellular and extracellular sources. (b) The inhibitory FcγRIIB interferes with these activating signaling pathways by recruiting phosphatases such as SHIP (SH2-containing inositide phosphatase) that hydrolyze phosphatidylinositol signaling intermediates necessary for recruitment of BTK and PLCγ, thus limiting cell activation. [Reprinted from Springer Seminars in Immunopathology (2006), 28:305–19, Copyright 2006 with kind permission of Springer Science and Business Media.]

usually coexpressed on the same cell, thereby setting a threshold for cell activation by simultaneously triggering counteracting signaling pathways (Figure 1b) (27). Crosslinking of activating FcγRs leads to the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) present in receptorassociated signaling adaptor proteins such as the common γ-chain (γc -chain) by Src family kinases. This phosphorylation induces several downstream signaling pathways, resulting in an increase in intracellular calcium levels and cell activation (Figure 1a). Simultaneous triggering of FcγRIIB leads to phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytosolic domain of this receptor, which in turn recruits phosphatases such as the SH2-domain containing inositol polyphosphate 5 phosphatase

(SHIP) that interfere with these activating signaling pathways at several stages (Figure 1). The individual receptors differ in their afﬁnity and speciﬁcity for the different IgG subclasses. In mice and humans, there is one high-afﬁnity receptor, FcγRI, which is the only FcγR that can bind to monomeric IgG. All other family members have a low to medium afﬁnity, in the micromolar range, and exclusively bind to antibodies bound to their respective antigen, including soluble proteins, microorganisms, and malignant or virus-infected cells in the form of immune complexes (IC). Thus, the relative expression level and afﬁnities of these different family members are important for regulating antibody activity in vivo (26). This ratio can be inﬂuenced by several cytokines and other pro- or anti-inﬂammatory stimuli, such www.annualreviews.org • Mechanism of IVIG Activity

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as lipopolysaccharide (LPS), several interleukins, transforming growth factor β (TGFβ), tumor necrosis factor α (TNF-α), and C5a (26). Effector functions that are triggered upon IC binding to FcγRs and regulated by balanced FcγR expression include phagocytosis, degranulation, antigen presentation, release of proinﬂammatory cytokines, and antibody-dependent cellular cytotoxicity (ADCC) (27–29). Although several IgG subclasses can efﬁciently activate the complement pathway and mediate target cell lysis in vitro, experimental evidence from in vivo model systems argues against an important role of the complement pathway for these antibody-mediated effector functions (26, 30). Because IVIG and the autoantibodies responsible for chronic inﬂammation and tissue destruction can bind to the same effector cells and molecules, one attractive possibility of IVIG activity is simply to compete for the same effector pathways as the pathogenic autoantibodies. This important point is addressed below.

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MECHANISMS OF IVIG ACTIVITY Several different models explain IVIG activity in various diseases and in vivo model systems. Most currently available in vivo data in mice and humans clearly point to a dominant role of the antibody Fc fragment as responsible for the anti-inﬂammatory activity of IVIG. There are, however, instances in which the Fab fragment may be involved in IVIG’s therapeutic activity. In many of these cases, whether both Fab and Fc fragments contribute to the antiinﬂammatory activity has not been addressed.

F(ab)2 -Dependent Effects Two main mechanisms for the Fab fragment’s anti-inﬂammatory function have been described in the literature. First, the F(ab)2 antibody portion may bind to and neutralize the potent proinﬂammatory activity of the C3aand C5a-anaphylatoxins in a mouse model of 518

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asthma and in a C5a-mediated shock model in pigs (31). However, allergic diseases are quite different from other autoimmune diseases, and IVIG treatment has not demonstrated beneﬁcial effects in two out of three randomized controlled studies with asthma patients. Furthermore, IVIG therapy is not recommended for patients with severe asthma by the Primary Immunodeﬁciency Committee of the American Academy of Allergy, Asthma and Immunology (32). Thus, it remains to be seen whether this is a relevant mechanism of action in other autoimmune diseases for which beneﬁcial effects of high-dose IVIG therapy have been described. Second, a number of studies have addressed whether antibodies with distinct speciﬁcities are present in the IVIG preparation. Indeed, several groups identiﬁed signiﬁcant binding to a variety of proteins and cell surface receptors that may explain the therapeutic activity of IVIG. These include anti-idiotypic, TCRα/β, Siglec-9, CD5, antiintegrin, Fas, cytokines, and cytokine receptor speciﬁcities (6, 33–38). It is beyond our scope to discuss all these possibilities, as in vivo evidence is not yet available to demonstrate a signiﬁcant contribution of many of these antigen-speciﬁc antibodies in the IVIG preparation. For special instances in which certain receptor-ligand interactions are the main cause for the disease, such as in myasthenia gravis or toxic epidermal necrolysis (TEN), these (auto-) antibody speciﬁcities may have an important therapeutic function (39, 40). TEN, for example, is the result of an adverse drug reaction and is characterized by the detachment of large sections of the epidermis from the dermis caused by widespread death of keratinocytes (40). The CD95- or Fas-mediated pathway of apoptosis is of central importance for keratinocyte death, and CD95-speciﬁc antibodies within the IVIG preparation can block this interaction by binding to CD95 (41). Depletion of the CD95-speciﬁc antibodies within the IVIG preparation results in a loss of activity, as measured in an in vitro assay with human

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keratinocytes. It is unclear, however, whether this is the sole mode of action of IVIG in TEN in vivo or if other Fc fragment–dependent activities are involved as well. Further complicating this scenario are reports that both agonistic and blocking anti-Fas antibodies are present in IVIG preparations (34, 42). Thus, apoptosis was induced upon incubation of human lymphocytes and monocytes with therapeutic preparations of IVIG, which may be responsible for eliminating or reducing effector cells involved in causing chronic inﬂammation (34). More studies are necessary to deﬁne the role of these antigen-speciﬁc antibodies in the IVIG preparation in suitable in vivo model systems.

Fc-Dependent Effects In contrast to the role of the Fab fragment, considerable evidence suggests that many autoimmune diseases for which IVIG has beneﬁcial effects involve the Fc fragment as playing the central role in IVIG’s anti-inﬂammatory activity. In a clinical trial with ITP patients and in many mouse autoimmune models, such as arthritis, nephrotoxic nephritis (NTN), and ITP, the Fc fragment was as effective as the whole IVIG preparation (43–46). The potential mechanisms for this activity are mirrored by the different effector pathways or receptors/ligands that can interact with the Fc fragment (Figure 2), including the complement pathway, FcRn, and classical activating and inhibitory FcγRs (23, 25, 26). As is now well established, the complement pathway is not signiﬁcantly involved in the activity of different cytotoxic and autoantibodies in most mouse in vivo model systems; therefore, we do not cover this topic in depth, although IVIG does bind to activated C3b and C4b and prevents the tissue deposition of these activated complement proteins (47–49). However, tissue deposition of activated complement proteins is not necessarily predictive of complementmediated tissue destruction. Indeed, in many mouse models of antibody-mediated inﬂammation, autoantibody activity and tissue de-

struction are abrogated in the absence of cellular FcγRs, although activated complement components are still present in high amounts in noninﬂamed tissues (50, 51). Similar results were reported for many other in vivo models of antibody-mediated inﬂammation in vivo (30). Direct evidence against a role for complement in the mechanism of IVIG activity comes from studies in which complement inactivation by injection of cobra venom factor had no impact on IVIG activity (52, 53). In addition to these well-described Fc fragment– dependent effector pathways, recent evidence points to a novel anti-inﬂammatory function of the Fc fragment–associated sugar moiety, which is discussed at the end of this review. IVIG-mediated saturation of FcRn. The FcRn is the crucial regulator of IgG half-life (Figure 2a). Therefore, one rationale to block autoantibody activity is to interfere with their interaction with FcRn, thus shortening their half-life and clearing them from the circulation before they can cause major damage. Indeed, a generation of antibody mutants with enhanced afﬁnity for FcRn or for blocking the IgG-FcRn interaction by antibodies speciﬁc for FcRn or its β2M subunit competes with serum antibodies for binding to FcRn and inducing their accelerated clearance (54– 56). One attractive possibility is that IVIG, by means of its high dose, may also mediate its anti-inﬂammatory activity by competing with autoantibodies for FcRn binding (Figure 2b) (57–61). Antibody half-life studies in ITP models in mice and rats demonstrated that IVIG at a dose of 1 g/kg can shorten the half-life of a platelet-speciﬁc autoantibody from 79 to 54 h (58). Importantly, however, the maximal levels of platelet depletion and of IVIG-mediated protection essentially occur immediately after autoantibody injection (within the ﬁrst 1–4 h) in mice and rats, which makes it rather unlikely that this change in antibody serum half-life will have an effect (46, 58, 62). Indeed, in this early phase hardly any difference in the serum level of the www.annualreviews.org • Mechanism of IVIG Activity

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Figure 2 Proposed Fc fragment–dependent mechanisms of IVIG activity. (a) The neonatal Fc receptor (FcRn) binds to serum IgG at low pH after it has been endocytosed and transported into acidic vesicles. FcRn-bound IgG molecules are then recycled to the cell surface and released into the circulation at physiologic pH. (b) Three different models have been proposed to mediate the anti-inﬂammatory activity of the IVIG Fc fragment in vivo. First, the high dose of IgG molecules present in the IVIG preparation may compete with autoantibodies for FcRn binding and thus result in their enhanced clearance. Second, immune complexes present in the IVIG preparation may bind to activating FcγRs and thereby prevent binding of autoantibody immune complexes. In the third model, IVIG activity is crucially dependent on the presence of the inhibitory FcγRIIB. (Reproduced from the Journal of Experimental Medicine, 2007, 204:11–15. Copyright 2007 Rockefeller University Press.) 520

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autoantibody in the presence or absence of IVIG was observed. Moreover, the therapeutic dose of IVIG can be reduced from roughly 25 mg per mouse to 600–700 μg if a speciﬁc glycovariant of the IVIG Fc fragment is used (45). This dramatically lower dose, while maintaining its anti-inﬂammatory activity, has no effect on the half-life of serum or autoantibodies and thus argues against a role for FcRn in these fast models of autoantibody-mediated platelet depletion (12, 45). In addition to ITP, two other mouse model systems of autoantibody-mediated arthritis and autoantibody-induced skin blistering diseases demonstrated a role for IVIG in mediating its activity via FcRn (57, 60). In the serum transfer arthritis model, serum from arthritogenic mice is injected into wild-type animals, which develop a transient joint swelling over the next 20 days (63). Treatment of mice with IVIG is very efﬁcient at blocking autoantibody-induced inﬂammation, which makes this model an attractive system to study the mechanism of IVIG activity. IVIG treatment resulted in a reduced serum half-life of the arthritogenic antibodies, and IVIG activity was reduced in mice deﬁcient in FcRn and the inhibitory FcγRIIB (57). The central role of FcγRIIB for IVIG activity in vivo has been shown in many model systems and is discussed later in this review (43, 45, 46, 50). The major problem with using FcRn-deﬁcient animals for these experiments is that the arthritogenic autoantibodies as well as IVIG have a dramatically reduced serum half-life. This necessitates very high doses of arthritogenic serum to obtain a level of inﬂammation that is still far below the response normally achieved in wildtype mice (57). In addition, the reduction in serum half-life of the autoantibodies was only investigated after administration of four consecutive 1 g/kg doses of IVIG, whereas one dose is normally sufﬁcient to block arthritis in this model, making it difﬁcult to evaluate whether the enhanced clearance rate induced by this high dose of IVIG is required for antiinﬂammatory activity.

Direct evidence against a role for FcRn in mediating IVIG activity in the serum transfer arthritis model comes from studies that analyzed the interaction of aglycosylated IgG with FcRn and the effect of the absence of this Fc fragment–attached sugar moiety on IVIG anti-inﬂammatory activity in vivo (45, 64). A mutation at position 297 that changes an arginine to an alanine (N297A) residue abrogated binding to classical FcγRs but kept its afﬁnity for FcRn (64). Thus, aglycosylated IVIG should still be functional without this sugar moiety if FcRn saturation and enhanced autoantibody degradation are the mechanisms of action. This is not the case, however, as aglycosylated IVIG loses its anti-inﬂammatory activity (45). In a similar serum transfer approach, the capacity of IVIG to suppress antibodymediated skin blistering diseases was investigated (60). Diseases such as bullous pemphigoid, pemphigus foliaceus, and pemphigus vulgaris are characterized by subepidermal or intraepidermal blisters and by autoantibodies of the IgG isotype speciﬁc for hemidesmosomal or epidermal proteins, which are able to transfer the disease if injected into newborn mice (65). In this model, injection of IVIG leads to a reduction in the serum halflife of the autoantibodies and efﬁciently prevents the development of blisters. In contrast to other model systems, in this model IVIG activity is independent of the inhibitory FcγRIIB. Moreover, deletion of this negative regulator of antibody activity does not further exacerbate the autoimmune symptoms, which again is not consistent with most of the results obtained in adult mouse model systems to date (26, 27). Thus, FcγRIIB deletion results in a lower threshold for B cell and innate immune effector cell activation, leading to the generation of class switched autoantibodies, spontaneous dendritic cell maturation, higher levels of autoantibody-mediated tissue destruction, and the development of autoimmune diseases on otherwise resistant genetic backgrounds (66–74). Indeed, the expression

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and functionality of activating and inhibitory FcRs have not been studied in depth in newborn mice, making it hard to predict whether these ﬁndings can be generalized and are comparable with results obtained in adult mice and the adult immune system. Taken together, technical difﬁculties in designing an unequivocal experimental setting make it hard to predict whether IVIGmediated FcRn saturation is responsible for its activity in vivo. Nonetheless, reducing autoantibody half-life by modulating the antibody-FcRn interaction may be a promising therapeutic strategy.

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Activating Fc receptor blockade and cytokine modulation. The second major theory for how IVIG mediates its antiinﬂammatory activity is via blockade of activating FcγRs (Figure 2b). Indeed, because FcγRs are centrally important in antibodymediated effector functions, blocking of individual or all activating FcRs results in the abrogation of antibody activity in a variety of autoimmune and tumor models, such as ITP, NTN, arthritis, and several models of antibody-mediated tumor cell destruction (26, 50, 51, 62, 63, 75–81). Similarly, blocking human FcγRIIIA in a transgenic mouse model and in human ITP patients interferes with platelet depletion (46, 82– 84). In contrast, blocking or deleting the high-afﬁnity FcγRI in vivo in human ITP patients or in mice was therapeutically ineffective, highlighting the importance of the lowafﬁnity FcγRs for antibody-mediated effector functions (62, 85). Thus, we focus on the low-afﬁnity FcγRs in discussing the possible impact of IVIG-mediated interaction with activating FcγRs. Low- or medium-afﬁnity receptors, such as mouse FcγRIIB, FcγRIII, and FcγRIV and even more so their human counterparts FcγRIIA/B/C and FcγRIIIA, cannot interact with monomeric IgG but can only bind to IgG in the form of an IC (27). Therefore, monomeric IgG, which constitutes more than 97% of the IVIG preparation, cannot 522

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be responsible for directly blocking activating FcγRs. As discussed above, however, different IVIG preparations may contain varying levels of dimeric or multimeric IgG molecules. Aged IVIG preparations with a higher content of IgG dimers have an enhanced antiinﬂammatory activity in a mouse model of ITP, although whether these dimeric molecules are able to block autoantibody binding to activating FcγRs has not been investigated (53). A more recent study reported that antibodies speciﬁc for soluble proteins in the presence of their antigen or antibodies speciﬁc for cell surface proteins on red blood cells, for example, can exhibit IVIG-like activity in ITP (52, 86). This ﬁnding is consistent with earlier data from human ITP patients treated with the so-called anti-D IgG, which is a pooled polyclonal IgG fraction that comes selectively from donors immunized to the rhesus D antigen [anti-Rh0(D)-positive]. Anti-D, which, compared with IVIG, is used at a 40,000-fold lower dose, also can prevent platelet consumption in human ITP, and this activity is lost in patients who do not express the rhesus D antigen (87–90). However, the use of a monoclonal anti-D antibody of the IgG1 subclass that can efﬁciently interact with human FcRs was not able to prevent platelet depletion in seven anti-Rh0(D)-positive patients, arguing against a simple IC-formation mechanism (91). Clearly, the use of anti-D or other hyperimmune sera in the presence of the respective antigen will create a high level of IC that may compete with the autoantibodyantigen complexes and inhibit their phagocytosis by a mechanism similar to directly blocking activating FcγRs with an FcγR-speciﬁc antibody. With respect to the many speciﬁcities present in the IVIG preparation, these complexes may form after administration into patients. Whether the level of IC formation is high enough to achieve a signiﬁcant level of binding to activating FcγRs is unclear. More importantly, the use of ICs poses quite some danger to the patient as they will systemically

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trigger activating FcγRs and may even enhance inﬂammation by inducing release of proinﬂammatory cytokines from innate immune effector cells. Recently, two other mechanisms of IVIG activity involving FcγRIII were proposed. First, triggering activating FcγRs on dendritic cells in vitro and then adoptively transferring them into mice can have IVIG-like effects and can ameliorate murine ITP; thus, investigators concluded that IVIG can be replaced by antibodies speciﬁc for activating FcγRs (92). Although an intriguing ﬁnding, it remains to be seen whether this is the actual mechanism of IVIG activity, as IVIG preparations with enhanced activity have a reduced afﬁnity for activating FcγRs in mice and humans (45, 93). Second, investigators suggested that IVIG mediates its anti-inﬂammatory activity by inhibiting the release of the proinﬂammatory cytokine IFN-γ from myeloid cells in an FcγRIII-dependent manner (94). Although many studies described cytokine-modulating effects by IVIG, the relevance of these ﬁndings for the therapeutic activity of IVIG in vivo remains unclear (95). Indeed, IVIG is fully functional in preventing platelet depletion in mouse strains deﬁcient in a variety of proinﬂammatory cytokines and cytokine receptors, such as the IFN-γR, CCL3, IL12β, and TNF-α (96). More direct evidence against a central role for FcγRIII in IVIG activity comes from a recent study using a model of accelerated NTN. In this model, autoantibody-mediated kidney inﬂammation is mainly mediated by the activating FcγRIV, and IVIG is very potent in suppressing kidney destruction (49). Thus, we could investigate the role of activating FcγRIII in IVIG activity without simultaneously interfering with the interaction between the autoantibody and its activating FcγR. Importantly, deletion of FcγRIII had no inﬂuence on IVIG activity in vivo, whereas the absence of the inhibitory FcγRIIB abrogated the anti-inﬂammatory effect of IVIG. Taken together, the data suggest that IVIG-like effects can be achieved by blocking

the responsible activating FcγRs either with competing ICs or with FcγR-speciﬁc monoclonal antibodies that have the capacity to block binding of ICs to activating FcγRs. This highlights the importance of FcγRs in murine and human autoimmune diseases. At present, however, it seems unlikely that this is the actual mechanism of IVIG activity. Upregulation of the inhibitory FcγRIIB. As indicated brieﬂy in the previous paragraphs, the other classical FcγR that has been associated repeatedly with the antiinﬂammatory activity of IVIG in vivo is the low-afﬁnity inhibitory FcγRIIB (Figure 2b). Animals deﬁcient in this protein are no longer protected by administration of IVIG in mouse models of ITP, rheumatoid arthritis, and NTN (43, 45, 46, 50, 57, 97). Moreover, IVIG therapy resulted in the upregulation of the inhibitory FcγRIIB on effector macrophages (43, 46, 50). In addition, a dramatic decrease in the expression of the triggering activating FcγRIV was observed in a model of NTN (50). This altered expression level of activating and inhibitory FcγRs will heighten the threshold for innate immune effector cell activation, which ultimately leads to a lower level of inﬂammation and platelet consumption. Consistent with the low afﬁnity of FcγRIIB, a direct interaction with IVIG seems rather unlikely. In addition, the IVIGmediated upregulation of FcγRIIB on effector macrophages seems to be indirect as determined by the loss of IVIG activity and increased expression of FcγRIIB on effector macrophages in op/op mice (43). These mice lack the hematopoietic growth factor colonystimulating factor (CSF)-1, which results in the loss of select monocyte and macrophage subpopulations (98). Because IVIG activity is lost but antibody-mediated inﬂammation is maintained, investigators suggested that a CSF-1-dependent macrophage population has a regulatory function and is responsible for the upregulation of the inhibitory FcγR on effector macrophages (Figure 3) (12). Although this provides some information about www.annualreviews.org • Mechanism of IVIG Activity

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Immune complex Autoantigen Autoantibody

Figure 3 The role of the inhibitory FcγRIIB for IVIG activity. Binding of SA-rich immunoglobulins in the IVIG preparation to an as yet unknown receptor on CSF-1-dependent regulatory macrophages results in the upregulation of the inhibitory FcγRIIB on effector macrophages, which leads to an increased threshold for cell activation and ultimately reduces or blocks chronic inﬂammatory reactions. (Reproduced from the Journal of Experimental Medicine, 2007, 204:11–15. Copyright 2007 Rockefeller University Press.)

the cell types that are involved in IVIG activity, the fact of this regulatory function does not explain why such high doses are required to achieve it. Rather, it suggests that only a minor component in the IVIG preparation is the active component. This, at ﬁrst sight, seems to be at odds with the limited heterogeneity within the IVIG preparation (which consists of only four different IgG subclasses) if one focuses on IgG Fc fragment–mediated effects. Differential antibody glycosylation. IgG antibodies are glycoproteins that contain a sugar moiety attached to each of the asparagine 297 (N297) residues in the two chains of the antibody Fc fragment (99). This glycan moiety is an integral structural component of the IgG molecule, forming part of the scaffold for FcγR binding 524

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(Figure 4). In addition, depending on the variable region sequences, roughly 20% of serum IgG antibodies have a Fab fragment– attached N-linked sugar side chain. These sugar moieties consist of a biantennary heptameric core sugar structure high in mannose and N-acetylglucosamine with variable amounts of branching and terminal sugar residues such as galactose, sialic acid (SA), N-acetylglucosamine, and fucose (Figure 4b) (100–102). Whereas Fab fragment–attached sugar moieties and the sugar domains of other serum proteins such as tranferrin or fetuin are usually fully processed (meaning that they contain all possible sugar residues), the Fc fragment–associated moiety is much more heterogeneous. In fact, more than 30 different antibody glycovariants have been detected in human serum, with about 25%–30% of

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FcγR

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Figure 4 The sugar domain of the antibody Fc fragment. (a) Shown is the crystal structure of the extracellular domain of human FcγRIIIA in complex with an immunoglobulin Fc fragment (CH2-CH3 domains). The Fc fragment–attached sugar moieties that protrude into the central cavity between the two antibody heavy chains are shown as a stick and ball model. (b) Cartoon of the antibody-attached sugar moiety. The heptameric core sugar structure consisting of mannose (man) and N-acetylglucosamine (GlcNAc) residues is shown in bold, and terminal or branching sugar residues are indicated. Most IgG glycoforms can be distinguished by the absence of terminal sialic acid (SA) or galactose (Gal) residues (IgG-G0 glycoform), the presence of one or two galactose residues (IgG-G1/2 glycoform), and the presence of one or two terminal SA residues (fully processed or native form).

them in the IgG-G0 glycoform (Figure 4b) without terminal SA or galactose residues (103). Thus, these 30 variants, multiplied by the four different IgG subclasses, result in more than 120 different glycoproteins in the IVIG preparation that could contain the active anti-inﬂammatory component. The im-

portance of this sugar moiety is highlighted by the loss of therapeutic activity of deglycosylated IVIG preparations (45). As discussed above, this excludes a simple FcRn competition model because the FcRn, unlike classical FcγRs, retains its afﬁnity for deglycosylated Fc fragments. The loss of classical www.annualreviews.org • Mechanism of IVIG Activity

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FcγR binding could indicate that a direct interaction with these molecules is important. However, there is strong evidence against this scenario, as IVIG preparations as well as isolated Fc fragments enriched for terminal SA residues have a more than 10fold higher anti-inﬂammatory activity. These SA-rich IgG glycovariants have a decreased afﬁnity for classical FcγRs in mice and humans, consistent with their strongly impaired activity in vivo (45, 93). This excludes the possibility that SA-rich IVIG preparations block access of autoantibody ICs to activating FcγRs and rather argues for a novel receptor on regulatory macrophages that can speciﬁcally recognize SA-rich IgG and promote an anti-inﬂammatory milieu. Data in favor of an important role of differential antibody glycosylation and especially sialylation in vivo come from human arthritis patients and mice with a variety of autoimmune diseases, such as systemic lupus erythematosus, arthritis, and NTN (45, 104–110). Especially during acute disease phases, a signiﬁcant reduction in terminal SA residues in serum and antigen-speciﬁc antibodies can be observed, and the IgG-G0 glycovariant constitutes more than 50% of serum IgG (106). Thus, IVIG infusion would restore the level of SA-rich IgG that is necessary to regain an anti-inﬂammatory environment that suppresses the activity of autoantibodies by increasing inhibitory FcγRIIB expression.

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SUMMARY AND OUTLOOK Despite the widespread use of IVIG, we have only recently begun to shed some light on the mechanism of IVIG activity in vivo. Elucidating the molecular details of this activity is crucially important for gaining some general insights into the regulation of pro- and anti-inﬂammatory immune responses as well as for optimizing IVIG activity or even replacing it by a recombinant product in the future.

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Although probably none represent the actual mechanism of IVIG activity, several possibilities have already been described, such as enhancing autoantibody clearance by blocking the neonatal FcR or interfering with the interaction of autoimmune complexes with activating FcγRs. Although attractive strategies, both have drawbacks: Continuous blocking of FcRn will result in a more rapid clearance not only of autoantibodies but also of all other serum IgG molecules that have protective functions against microbial infections. Blocking activating FcγRs is, of course, the most direct strategy to interfere with innate effector cell activation, but it is far downstream in the inﬂammatory cascade and may also pose some signiﬁcant dangers as the blocking antibodies or competing soluble IC might result in unspeciﬁc cell activation and the massive systemic release of proinﬂammatory cytokines, a so-called cytokine storm. Thus, potential FcγR-blocking antibodies or soluble ICs have to be carefully evaluated to prevent these severe side effects. In contrast, restoring a balanced immune response might be a very promising strategy in the longterm. Although we are just at the very beginning, the recent identiﬁcation of antibody glycovariants with immunoregulatory activity might enable us to pursue more efﬁcient and long-lasting therapeutic responses, especially in combination with therapies that aim at a speciﬁc depletion of autoantibody-producing B cells. Important topics to be addressed include determining whether genetic polymorphisms exist in the population that result in a differential antibody glycosylation pattern and which signals regulate antibody glycosylation in B cells during the steady state and during immune or autoimmune responses. A better understanding of the molecular details of differential antibody glycosylation might enable us to speciﬁcally modify these pathways during autoimmune responses and interfere with chronic inﬂammatory processes.

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SUMMARY POINTS 1. The two major clinical uses of IVIG are Ig replacement and anti-inﬂammatory therapy. 2. IVIG is produced from the pooled serum of thousands of donors enriched for the IgG fraction. 3. The anti-inﬂammatory activity of IVIG requires a high-dose treatment regimen. 4. IVIG contains autoreactive antibody species of varying speciﬁcity.

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5. A F(ab)2 -mediated mechanism may account for IVIG activity for select diseases. 6. Most in vivo evidence suggests that the Fc fragment contains the anti-inﬂammatory activity. 7. Blocking FcRn or activating FcγRs results in an IVIG-like activity by interfering with autoantibody half-life or binding to triggering activating FcγRs. 8. The inhibitory FcγRIIB is required for IVIG activity in vivo. 9. More than 30 different IgG glycovariants can be detected in human serum. 10. IgG antibodies from human arthritis patients and autoimmune mouse strains have a reduced level of terminal SA residues. 11. The SA-rich IgG fraction of IVIG has an enhanced anti-inﬂammatory activity.

FUTURE ISSUES 1. Why do autoimmune patients have aberrantly glycosylated antibodies? 2. Are there genetic factors that impact antibody glycosylation? 3. How is antibody glycosylation regulated? 4. Can recombinantly produced SA-rich IgG replace IVIG? 5. What is the cellular receptor that can recognize SA-rich IgG?

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS This work was sponsored by grants from the NIH to J.V.R. and from the German Research Foundation (DFG) and the Bavarian Genome Research Network (BayGene) to F.N. We are especially grateful to Peter Sondermann (Roche) for providing the crystal structure shown in Figure 4. We apologize to all colleagues whose important work was not directly cited. These references can be found in the review articles cited in this paper.

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94. Park-Min KH, Serbina NV, Yang W, Ma X, Krystal G, et al. 2007. FcγRIII-dependent inhibition of interferon-γ responses mediates suppressive effects of intravenous immune globulin. Immunity 26:67–78 95. Zhou B, Zhao H, Yang RC, Han ZC. 2005. Multi-dysfunctional pathophysiology in ITP. Crit. Rev. Oncol. Hematol. 54:107–16 96. Crow AR, Song S, Semple JW, Freedman J, Lazarus AH. 2007. A role for IL-1 receptor antagonist or other cytokines in the acute therapeutic effects of IVIg? Blood 109:155–58 97. Crow AR, Song S, Freedman J, Helgason CD, Humphries RK, et al. 2003. IVIg-mediated amelioration of murine ITP via FcγRIIB is independent of SHIP1, SHP-1, and Btk activity. Blood 102:558–60 98. Stanley ER, Berg KL, Einstein DB, Lee PS, Yeung YG. 1994. The biology and action of colony stimulating factor-1. Stem. Cells 12(Suppl. 1):15–25 99. Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA. 2006. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25:21–50 100. Butler M, Quelhas D, Critchley AJ, Carchon H, Hebestreit HF, et al. 2003. Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the speciﬁc defective glycan processing step and provides an insight into pathogenesis. Glycobiology 13:601–22 101. Jefferis R, Lund J, Mizutani H, Nakagawa H, Kawazoe Y, et al. 1990. A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins. Biochem. J. 268:529–37 102. Wormald MR, Rudd PM, Harvey DJ, Chang SC, Scragg IG, Dwek RA. 1997. Variations in oligosaccharide-protein interactions in immunoglobulin G determine the site-speciﬁc glycosylation proﬁles and modulate the dynamic motion of the Fc oligosaccharides. Biochemistry 36:1370–80 103. Arnold JN, Dwek RA, Rudd PM, Sim RB. 2006. Mannan binding lectin and its interaction with immunoglobulins in health and in disease. Immunol. Lett. 106:103–10 104. Nimmerjahn F, Anthony RM, Ravetch JV. 2007. Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity. Proc. Natl. Acad. Sci. USA 104:8433–37 105. Bond A, Cooke A, Hay FC. 1990. Glycosylation of IgG, immune complexes and IgG subclasses in the MRL-lpr/lpr mouse model of rheumatoid arthritis. Eur. J. Immunol. 20:2229–33 106. Malhotra R, Wormald MR, Rudd PM, Fischer PB, Dwek RA, Sim RB. 1995. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nat. Med. 1:237–43 107. Matsumoto A, Shikata K, Takeuchi F, Kojima N, Mizuochi T. 2000. Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation. J. Biochem. 128:621–28 108. Mizuochi T, Hamako J, Nose M, Titani K. 1990. Structural changes in the oligosaccharide chains of IgG in autoimmune MRL/Mp-lpr/lpr mice. J. Immunol. 145:1794–98 109. Rademacher TW, Williams P, Dwek RA. 1994. Agalactosyl glycoforms of IgG autoantibodies are pathogenic. Proc. Natl. Acad. Sci. USA 91:6123–27 110. Rook GA, Steele J, Brealey R, Whyte A, Isenberg D, et al. 1991. Changes in IgG glycoform levels are associated with remission of arthritis during pregnancy. J. Autoimmun. 4:779–94

www.annualreviews.org • Mechanism of IVIG Activity

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:513-533. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi Department of Immunology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; email: [email protected]

Annu. Rev. Immunol. 2008. 26:535–84

Key Words

First published online as a Review in Advance on December 20, 2007

interferon regulatory factor, transcriptional regulation, innate immunity, immune cell development, oncogenesis

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090400 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0535$20.00

Abstract The interferon regulatory factor (IRF) family, consisting of nine members in mammals, was identiﬁed in the late 1980s in the context of research into the type I interferon system. Subsequent studies over the past two decades have revealed the versatile and critical functions performed by this transcription factor family. Indeed, many IRF members play central roles in the cellular differentiation of hematopoietic cells and in the regulation of gene expression in response to pathogen-derived danger signals. In particular, the advances made in understanding the immunobiology of Toll-like and other pattern-recognition receptors have recently generated new momentum for the study of IRFs. Moreover, the role of several IRF family members in the regulation of the cell cycle and apoptosis has important implications for understanding susceptibility to and progression of several cancers.

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INTRODUCTION IRF: interferon regulatory factor

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Type I interferons: a family of cytokines that includes IFN-α (encoded by 13 functional genes in humans and 14 in mice) and IFN-β IFN-stimulated response element (ISRE): a common DNA motif found in the promoters of genes that are activated by type I IFNs Patternrecognition receptor (PRR): a receptor that recognizes pathogen-associated molecular patterns (PAMPs) to initiate innate immune responses

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Host defense serves two main functions: the generation of immune responses to invading pathogens and the suppression of tumor development. These are achieved by the efﬁcient coordination of cellular responses by genetic regulatory networks in which a given transcription factor controls the expression of a diverse set of target genes, depending on the cell type and/or the nature of cellular stimuli. We review the current literature on how interferon (IFN) regulatory factors (IRFs) orchestrate and control homeostatic mechanisms of host defense. Although IRFs were ﬁrst and best characterized as transcriptional regulators of type I IFNs and IFN-inducible genes, this family is now recognized as playing a pivotal part in the regulation of many facets of innate and adaptive immune responses. In addition to its contributions to immunity, the role of IRFs in the regulation of oncogenesis is also discussed herein. The IRF family of transcription factors, the ﬁrst member of which, IRF1, was discovered in 1988, consists of nine members in humans and mice: IRF1, IRF2, IRF3, IRF4 (also known as PIP, LSIRF, or ICSAT), IRF5, IRF6, IRF7, IRF8 (also known as ICSBP), and IRF9 (also known as ISGF3γ) (Table 1) (1, 2). In addition, IRF10 was identiﬁed in chickens, although it is absent in humans and mice (3). Each IRF contains a well-conserved N-terminal DNA-binding domain (DBD) of ∼120 amino acids that possesses ﬁve conserved tryptophan repeats and bears a resemblance to the DBD of Myb transcription factors (2, 4). The IRF DBD forms a helix-turn-helix domain and recognizes a DNA sequence corresponding to the IFN-stimulated response element (ISRE, A /G NGAAANNGAAACT) (5). An analysis of the crystal structure of the DBD of IRF1 bound to the PRDI of the IFN-β enhancer revealed that 5 -GAAA-3 is the core recognition sequence of the helix-turn-helix motif (6). A subsequent analysis of the crystal structure of the IRF2 DBD in complex with a tandem

Tamura et al.

repeat of GAAA shows that 5 -AANNGAAA3 is the consensus IRF recognition sequence (7). The C-terminal regions of IRFs, except IRF1 and IRF2, carry an IRF association domain (IAD) that is responsible for homo- and heteromeric interactions with other family members or other transcription factors such as PU.1 and signal transducer and activator of transcription (STAT) (1, 2). The IADs share structural similarities with the Madhomology 2 (MH2) domains of the Smad family of transcription factors, which also mediate protein-protein interactions (8, 9). Interaction of IRFs with other transcription factors can further deﬁne the nucleotide sequences adjacent to the core IRF-binding motif to which the protein complex binds, as exempliﬁed by the binding of the PU.1-IRF4 or the PU.1-IRF8 complex. Gene-targeting studies performed on all nine IRFs had been carried out by 2006 and have revealed the markedly diverse roles played by this family. Since our 2001 review in the Annual Review of Immunology (2), the crucial involvement of many IRFs (IRF1, IRF3, IRF4, IRF5, IRF7, and IRF8) in innate immune responses elicited by patternrecognition receptors (PRRs) has been clariﬁed in greater detail. Major progress has also been made in understanding the role of IRFs (IRF1, IRF2, IRF4, and IRF8) in the development of various immune cells. Moreover, evidence has accumulated on the critical role of several IRFs (IRF1, IRF3, IRF5, and IRF8) in the control of cell growth, cell survival, and oncogenesis. We refer the reader to related review articles on IRFs published elsewhere (10–13).

REGULATION OF INNATE IMMUNE RESPONSES Recognition of invading pathogens is central to the host immune system. The innate immune system depends on a limited number of germline-encoded receptors called PRRs

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to recognize pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and viral nucleic acids (14). Depending on the nature of the pathogen and cell type involved, signal transduction pathways are activated by PAMP-engaged PRRs so as to elicit antimicrobial responses by inducing various target genes that include those encoding type I IFNs, proinﬂammatory cytokines, and chemokines. One of the major and best characterized of the players that transmit PRR signals to the nucleus is nuclear factor κB (NF-κB), activated by virtually all PRRs. In the past few years, however, extensive studies have revealed that IRFs are also widely involved in most PRR signaling events, thereby conferring a more diverse immune activation capability on the platform that links innate and adaptive immunities. IRF3 and IRF7 are primarily responsible for the activation of type I IFN genes downstream of PRR activation, although IRF1, IRF5, and IRF8 can also contribute to it. IRF1, IRF3, IRF5, and IRF8 also induce the expression of proinﬂammatory cytokines and other genes in response to PRR activation. Two classes of PRRs, cytosolic PRRs and membrane-bound Toll-like receptors (TLRs), have been identiﬁed (14, 15). In this section, we summarize the recent progress made in understanding the role of IRFs in the various PRR signaling pathways.

IRFs and Cytosolic PRR Signaling The cytosolic PRRs include the retinoic acid inducible gene I (RIG-I) family, IFNinducible double-stranded RNA (dsRNA)dependent protein kinase (PKR), DNAdependent activator of IRFs (DAI), and nucleotide-binding oligomerization domain (NOD) proteins (14–16). PKR is the ﬁrst sensor found to detect intracellular dsRNA and is produced during replication of certain RNA viruses. However, gene-targeting studies in mice show that although PKR contributes to type I IFN production in response to polyriboinosinic polyribocytidylic

acid (poly(rI:rC)), a synthetic dsRNA (17), it only plays a minor role in type I IFN gene induction in response to viral infection (18). The RIG-I family of proteins, however, recognizes cytoplasmic viral RNA and is vital to the activation of IRFs. Several NOD family members recognize cytoplasmic bacterial PAMPs and are implicated in the activation of NF-κB and caspases, but an involvement of IRF members downstream of the NOD pathways has not been identiﬁed. DAI is a newly identiﬁed sensor for cytoplasmic DNA whose signaling pathway is still being elucidated. In this section, we describe the activation mechanisms and biological functions—with an emphasis on type I IFN gene induction— of IRF7, IRF3, and IRF5 in the cytosolic PRR signaling mediated by the RIG-I family and by DAI (Figure 1).

TLR: Toll-like receptor

Activation of IRFs by the RIG-I/MDA5 signaling pathway. Two RNA helicase enzymes, RIG-I (also called DDX58) and MDA5 (also called Helicard), have been identiﬁed as essential cytosolic receptors for intracellular viral RNA to induce type I IFN genes in various cell types, except plasmacytoid dendritic cells (pDCs) (19–22). This functional distinction is important because in vivo experiments show that cells other than pDCs also act as essential IFN-producing cells in response to many viruses (17, 23). RIG-I and MDA5 contain a C-terminal DExD/H box RNA helicase domain as well as two N-terminal caspase recruitment domains (CARDs) (24). The helicase domain is responsible for the detection of viral RNA, whereas the CARDs activate downstream signaling pathways. The in vivo relevance of RIG-I and MDA5 was demonstrated by studies using mice deﬁcient in RIG-I or MDA5 (20, 21, 25). On the one hand, RIG-I mounts antiviral responses against a set of negative-strand single-stranded (ss)RNA viruses such as Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), Sendai virus, and inﬂuenza virus, as well as some positive-strand ssRNA viruses such as Japanese encephalitis virus. www.annualreviews.org • The IRF Family Transcription Factors

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538 • Stimulates expression of IFN-inducible genes (GBP, iNOS, Caspase-1, Cox-2, CIITA, TAP1, and LMP2) • Binds to MyD88 and enhances TLR-dependent gene induction in IFN-γ-treated cells (IFN-β, iNOS, IL-12p35, and IL-12p40) • Attenuates type I IFN responses by antagonizing IRF1 and IRF9 • In some cases cooperates with IRF1 to activate transcription (IL-12p40 and Cox-2)

• Induces type I IFNs (IFN-α4 and IFN-β) and chemokines (CXCL10) upon virus infection, TLR stimulation, and DNA stimulation

• Binds to MyD88 and negatively regulates TLR-dependent induction of proinﬂammatory cytokine genes

Expression

• Constitutive and IFN-inducible in various cell types • Inducible by DNA damage at transcriptional and posttranslational levels • Mainly in the nucleus and partially in the cytoplasm • Modiﬁed by TLR signaling to efﬁciently translocate to the nucleus

• Constitutive and IFN-inducible in various cell types

• Constitutive in various cell types • Mainly in the cytoplasm • Phosphorylated upon virus infection, TRIF-dependent signaling, cytosolic PRR signaling, and DNA damage, and then translocates to the nucleus

• Constitutive in B cells, Ms, and CD11b+ DCs and inducible by antigen stimulation in T cells and by TLR signaling in Ms • Mainly in the nucleus and partially in the cytoplasm

IRF

IRF1

IRF2

IRF3

IRF4

Role in immune cell function (target genes)

A summary of IRF family member functions

Tamura et al. • Required for differentiation of CD4+ DCs • Supports B cell development (Ig light chains) • Required for plasma cell differentiation (Blimp-1 and AID) and GC formation • Required for Th2 differentiation (IL-4)

Unknown

Role in cell growth

• May possess oncogenic potential

• Stimulates apoptosis in Ms upon bacterial infection • May promote DNA damage–induced apoptosis

• Suppresses oncogene-induced transformation (Lysyl oxidase) • Required for DNA damage–induced growth arrest (p21/WAF1/CIP1) • Required for DNA damage–induced apoptosis

References

133, 135, 137, 138, 238, 240, 244, 246, 253, 261, 262, 266

36, 39, 51, 54, 56, 59, 61, 99, 105, 350

48, 53, 179, 180, 220, 224, 228, 234, 252, 260, 271, 336

2, 53, 134, 220–222, 231–233, 249, 250, 256, 260, 271, 272, 278, 280, 294, 295, 349

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• Required for differentiation of CD4+ DCs • Required for NK cell development • Suppresses basophil expansion • Promotes Th1 differentiation (IL-12 in Ms) • Suppresses Th2 differentiation (represses IL-4)

• Required for NK cell development (IL-15 in bone marrow stromal cells) • Required for differentiation of CD8+ T cells • Promotes Th1 differentiation through NK cells (IL-15), Ms/DCs (IL-12), and a T cell–intrinsic mechanism • Suppresses Th2 differentiation (represses IL-4)

Role in development of immune cells and other cells

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Table 1

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• Binds to MyD88 and positively regulates TLR-dependent induction of proinﬂammatory cytokine genes (IL-12p40, IL-6, and TNF-α) • Induces type I IFNs and proinﬂammatory cytokines upon virus infection (type I IFNs, IL-6, and TNF-α) Unknown • Binds to MyD88 and positively regulates TLR-dependent induction of type I IFNs (IFN-α/β)

• Binds to TRAF6 and is required for TLR9 signaling in DCs and Ms • Promotes type I IFN production in DCs (IFN-α/β) • Stimulates IFN-γ- and PAMP-inducible genes (IL-12p40, iNOS, FcγRI, PML, and others)

• Binds to STAT1 and STAT2 to form ISGF3 and stimulates type I IFN–inducible genes (OAS, PKR, IRF7, and many others)

• Constitutive in B cells and DCs, and inducible by type I IFNs and TLR signaling • Mainly in the cytoplasm • Phosphorylated upon virus infection, TLR-dependent signaling, and DNA damage, and then translocates to the nucleus

• Constitutive in skin

• Constitutive in B cells, pDCs, and monocytes and inducible by type I IFNs in various cell types • Mainly in the cytoplasm • Phosphorylated upon virus infection and TLR-dependent signaling, and then translocates to the nucleus

• Constitutive in B cells, Ms, and CD11b− DCs and further inducible by IFN-γ in Ms and by antigen stimulation in T cells • Mainly in the nucleus and partially in the cytoplasm

• Constitutive and inducible by IFN-γ in various cell types • Mainly in the nucleus

IRF5

IRF6

IRF7

IRF8

IRF9

Unknown

• Required for differentiation of CD8α+ DCs and pDCs • Stimulates M differentiation (Blimp-1, METS, and lysosomal/endosomal enzyme-related genes; represses disabled-2) • Supports B cell development (Ig light chains) • Stimulates the GC program (BCL6 and AID) • Promotes Th1 differentiation through Ms/DCs (IL-12)

• Mediates type I IFN induction of p53 (p53)

• Inhibits myeloid cell growth (Blimp-1, METS, and p15/INK4B) • Promotes apoptosis in myeloid cells • Its absence leads to CML-like disease

Unknown

4, 331, 353, 354

52, 140, 142, 173, 175, 187, 189, 190, 193, 199, 202, 214, 215, 238, 243, 352

44, 45, 49, 50, 64, 115, 123, 124, 128, 351

267, 268

46, 65, 130, 314, 315

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Unknown

Unknown

• Suppresses oncogene-induced transformation • Required for DNA damage–induced apoptosis

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• Required for keratinocyte differentiation

Unknown

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www.annualreviews.org • The IRF Family Transcription Factors

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On the other hand, MDA5 senses the presence of positive-strand ssRNA picornaviruses such as encephalomyocarditis virus (EMCV), Theiler’s virus, and Mengo virus. The molecular basis for the differential viral recognition by these two RNA helicases was recently reported. RIG-I senses the uncapped

5 -triphosphate end of RNA, a molecular signature that is absent in the cytosolic RNA of eukaryotic cells and in viruses detected by MDA5 (26, 27). MDA5, in contrast, recognizes dsRNA including poly(rI:rC) (21, 25). In addition, evidence has been provided that RIG-I and MDA5 mediate the induction of

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RNA virus

DNA virus

Intracellular bacterium

V protein Phagosome

RIG-I or MDA5 Helicase dsRNA or 5'P-RNA NS3/4A

CARDs

PPP

dsDNA L. monocytogenes

VISA

DAI and others

TRAF3 IKKγ

Mitochondrion RIP1 IKKγ IKKα IKKβ

IRF3

FADD

IKKγ

TBK1 or IKKε

TBK1

TRAF6

IRF7 IKKα IKKβ

IRF7 IRF3 I B

I B

NF- B p50

p65

IRF7

IRF5 IRF8

Pro-inflammatory cytokines 540

Tamura et al.

IRF3

P

Type I IFNs

P

IRF7 IRF3

P

P

Pro-inflammatory cytokines

p50

p65

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IFN responses by small self-RNAs generated by RNaseL (28). The adaptor molecule that links the sensing of viral RNA by RIG-I or MDA5 to downstream signaling was identiﬁed by multiple groups and variously named virus-induced signaling adaptor (VISA) (29), mitochondrial antiviral signaling protein (MAVS) (30), IFNβ-promoter stimulator 1 (IPS-1) (31), and CARD adaptor inducing IFN (Cardif ) (32); we refer to it as IPS-1 throughout this review for convenience. IPS-1 contains an Nterminal CARD that shares homology with and mediates CARD-CARD interactions between the CARDs of RIG-I and MDA5 to transmit downstream signaling. IPS-1 also contains a transmembrane region that targets itself to the mitochondrial outer membrane. This localization is essential for triggering downstream signaling and indicates a link between mitochondria and antiviral immunity. Importantly, mice deﬁcient in IPS-1 are defective in RIG-I- and MDA5-, but not TLR-, B-DNA-, or DNA virus–mediated activation of IRFs and NF-κB; these mice are also susceptible to RNA virus infection (33, 34). How IPS-1 relays the signals from RIG-I and MDA5 to IRFs and NF-κB is still incom-

pletely understood; however, a recent study suggests that NEMO/IKKγ acts downstream of IPS-1 and upstream of IRF and NF-κB activation (35). NEMO/IKKγ, through TANK, interacts with the kinases that phosphorylate IRF3, IRF7, and IRF5, namely TANKbinding kinase 1 (TBK1, also known as T2K and NAK) and IKKε (IKKi) to activate IRFs (36–39). At the same time, NEMO/IKKγ presumably forms a complex with canonical IKKs (IKKα and IKKβ) to activate the NF-κB pathway. How IPS-1 transmits signals to NEMO/IKKγ remains unknown, but it interacts with several signaling proteins such as TRAF3 and TRAF6, the adaptor FADD, and the kinase RIP-1 (29, 31). Among them, TRAF3 (40, 41) interacts with both IPS-1 and TBK1, raising the possibility that TRAF3 links IPS-1 to a complex containing NEMO/IKKγ, TANK, and TBK1/IKKε. Although both TBK1 and IKKε can phosphorylate serine residues in the C-terminal (regulatory) region of IRF3 and IRF7 in vitro, gene-targeting studies show that TBK1 is the main contributor to type I IFN gene induction in virus-infected mouse embryonic ﬁbroblasts (MEFs) (42, 43). As discussed in more detail below, TBK1 also functions downstream of

MEF: mouse embryonic ﬁbroblast

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1 Activation of IRFs by cytosolic PRRs. The presence of RNA or dsDNA in the cytosol triggers host responses via a speciﬁc cytoplasmic pattern-recognition system. The binding of uncapped 5 -triphosphate RNA or dsRNA to the helicase domain of RIG-I/MDA5 induces the interaction between the CARD of RIG-I or MDA5 and the CARD-like domain of the adaptor protein IPS-1, which is located on the mitochondrial membrane. This receptor-adaptor interaction results in the activation of TBK1 and IKKε. Activated TBK1 induces the phosphorylation of the speciﬁc serine residues of IRF3 and IRF7. These IRFs then translocate into the nucleus and activate the type I IFN genes. In some cases, IRF5 or IRF8 participate in this IFN gene induction pathway. TRAF3, IKKγ/NEMO, and TANK positively regulate TBK1-mediated IRF activation. FADD, RIP-1, and TRAF6 interact with IPS-1 and are implicated in the NF-κB activation pathway, resulting in the induction of proinﬂammatory cytokine genes. dsDNA such as B-DNA is recognized by DAI. IRF3 and TBK1 interact with DAI, resulting in the induction of type I IFN genes. IRF7 is also involved in this pathway. Recognition of dsDNA by DAI activates NF-κB as well, which results in the induction of proinﬂammatory cytokine genes. The HCV NS3/4A protein and V proteins of paramyxovirus inhibit this pathway by cleaving IPS-1 and by binding to MDA5, respectively. (Abbreviations: IFN, interferon; IRF, IFN regulatory factor; RIG-I, retinoic acid inducible gene I; MDA5, melanoma differentiation-associated gene 5; CARD, caspase recruitment domain; IPS-1, IFN-β-promoter stimulator 1; TRAF, tumor necrosis factor receptor–associated factor; TANK, TRAF family member–associated NF-κB activator; TBK1, TANK-binding kinase 1; IKK, IκB kinase; NEMO, NF-κB essential modulator; FADD, FAS-associated via death domain; RIP-1, receptor interacting protein-1; DAI, DNA-dependent activator of IFN regulatory factors; HCV, hepatitis C virus.) www.annualreviews.org • The IRF Family Transcription Factors

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Contributions of IRFs to RIG-I/MDA5 signaling. Five IRFs (IRF1, IRF3, IRF5, IRF7, and IRF8) have been implicated as positive regulators of type I IFN gene induction (2, 46–52). Although IRF1 was the ﬁrst family member discovered capable of activating type I IFN genes in vitro, NDVinduced type I IFN levels were normal in Irf1−/− MEFs, indicating that IRF1 is not essential for the induction of type I IFN genes by the virus-activated cytosolic pathway (53). The two IRF family members that have the greatest structural homology, IRF7 and IRF3, have been subsequently shown to be essential for the RIG-I/MDA5-mediated type I IFN gene induction pathway. Both reside in the cytosol in a latent form in unstimulated cells. Upon viral infection, speciﬁc serine residues in the C-terminal (regulatory) regions are phosphorylated, which causes the IRFs to undergo nuclear translocation. A two-step phosphorylation model by TBK1 is suggested for IRF3. First, phosphorylation at Ser396 to Ser405 in site 2 alleviates auto-inhibition to allow interaction with the coactivator cyclic-AMP-responsive-elementbinding protein (CREB)-binding protein (CBP). CBP facilitates the phosphorylation of Ser385 or Ser386 at site 1, which then allows for IRF dimerization (54, 55). Thus, the holocomplex containing dimerized IRF3 and IRF7 (either as a homodimer or a heterodimer) and coactivators such as CBP or p300 is formed in the nucleus (51, 56–59) and then binds to target ISRE DNA sequences within the promoters of type I IFN genes and certain chemokine genes. The histone acetyltransferase activities of the coactivators presumably lead to alterations in the local chromatin, resulting in the recruitment of basal transcription factors. In the case of the IFN-β gene promoter, other transcription factors such as NF-κB and activator protein 1 (AP1) are also recruited to 542

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form an enhanceosome to initiate efﬁcient transcription (1, 2, 60). The initial hypothesis for the mode of IRF3/IRF7 induction of type I IFN genes was the positive-feedback model, in which IRF3 is primarily responsible for the induction of IFN-β in the early phase of a response, whereas IRF7, whose mRNA expression is induced by IFN-β, functions in the later phase of a response to induce IFN-α expression (61, 62). Indeed, IFN-α gene induction is reduced in Ifnb−/− MEFs (63), and type I IFN mRNA induction is impaired in Irf3−/− MEFs upon NDV infection (61). However, a subsequent study performed on Irf 7−/− MEFs revealed that both the early and later phases of type I IFN gene induction upon infection by ssRNA viruses (VSV and EMCV) are abolished in the absence of IRF7 (64). Thus, although IRF7 is initially expressed at a low level, the formation of a heterodimer between IRF7 and IRF3, rather than an IRF3 homodimer, is presumed to be more crucial for the production of IFN-α and IFN-β. Positive-feedback regulation of IRF7 then comes into effect to achieve the full induction of type I IFN genes during the later phases of the response. Recently, IRF5 has been shown to be involved in the RIG-I signaling pathway (65). Irf5−/− mice are highly vulnerable to VSV infection, accompanied by a decrease in type I IFN induction in the sera. The requirement of IRF5 is cell type selective; Irf5−/− macrophages (Ms) are defective in the production of type I IFNs by these viruses, whereas Irf5−/− MEFs are not, possibly because IRF5 is more highly expressed in hematopoietic cells. In addition, Irf5−/− mice show reduced levels of proinﬂammatory cytokines, such as IL-6, in the sera and in M cultures upon infection with these viruses, suggesting that IRF5 is also involved in the induction of proinﬂammatory cytokine genes. Although IRF5, like IRF3 and IRF7, can be phosphorylated by TBK1 (66), the detailed activation mechanism of IRF5 in the RIG-I signaling pathway, as well as how IRF5 participates in the transcriptional regulation

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of type I IFNs and proinﬂammatory cytokine genes, are still incompletely understood (also see below). Recently, it was reported that IRF8 is required for type I IFN induction in virus-stimulated DCs (52). IRF8 binds to the promoters of type I IFN genes and may participate in the subsequent IRF7-mediated amplifying phase of IFN transcription. Cytosolic DNA sensors. In addition to the cytosolic RNA-sensing mechanisms, recent attention has been focused on characterizing cytosolic DNA-sensing systems, as they also evoke protective and pathological immune responses. Indeed, the induction of type I IFN genes by DNA was reported more than four decades ago (67, 68). More recently, this issue was revisited in relation to TLR signaling, in particular TLR9 because it is activated by hypomethylated DNA (14, 15). However, a TLR9-independent mechanism(s) of type I IFN gene expression has also been described in which herpes simplex virus 1 (HSV-1) DNA virus can induce type I IFNs in cells deﬁcient in TLR9 signaling (69). Furthermore, IFNβ induced via the TBK1-IRF3 pathway (70) by the obligate cytoplasmic bacteria Listeria monocytogenes is mainly mediated by DNA and is also independent of TLR signaling (71). Finally, cytosolic delivery of synthetic dsDNA into cells results in the expression of type I IFNs, and this induction also occurs independently of TLR signaling (72). These observations in toto indicate the presence of a cytosolic DNA sensor(s) that can initiate innate immune responses, including the induction of type I IFN genes, independent of TLR signaling. Recently, a candidate DNA sensor called DAI was identiﬁed (73). This protein, ﬁrst identiﬁed as DLM-1 in tumor stromal tissue (74), possesses two DBDs for left-handed Zform DNA (Z-DNA) and, hence, is also referred to as Z-DNA-binding protein (ZBP1) (75, 76). With the discovery of its biological function as a DNA sensor, the name DAI has been proposed (73). A synthetic dsDNA, poly(dA-dT) poly (dT-dA), termed B-DNA because it likely

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takes on a right-handed B-form conﬁguration, can strongly evoke innate immune responses (72). On the one hand, when DAI is artiﬁcially expressed in a murine ﬁbroblast cell line, B-DNA induction of genes encoding type I IFNs, IL-6, and CXCL10 is signiﬁcantly augmented. On the other hand, knockdown of DAI expression by RNA interference inhibits the activation of these genes upon BDNA, but not upon poly(rI:rC), treatment, indicating that DAI selectively controls this DNA-mediated signaling pathway. Such an effect of DAI has also been observed for other pathogen-derived DNAs such as bacterial and viral DNAs, suggesting the involvement of DAI in the host defense against pathogens. Can DAI directly recognize DNA and, if so, how does it transmit signals? Investigators have shown that DAI, which mainly resides in the cytosol, directly interacts with B-DNA in the cell and that this DNA-DAI interaction requires the N-terminal half of the protein that contains two known DBDs (75, 76) and a newly identiﬁed DBD (73). The in vitro interaction of recombinant DAI with B-DNA has recently been observed (T. Taniguchi, unpublished observation). Furthermore, DAI interaction with IRF3 and TBK1 through its C-terminal region is enhanced when cells are stimulated with B-DNA. Indeed, DAI requires both the N-terminal DNA-binding region and C-terminal TBK1/IRF-binding region for the B-DNA-mediated induction of type I IFN genes, supporting the notion that DNA is not only critical to initiate but also to sustain the active signaling complex (73). The requirement of IRF3 was demonstrated when B-DNA induction of IFN-β was abolished in Irf3−/− MEFs, but was normal in Irf 7−/− or Irf5−/− MEFs (71–73). The full induction of IFN-α, however, requires both IRF3 and IRF7 because both Irf3−/− and Irf 7−/− MEFs show impairment in its induction. DAI is likely to function redundantly with other DNA-sensing molecules. The DNAmediated induction of type I IFNs and other genes is not completely abolished in www.annualreviews.org • The IRF Family Transcription Factors

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DAI-knockdown cells (73; T. Taniguchi, unpublished observation), and activation of target genes by IFN-stimulatory DNA (ISD) is cell type dependent (71, 73).

IRFs and TLR Signaling To date, 13 functional TLRs (10 in human and 12 in mice) have been identiﬁed and found to recognize a variety of PAMPs derived from bacteria, viruses, fungi, and/or protozoa to trigger immune responses, including the induction of proinﬂammatory and type I IFN genes (77–79). All TLRs contain an intracellular Toll/IL-1 receptor (TIR) domain that transmits downstream signals by recruiting one or more TIR-containing adaptor proteins (14, 15, 80). These adaptors include MyD88 (myeloid differentiation primary-response protein 88), TIRAP (TIR domain–containing adaptor protein; also called MAL), TRIF (TIR domain– containing adaptor inducing IFN-β; also called TICAM1), and TRAM (TRIF-related adaptor molecule; also called TICAM2) (15, 80–87). Signaling through TLRs can be broadly categorized into two pathways: the MyD88-dependent pathway and the TRIFdependent pathway (or MyD88-independent pathway) (88, 89). All TLRs except TLR3 activate the MyD88-dependent pathway. By contrast, TLR3 and TLR4 activate the TRIFdependent pathway. Although most TLRs directly associate with either MyD88 or TRIF upon PAMP stimuli, TLR4 requires the additional adaptors TIRAP (81) and TRAM (82, 90) for the recruitment of MyD88 and TRIF, respectively. Both the MyD88-dependent and TRIFdependent pathways lead to the activation of at least three major downstream molecules: NF-κB, mitogen-activated protein kinases (MAPKs), and IRFs (11, 15, 77, 80, 91, 92). In the MyD88-dependent pathway, MyD88 ﬁrst recruits IL-1 receptor-associated kinases 1/4 (IRAK1/4) and then TRAF6 and transforming growth factor (TGF)-β-activated kinase 1 (TAK1) (15, 83, 86, 92–94). TAK1 activates Tamura et al.

NF-κB and MAPKs through the phosphorylation of IKKβ and MAPK kinase 6 (MKK6), respectively (95). In addition, multiple IRFs are also recruited to the multimolecular complex containing MyD88 and TRAF6. In the following sections, we discuss the role of IRFs in the TRIF-dependent pathway and then summarize the contribution of each IRF to the MyD88-dependent pathway (Figure 2). IRF3 and IRF7 in the TRIF-dependent pathway. Both TLR4 and TLR3 utilize the TRIF-dependent pathway; TLR4 recruits TRAM-TRIF to activate IRF3 to induce IFN-β (TLR4 also activates the MyD88dependent signaling pathway), and TLR3 directly recruits TRIF to activate IRF3 and IRF7 to induce both IFN-α and IFN-β (11, 15). TLR4 is a cell surface receptor that recognizes non-nucleic acid ligands such as LPS from Gram-negative bacteria, and F (fusion) protein of the respiratory syncytial virus (RSV) and Moloney murine leukemia virus (96–98). TLR4 signaling results in the induction of the gene encoding IFN-β, but not the genes encoding IFN-α except IFN-α4 (99, 100, 101). The induction of the Ifnb gene by TLR4 is mostly MyD88-independent and TRAM-TRIF-dependent, whereas the induction of genes encoding proinﬂammatory cytokine genes or chemokines, such as IL-6, TNF-α, CCL5/RANTES, CXCL10, or CCL2, depends on MyD88 and TRAM-TRIF (102, 103). TRAM-TRIF recruits TRAF3, NAP1, and then the IRF kinase TBK1 (37, 38, 40, 104). The induction of IFN-β in response to LPS is abolished in Irf3−/− DCs, whereas this induction is almost normal in Irf 7−/− cells (64, 99, 105). Consistent with this role, Irf3−/− mice exhibit resistance to LPS-induced endotoxin shock (99) for which IFN-β induction is critical (106). Thus, IFN-β induction by TLR4 is mainly mediated by IRF3 rather than IRF7 via its phosphorylation by TBK1, a mechanism similar to that of the RIG-I pathway. It is not clear why TLR4-mediated activation of TBK1 via the TRAM-TRIF

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Figure 2 TLR-mediated IRF activation pathways. (a) TLR4 signals through at least four adaptors, MyD88, TIRAP, TRIF, and TRAM. Among them, TRAM and TRIF mediate the activation of IRF3. TRIF associates with TBK1 through NAP1 and TRAF3, which mediates the phosphorylation (P) of IRF3 (and IRF7, if present). Phosphorylated IRF3 forms homodimers and induces expression of genes encoding IFN-β and CXCL10. (b) IRF7 directly binds to MyD88 in the endosomal compartment and regulates the type I IFN gene induction program. Upon TLR9 stimulation, IRF7 interacts with MyD88 and is activated/phosphorylated by IRAK1 and IKKα. TRAF3 and TRAF6 interact with MyD88 and IRAK1 and are also involved in IRF7 activation. (c) IRF5 interacts with and is activated by MyD88 and TRAF6 by a still unknown mechanism. Activated IRF5 translocates to the nucleus to activate proinﬂammatory cytokine gene transcription, presumably in cooperation with NF-κB. IRF8 is also involved in TLR9-mediated signaling by directly activating proinﬂammatory cytokine genes and/or by the activation of NF-κB. IRF4 binds to MyD88 in a region overlapping with that of IRF5 and inhibits the further binding of IRF5 to MyD88, thereby attenuating the induction of proinﬂammatory cytokine genes. (d ) IFN-γ stimulation induces the expression of IRF1 via the formation of homodimers of phosphorylated STAT1. Induced IRF1 interacts with and is activated by MyD88 by an unknown mechanism and translocates to the nucleus to induce genes encoding IFN-β, iNOS, and IL-12p35. Note that the pathways depicted here operate in a cell type–speciﬁc manner. (Abbreviations: MyD88, myeloid differentiation primary-response protein 88; NAP1, nucleosome assembly protein 1; TIR, Toll/IL-1 receptor; TIRAP, TIR domain–containing adaptor protein; TRIF, TIR domain–containing adaptor inducing IFN-β; TRAM, TRIF-related adaptor molecule; IRAK, IL-1 receptor–associated kinase; TRAF, TNF receptor–associated factor; STAT1, signal transducer and activator of transcription 1; iNOS, inducible nitric oxide synthase.)

pathway is linked to the activation of IRF3 and not IRF7. Interestingly, however, when IRF7 is upregulated by pretreatment with recombinant IFN-β, LPS induction of IFNβ mRNA can be observed in Irf3−/− DCs (99). Therefore, under certain conditions

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TLR3 is expressed within the membrane of endosomes and phagosomes or at the cell surface, in the case of endothelial and natural killer (NK) cells. TLR3 recognizes the synthetic dsRNA analog poly(rI:rC) and probably recognizes viral dsRNA derived from either dsRNA viruses, such as reovirus, or as replication intermediates from ssRNA viruses, such as West Nile virus and RSV (107–109). In addition, TLR3 is involved in the defense against infection by HSV and murine cytomegalovirus (dsDNA viruses) as well as the parasites Leishmania donovani and Schistosoma mansoni (110–113), indicating that ligands other than dsRNA might also activate TLR3. Similar to TLR4, the activation of TLR3 can induce type I IFN gene expression via a MyD88-independent, TRIF- and TBK1-dependent signaling pathway (84, 87, 102, 103, 107). Indeed, the induction of type I IFN genes by poly(rI:rC) stimulation is severely impaired in Trif−/− or Tbk1−/− cells (102, 103). Although IRF3 plays an essential role in this induction, the poly(rI:rC)mediated induction of type I IFN mRNAs is still observed in Irf3−/− DCs. This residual induction is completely abolished in IRF3 and IRF7 doubly deﬁcient DCs (H. Yanai & T. Taniguchi, unpublished observation). Therefore, in contrast to TLR4 signaling, IRF3 and IRF7 are both required for the maximal, TRIF-mediated induction of type I IFN genes. Although kinases TBK1 and IKKε can both phosphorylate IRF3 and IRF7 (36, 39), a gene-targeting study has revealed that TBK1 is most critical to the activation of IRF3 and IRF7 (37).

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Activation of IRF7 by the MyD88dependent signaling pathway. Recently, much attention has focused on the highlevel induction of type I IFN genes by pDCs (114–116). In contrast to conventional DCs (cDCs) and MEFs, pDCs express high levels of TLR7 and TLR9 in endosomes and produce large amounts of type I IFNs in response to corresponding TLR ligands. TLR7 recognizes ssRNA derived from genomic RNA 546

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of viruses such as inﬂuenza virus and VSV, whereas TLR9 recognizes hypomethylated CpG DNA motifs in bacteria or DNA viruses such as HSV (113, 117–121). In contrast to TLR3- or TLR4-mediated, TRIF-dependent IFN gene induction, TLR9 and TLR7 exclusively utilize MyD88 as its signaling adaptor (15). Indeed, type I IFN expression is abolished when MyD88-deﬁcient pDCs are stimulated with ligands to these TLRs (114–119, 122). Because the induction of type I IFNs is crucially dependent on the activation of IRFs, this raises the question of how these TLRs can activate IRFs through the MyD88 adaptor protein. Investigators have shown that IRF7, but not IRF3, directly interacts with the death domain of MyD88 (123, 124). Splenic pDCs derived from Irf 7−/− mice exhibited a profound defect in type I IFN gene induction either upon infection by viruses (HSV and VSV) or treatment with synthetic TLR ligands (CpG-A and ssRNA), whereas the induction was normal in Irf3−/− pDCs (64). Therefore, in contrast to the IRF3-dependent type I IFN gene induction by the TRIF pathway, robust MyD88-dependent IFN gene induction in pDCs depends on IRF7. IRF7 also interacts with TRAF6 such that the overexpression of TRAF6-induced type I IFN genes occurs through the activation of IRF7 (123). The IRAK family of serine/threonine kinases, which are signal transducers between MyD88 and TRAF6, are also involved where IRAK1 and IRAK4 (like IRF7) bind to the death domain of MyD88, and pDCs derived from Irak4−/− or Irak1−/− mice have a defect in IFN-α production upon stimulation of TLR9 or TLR7 (45, 123). A more recent study has shown that IKKα is essential for the phosphorylation of IRF7 (44). Therefore, the IRAK4-IRAK1-IKKα kinase cascade, known to be operational in the NFκB activation pathway, likely also leads to IRF7 activation. In addition, TRAF3 and osteopontin are also involved in this pathway, but their exact functions are unknown (40, 125).

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Spatiotemporal regulation of the MyD88IRF7 pathway in pDCs. Although the essential role of IRF7 in MyD88-dependent IFN gene induction is clear, another interesting issue is why pDCs, but not other cell types such as cDCs, produce large amounts of type I IFNs in response to the same TLR9 ligands. Though posited as a potential explanation, the higher constitutive expression level of IRF7 in pDCs compared with other cell types (126) does not fully account for the robust production of type I IFNs by pDCs. Instead, the spatiotemporal regulation of TLR9 (and probably TLR7) signaling appears to be critical for this speciﬁc feature of pDCs. In pDCs, the IFN-inducing TLR9 ligand CpG-A preferentially colocalizes to the MyD88-IRF7-containing endosomal compartment (127, 128). In contrast, the same ligand mainly localizes to lysosomes in cDCs and Ms. Further underscoring the importance of this selective localization, targeting CpG-A to the endosome resulted in the robust production of type I IFNs in cDCs and Ms (128, 129). Thus, pDCs have a unique mechanism for retaining CpG-A in the endosome where MyD88 and IRF7 are localized; the prolonged signaling of TLR by CpG-A stimulation in endosomal compartments results in the robust production of type I IFNs through a sustained positive-feedback loop, likely involving the accumulation and activation of IFN-induced, de novo synthesized IRF7. Consistent with these data, pDCs from mice deﬁcient in type I IFN receptor signaling (Ifnar1−/− ) are defective in robust type I IFN gene induction by CpG-A (64). Because CpG-B, which more strongly induces proinﬂammatory cytokines than does CpG-A, undergoes rapid lysosomal trafﬁcking, the lysosomal TLR9 signaling may be crucial for effectively evoking this response. TLR signaling and IRF5. Similar to IRF7, IRF5 also interacts with MyD88 and TRAF6 (130). However, unlike IRF7, which binds to the death domain of MyD88, IRF5 interacts with the central region (the intermediary

domain and part of the TIR domain) of MyD88. Following TLR9 activation, IRF5 translocates to the nucleus and binds to the promoter of target genes, such as Il12b, that contain an ISRE. Indeed, the induction of proinﬂammatory cytokines, such as IL-12, TNF-α, and IL-6, is impaired in Irf5−/− Ms after stimulation with poly(rI:rC), LPS, ﬂagellin, ssRNA, and CpG-B. Other genes such as those encoding IκBζ and CXCL2 are also regulated by IRF5 following TLR9 activation (130, 131), although whether IRF5 regulates these genes directly or indirectly is not clear. In accordance with the above observation, Irf5−/− mice show resistance to lethal endotoxin shock induced by CpG-B or LPS. However, recent studies have indicated a cell type dependency for the role of IRF5 in cytokine gene induction (132, 133). Irf5−/− bone marrow–derived Ms and DCs show normal production of proinﬂammatory cytokines in response to LPS or peptide glycan (PGN) (132), whereas DCs but not Ms display an impairment when stimulated with poly(rI:rC) (133). In addition, type I IFN gene induction in Irf5−/− splenic pDCs by CpG-A stimulation is almost normal (130) but is diminished in bone marrow–derived Irf5−/− pDCs (132). The activation mechanism of IRF5, a more detailed mode of IRF5 action in the nucleus, and the relationship between IRF5 and other mediators such as NF-κB and IRF7 are important issues to be clariﬁed in the future. MyD88 signaling pathway for IRF1 activation. In addition to IRF7 and IRF5, IRF1 also directly interacts with MyD88 (134). Although IRF1 is strongly induced by type II IFN (IFN-γ), IFN-γ signaling itself cannot fully activate IRF1. Instead, TLR9 engagement causes MyD88-associated IRF1 to undergo posttranslational modiﬁcations and migrate into the nucleus more efﬁciently than does non-MyD88-associated IRF1. The critical role of MyD88-dependent “IRF1 licensing” is underscored by the observation that the induction of a speciﬁc gene subset downstream of the TLR-MyD88 pathway—such www.annualreviews.org • The IRF Family Transcription Factors

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as those genes encoding IFN-β, inducible nitric oxide synthase (iNOS), and IL-12p35— is impaired in Irf1−/− cDCs and Ms stimulated with IFN-γ plus CpG (134). Thus, IRF1 is critical for the IFN-γ enhancement of the TLR-dependent gene induction program.

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IRF4 and IRF8 in TLR signaling. Further studies on other IRF family members revealed that IRF4 also forms a complex with MyD88 (133). Although IRF4 resides mostly in the nucleus, a signiﬁcant fraction is also present in the cytoplasm, where it colocalizes with MyD88. Unlike the MyD88-interacting IRFs described above, IRF4 negatively regulates TLR signaling, as TLR-induced proinﬂammatory cytokines are enhanced in Irf4−/− cells (133, 135). Furthermore, Irf4−/− mice are highly sensitive to endotoxin shock induced by CpG-B, a potent inducer of proinﬂammatory cytokines (122, 136). Importantly, Irf4 mRNA is induced upon TLR activation; because IRF4 and IRF5 bind to the same region of MyD88, TLR-induced IRF4 can compete with and inhibit the sustained binding of IRF5 to MyD88. This suggests a role for IRF4 in the negative-feedback regulation of TLR signaling. As the expression of IRF4 is restricted to immune cells, particularly B cells, T cells, Ms, and DCs (12, 137, 138), IRF4 may selectively control MyD88-dependent gene regulation in a cell type–speciﬁc manner. IRF8 is another immune cell–speciﬁc IRF family member and has homology with IRF4. Recent studies have shown that IRF8 participates in the TLR9-MyD88-dependent signaling pathway. For example, Irf8−/− DCs fail to produce proinﬂammatory cytokines such as TNF-α and IL-6 upon stimulation with CpG, whereas normal levels of these cytokines are produced in response to LPS. This defect coincides with the inability of Irf8−/− DCs to activate NF-κB and MAPKs in response to TLR9 stimulation (139), suggesting that IRF8 can act upstream of NF-κB. Although IRF8 does not bind to MyD88 (133), IRF8 does interact with TRAF6 (140), indicating that IRF8 may function in the cytosol. In the nucleus, 548

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IRF8 is required for the expression of the gene encoding IL-12p40 upon various PAMP stimuli in Ms and DCs (141–145) and for the induction of type I IFN genes by viruses and TLR ligands in DCs (52) (also see below). Although the precise mode of IRF8 action is still incompletely understood (for example, it is unknown if IRF8 translocates from the cytoplasm to the nucleus upon PAMP stimulation), IRF8’s participation in the MyD88dependent signaling pathway may add to the diversity of the signaling cascade downstream of TLRs and enable the cell type– and ligandspeciﬁc activation of target genes.

Viral Factors Affecting IRF Activation Pathways Many viruses have evolved mechanisms to dispose of the host immune response. Given the diverse and potent effects of IRFs on the immune system, it is not surprising that these transcription factors and their activation pathways are the target of viral immune disturbance. For example, the V proteins of paramyxoviruses associate with MDA5 and inhibit dsRNA-induced activation of the IFNβ promoter (19). Vaccinia virus produces N1L to antagonize TLR signaling at the level of IKKs and TBK1 (146). Hepatitis C virus (HCV) encodes nonstructural proteins 3 and 4A (NS3/4A) protease that cleave IPS-1 and TRIF, thereby inhibiting the activation of IRF3 and/or IRF7 during HCV infection (147, 148). Infected cell protein 0 (ICP0) from HSV-1 inhibits IRF3 activation (149). Rotavirus nonstructural protein 1 (NSP1) mediates the degradation of IRF3, IRF5, and IRF7 (150, 151). Kaposi’s sarcoma–associated herpesvirus/human herpes virus 8 encodes replication and transcription activator, a ubiquitin E3 ligase that promotes IRF7 ubiquitination and proteasome-mediated degradation (152). Furthermore, Kaposi’s sarcoma–associated herpesvirus encodes a cluster of three viral IRFs, vIRF1, vIRF2, and vIRF3/latencyassociated nuclear antigen 2 (LANA2)

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(153–155). Although vIRF1 displays a high sequence similarity to IRF8 (156), all three vIRFs show homology in their N-terminal regions to the DBD of IRFs. However, these factors lack several of the tryptophan residues that are essential for DNA binding and thus, in contrast to IRFs, are presumed to be unable to bind directly to DNA. Although its precise mechanism has not been elucidated, vIRF1 functions as a repressor of virus-mediated induction of type I IFN genes in a transient transfection assay (157–159). Additionally, vIRF2 and vIRF3/LANA2 also inhibit the activation of promoters of type I IFN genes, which may involve interference with host IRFs. Indeed, vIRF3/LANA2 binds to and inhibits the DNA-binding activity of IRF7 (160, 161). The fact that viruses have exploited derivatives of IRFs to compromise antiviral immunity underscores the importance of the IRF systems in immunity against viral infections and highlights the coevolution of vertebrates and viruses.

Implications for Autoimmune Diseases The accurate discrimination between self and nonself molecules is critical for host defense. Besides the beneﬁcial aspects of the host defense system against nonself pathogenderived molecules via PRRs, ample evidence suggests that the aberrant activation of immune systems, evoked by the recognition of self molecules such as self-DNA or -RNA, contributes to the development of autoimmune diseases. Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by the loss of tolerance to diverse self nucleic acid–containing macromolecules, such as U1 small nuclear RNA with proteins (snRNPs) and DNA and histones (162–166). SLE patients make high-titer auto-antibodies to chromatin, leading to the formation of immune complexes that can bind to Fc receptors and stimulate PRRs, in particular TLR7, TLR8, or TLR9 (162–164, 166). Therefore, the aberrant activation of TLR7/8/9-

mediated IRFs by immunogenic self-RNA or self-DNA might contribute to the pathogenesis of SLE. Supporting this notion, two recent studies reported an allelic association between the IRF5/rs2004640 T allele and SLE (167, 168), although further studies are needed to address fully how IRF5 contributes to SLE. A better understanding of the IRF activation pathways may provide a platform for developing speciﬁc therapeutics for autoimmune diseases such as SLE.

REGULATION OF IMMUNE CELL DEVELOPMENT In addition to the functions assigned to IRFs in differentiated immune cells, multiple IRFs (IRF1, IRF2, IRF4, and IRF8) have pivotal roles in the development of immune cells, such as dendritic, myeloid, NK, B, and T cells, as recent studies have revealed. These ﬁndings have added to our understanding of the molecular mechanism for how the immune system is assembled from numerous and highly specialized cell types.

Systemic lupus erythematosus (SLE): an autoimmune disease characterized by the loss of tolerance to a diverse set of self-antigens and the consequent production of autoantibodies Antigen-presenting cell (APC): a cell that displays antigens to T cells via the major histocompatibility complex (MHC) Th: T helper or effector T cells; express CD4 and include Th1, Th2, Th17, and follicular B helper T

DC Subset Development Governed by IRF8, IRF4, IRF2, and IRF1 DCs are professional antigen-presenting cells (APCs) that are crucial in initiating innate and adaptive immune responses (169). These cells sense invading pathogens through PRRs, as described above, and respond by secreting various cytokines and by upregulating the expression of major histocompatibility complex (MHC) II and costimulatory molecules on their cell surface. DCs also capture and process antigens and present antigenic peptides on MHC molecules to T cells, thereby eliciting Th1 and Th2 responses or inducing tolerance. Importantly, DCs are a heterogeneous population of multiple subtypes with diverse functions (170, 171). For instance, mouse splenic DCs can be classiﬁed into at least four subsets: CD4+ DCs, CD8α+ DCs, CD4− CD8α− (double negative, DN) DCs, and pDCs. These subsets express different www.annualreviews.org • The IRF Family Transcription Factors

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sets of genes and manifest distinct functions (172). Generation of DC subsets regulated by IRF8 and IRF4. The expression of two structurally related IRFs, IRF8 and IRF4, is subset-selective (138, 173, 174). CD8α+ DCs and pDCs express high levels of IRF8, but no and low levels of IRF4, respectively. Conversely, IRF4 expression is high in DN DCs and CD4+ DCs, but IRF8 expression is low and absent, respectively. Analysis of Irf8−/− , Irf4−/− , and Irf8−/− Irf4−/− mice reveals that the above pattern of IRF8/IRF4 expression correlates with their requirement for DC subtype development (Figure 3). Thus, CD8α+ DCs and pDCs are largely missing in Irf8−/− mice, whereas the number of CD4+ DCs is severely reduced in Irf4−/− mice (138, 142, 173–175). Irf8−/− Irf4−/− mice have only a few DN DCs and totally lack other DC subtypes in the spleen (173). The few residual CD11c+ DC-like cells arising in Irf8−/− Irf4−/− mice are devoid of CIITA and MHC II expression at the mRNA levels and are unable to stimuDC progenitors IRF4

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Figure 3 Regulation of DC development by IRF family members. IRF8 is required for the development of CD8α+ DCs and pDCs (142, 175). IRF4 is critical for the development of CD4+ DCs (138, 173). The differentiation of DN DCs requires IRF8 or IRF4 (173). pDC development is also partially regulated by IRF4. The requirement of IRF8 and IRF4 for DC subset development correlates with their subset-selective expression. IRF8 is also important for functional differentiation of Langerhans cells (LCs) (176). IRF2 is required for the development of splenic CD4+ DCs as well as CD4+ LCs through negatively regulating type I IFN signaling (179, 180). IRF1 positively regulates CD8α+ DC differentiation, whereas it negatively regulates pDC differentiation (182). 550

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late T cell proliferative responses (173). Granulocyte macrophage colony-stimulating factor (GM-CSF)-mediated DC differentiation depends on IRF4, whereas Fms-like tyrosine kinase 3 (Flt3) ligand-mediated differentiation depends largely on IRF8 (138, 173). Furthermore, the full differentiation and function of epidermal Langerhans cells (LCs) and dermal DCs require IRF8 (176). Gene transfer experiments into Irf8−/− Irf4−/− bone marrow progenitor cells in vitro demonstrated that both IRFs have an overlapping activity to drive common processes of DC development, such as the induction of Ciita (encoding CIITA), while also possessing distinct activities in order to stimulate subset-speciﬁc gene expression, leading to the generation of functionally divergent DCs (173). It is tempting to speculate that DN DCs might represent a “prototype” DC subset generated via a default developmental pathway and that the emergence of IRFs permitted the acquisition of more diverse DC subtypes and functions. DC function regulated by IRF8. The above studies also indicate the speciﬁc role of IRF8 in DC function, i.e., DCs’ response to immune stimuli. Irf8−/− DCs, but not Irf4−/− DCs, fail to produce IL-12p40 or IFN-α in response to various PAMPs or NDV (142, 143, 173, 174). Moreover, the introduction of IRF8, but not IRF4, can confer upon Irf8−/− Irf4−/− DCs the ability to produce these cytokines in response to several PAMPs (173). The capability of IRF8 to support the production of type I IFNs and IL12 is achieved not only by the regulation of DC development but also by a direct involvement of IRF8 in the transcriptional regulation of the genes encoding these cytokines. It has been repeatedly documented that IRF8 serves as a transcriptional activator of IL-12p40 and IL-12p35 genes (141, 144, 145, 177). A recent study demonstrates the active participation of IRF8 in transcription of type I IFN genes (52). In addition, the knockdown of Irf8 expression shows that IRF8 is required for the positive

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regulation of Indo (encoding indoleamine 2,3 dioxygenase; IDO) and the negative regulation of Tyrobp (encoding DAP12) in tolerogenic DCs (IFN-γ-treated mouse CD8α+ DCs and LPS-matured, IFN-γ-treated human monocyte-derived DCs) (178). DC development regulated by IRF2 and IRF1. IRF2 and IRF1 also regulate DC subset development. Irf2−/− mice exhibit a selective loss of splenic and epidermal CD4+ CD8α− DCs. This defect is due to abnormally augmented type I IFN signaling; the deﬁciency was rescued by the introduction into Irf2−/− mice of a null mutation for the IFN receptor complex (thus, Irf2−/− Ifnar1−/− ) (179, 180). Interestingly, although type I IFNs can promote the latestage differentiation or maturation of DCs (181), these cytokines attenuate differentiation if added during the early stages of DC development (18, 179). Irf1−/− mice, in contrast, exhibit a modest but constant increase in pDC and decrease in CD8α+ DC numbers (182). Irf1−/− DCs express much higher levels of IL-10, TGFβ, and the tolerogenic enzyme IDO, while being defective in IL-12p40 production. As a consequence, Irf1−/− DCs fail to mature fully, are unable to stimulate the proliferation of allogeneic T cells, and induce an IL10-mediated suppressive activity in allogeneic CD4+ CD25+ regulatory T cells, suggesting a novel role of IRF1 in regulating the tolerogenic features of DCs (182). Altogether, multiple IRFs are critically involved in the regulation of DC development and function. Through their speciﬁc and common activities, IRFs appear to equip DCs with the diversity required for directing optimal immune responses. The role of IRFs in human DC subtypes is less clear than it is in mouse DCs at present. However, transcripts from the genes encoding IRFs are expressed in human DCs (126, 183–185). Future studies for identifying IRF target genes, interacting proteins, and the relationship with other transcription factors known to regulate DC

development such as PU.1 and Spi-B, both of which may be common partners for IRF8 and IRF4, as well as RelB, STAT3, Gﬁ1, Ikaros, Runx3, and Id2 (186), would further clarify the molecular programs required for the development and function of DC subtypes.

Myeloid Cells: MΦ and Granulocyte Differentiation Regulated by IRF8 and Other IRFs Role of IRF8 in myeloid cell development. An unexpected role of IRF8 in myeloid cell development was ﬁrst suggested by the observation that Irf8−/− mice exhibit a systemic expansion of neutrophils followed by a fatal blast crisis, resembling human chronic myelogenous leukemia (CML) (187). In addition, a mutation in the Irf8 gene causes a CML-like syndrome in BXH-2 mice (188). Subsequent studies have revealed that there is a cell-intrinsic function of IRF8 in the differentiation, growth, and apoptosis of myeloid cells. Irf8−/− mice harbor increased numbers of progenitor cells that are hyperresponsive to both GM-CSF and granulocyte colonystimulating factor (G-CSF) (189). Cell transfer studies show an intrinsic leukemogenic potential and long-term reconstitution capability of Irf8−/− progenitors. In contrast, their response to macrophage colony-stimulating factor (M-CSF) was strongly reduced, and, surprisingly, most of the Irf8−/− progenitor cells differentiated into granulocytes even in the presence of M-CSF. Consistent with these observations, there are signiﬁcantly fewer cells of the M lineage in Irf8−/− bone marrow than in wild-type counterparts (189). Myeloid progenitor cells give rise to granulocytes and Ms. Studies with Irf8−/− myeloid progenitor cell lines and freshly isolated bone marrow progenitor cells from Irf8−/− mice have indicated IRF8’s role in the lineage selection of myeloid progenitors; IRF8 drives their differentiation toward Ms and inhibits granulocytic (neutrophilic) differentiation (190, 191) (Figure 4). Moreover, in both cases IRF8 strongly inhibits cell www.annualreviews.org • The IRF Family Transcription Factors

CML: chronic myelogenous leukemia Apoptosis: also called programmed cell death, a highly regulated mechanism of altruistic suicide of a cell that occurs during development and host defense in multicellular organisms

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Granulocyte /monocyte progenitors

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Basophils Neutrophils

MΦs Figure 4

The role of IRFs in myeloid cell development. IRF8 controls the fate of myeloid progenitor cells by directing M differentiation and inhibiting neutrophilic differentiation (189–191). In addition, IRF8 negatively regulates proliferation and promotes apoptosis in myeloid cells. As a result, Irf8−/− mice develop a CML-like syndrome (187). Importantly, IRF8 expression is severely diminished in human CML patients (197). IRF1 is required for the full maturation of neutrophils and Ms (219). IRF2 negatively regulates the expansion of basophils (224).

growth. Consistent with these observations, IRF8 expression is detected in mouse and human hematopoietic progenitor cell populations and persists in Ms, whereas it declines in granulocytes (191, 192). Also, IRF8 positively regulates apoptosis in myeloid cells (193). Irf8−/− Ms terminate M-CSF signaling more rapidly than do wild-type cells owing to accumulation of c-Cbl, a ubiquitin ligase that targets an activated M-CSF receptor for degradation (194). These results demonstrate IRF8’s critical role in the development of myeloid cells and illustrate why the loss of IRF8 leads to a CML-like syndrome. Given that IRF8 transcript levels are severely reduced in cells from human CML patients (195–197), CML cells may have a previously unrecognized defect in the IRF8regulated myeloid differentiation program, i.e., a disproportional differentiation toward granulocytes.

IRF8 target genes and MΦ differentiation. IRF8 acts either as an activator or a repressor depending on its interacting factors and 552

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target DNA elements (198, 199). On the one hand, IRF8 associates with IRF1, IRF2, and IRF4 and the Ets repressor TEL on ISREs that have a tandem repeat of the core IRFbinding motif GAAA (200). In this case, the overexpression of IRF8 generally represses ISRE-driven transcription, although only a limited number of genes such as Isg15 are known to be affected by endogenous IRF8. On the other hand, IRF8 also interacts with an Ets transcription factor PU.1, the master regulator of B cell and M differentiation. Here, IRF8 activates transcription of genes that carry the Ets-IRF composite element (EICE, GGAANNGAAA), the Ets/IRF response element (EIRE, GGAAANNGAAA, a subset of the ISREs), or the IRF-Ets composite sequence (IECS, GAAANN(N)GGAA). The EICE is especially critical in the B cell lineage, whereas during M differentiation the IECS is responsible for multiple IRF8 target genes such as those encoding Cathepsin C and Cystatin C (201). The EICE and EIRE are also active in cells stimulated by IFN-γ, which strongly induces IRF8 gene expression. Other DNA elements that do not fall into the above consensus sequences, for instance those found in Cdkn2b and Nf1 genes, are also targeted by IRF8 during M differentiation (202, 203). Overall, studies of IRF8 target genes show that IRF8 controls several key genes that repress cell growth and apoptosis in myeloid cells, while it also induces many genes important for M function. During growth control, IRF8 directly induces Prdm1 and Etv3, genes that encode for Blimp-1 and METS, respectively (204). Both genes are transcriptional repressors of Myc and are capable of inhibiting cell growth. In addition, IRF8 directly activates the transcription of Cdkn2b, which encodes an inhibitor for cyclin-dependent kinase p15Ink4b (202). Furthermore, IRF8 represses antiapoptotic genes Bcl2l1 (encoding Bcl-XL ) and BCL2 and thereby controls apoptosis (193, 205). Interestingly, the Nf1 gene, which encodes the Ras-GAP neuroﬁbromatosis 1 (NF1) protein, is a direct target gene for IRF8 (203, 206). NF1 inactivates

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Ras in hematopoietic cells, and Nf1−/− hematopoietic cells cause myeloproliferative symptoms because of hypersensitivity to GMCSF. These authors show that IRF8 induces Nf1 to suppress Ras signaling and cell proliferation of myeloid cells in response to GM-CSF or M-CSF. Another recent ﬁnding describes IRF8 as indispensable for the expression of the Pml gene and the formation of nuclear bodies in myeloid cells (207). Given that the promyelocytic leukemia protein (PML) is important for a variety of cell functions such as cell cycle regulation, apoptosis, DNA repair, and tumor suppression, PML may be responsible for some of the leukemia suppressor activity of IRF8. Although the mechanism of IRF8 stimulation of M differentiation and function has been clariﬁed to the degree described above, the mechanism of how IRF8 represses granulocytic differentiation is still poorly deﬁned.

IRF8 target genes for MΦ function. IRF8 also directly induces several endosomal/lysosomal enzyme-related genes such as those encoding cathepsin C, lysozyme, cystatin C, and prosaposin (201) and represses the Dab2 gene encoding disabled-2 that stimulates M adhesion and spreading (208), enabling Ms to establish their proper functionality. Moreover, IRF8 contributes to the induction of numerous genes in IFN-γtreated cells. In addition to the target DNA elements described above, IRF8 can stimulate transcription of genes carrying a subset of the IFN-γ activation site (GAS) via indirect DNA binding (209). IRF8 target genes in cells treated with IFN-γ and PAMP are regulated by multiprotein complexes containing IRF8 and other transcription factors, especially PU.1 and in some cases IRF1; these genes include those encoding IL-12p40, IL-12p35, gp91phox , p67phox , TLR4, TLR9, iNOS, Fcγ receptor I (FcγRI), IRF8 itself, IL-18, CCL5/RANTES, and Nramp1 (198, 210–212).

Owing to the defects in the development and function of DCs and Ms described above, Irf8−/− mice are highly susceptible to various pathogens such as Listeria monocytogenes (213), vaccinia virus (187), lymphocytic choriomeningitis virus (LCMV) (187), Toxoplasma gondii (214), Leishmania major (215), Yersinia enterocolitica (216), and Brucella abortus (217). Regulation of myeloid differentiation by IRF1 and IRF2. Irf1 expression is upregulated during myeloid differentiation (218). Irf1−/− bone marrow cells exhibit an increased number of immature granulocytic precursors while their colony-forming ability in response to both G-CSF and M-CSF is decreased, suggesting a defective maturation process (219) (Figure 4). As an IFN-inducible transcriptional activator for the ISRE-containing genes, IRF1 transcriptionally targets many genes, including those encoding GBP, iNOS, Caspase-1, Cox-2, CIITA, and gp91phox in IFN-stimulated Ms (2, 220–223). In IFNγ-primed PAMP-stimulated cells, IRF1 is further activated by the TLR-MyD88 pathway, as already discussed, and induces a subset of genes such as those encoding IL-12p40, IL-12p35, iNOS, IL-18, and IFN-β (2, 134, 223). Irf2−/− mice display an expansion of basophils, resulting in an increase in IL-4 production (224) (Figure 4). The same study shows that these abnormalities are responsible for the excess Th2 polarization observed in naive Irf2−/− mice. An in vitro ectopic expression study shows that IRF2 is inhibitory to granulocytic differentiation and stimulates megakaryotic differentiation (225). In Irf2−/− Ms, the expression of the gene encoding Caspase-1 is enhanced, suggesting that IRF2 counteracts IRF1 (226). This augmented Caspase-1 expression may be responsible for the accelerated apoptosis observed in Irf2−/− Ms treated with a fungal metabolite gliotoxin or IFN-γ plus LPS (226, 227). Unexpectedly, the induction of genes encoding IL-12p40 and Cox-2 has been found to be www.annualreviews.org • The IRF Family Transcription Factors

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Germinal center (GC): a site within secondary lymphoid organs where B cells perform somatic hypermutation and class switch recombination following antigen and T cell stimulation

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diminished in Irf2−/− Ms treated with IFN-γ and/or LPS, suggesting that IRF2 cooperates with, rather than antagonizes, IRF1 to induce these genes (220, 228, 229).

The Development of Natural Killer Cells Regulated by IRF1 and IRF2 by Distinct Mechanisms Several studies have shown that the numbers of NK (NK1.1+ TCRα/β− ) cells are dramatically reduced, and NK cell activities such as cytotoxicity and IL-12-dependent IFN-γ production are absent in Irf1−/− mice (230– 233). The lack of IRF1 selectively affects the bone marrow stromal cells that constitute the microenvironment for NK cell development, but not NK progenitors, per se (Figure 5). Indeed, IRF1 in stromal cells is required for transcription of the gene encoding IL-15, a cytokine essential for NK cell development (231). This defect of Irf1−/− mice in IL-15 production also causes impaired development

IRF2

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Bone marrow stroma cells Figure 5 Regulation of NK cell development by IRF1 and IRF2. IRF1 is indispensable for NK cell development by regulating the expression of IL-15, a cytokine that supports NK cell development in bone marrow stromal cells (231). Although not shown in this ﬁgure, IRF1 is also essential for the development of NKT cells and intestinal intraepithelial T cells (232), because these cells also require IL-15 for their development. IRF2, on the other hand, supports NK cell development in an NK cell–intrinsic manner (234). 554

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of NKT (NK1.1+ TCRα/β+ ) cells and intestinal intraepithelial T cells (232). Irf2−/− mice also carry an impairment in NK cell development and function (234) (Figure 5). Unlike the case of IRF1, however, IRF2 affects NK cell development in an NK cell–intrinsic manner. Irf2−/− mice selectively lack mature CD11bhigh Dx5high NK cells, likely because of accelerated apoptosis (235).

B and Plasma Cells: Multiple Developmental Steps Involving IRF4 and IRF8 Pre-B to B cell transition regulated by IRF4 and IRF8. The cooperative regulation of immune cell development by IRF4 and IRF8 also operates in B cell differentiation. Both IRFs are expressed in immature states of B cells, including pre-B cells in the bone marrow (236, 237). A redundant role of IRF4 and IRF8 in promoting pre-B to IgM+ B cell transition was revealed by the observation that B cells in Irf4−/− Irf8−/− but not in Irf4−/− or Irf8−/− mice are arrested at the cycling preB stage (238) (Figure 6). These IRFs activate conventional immunoglobulin (Ig) light chain (i.e., κ and λ) gene transcription and rearrangement, while they downregulate the preantigen receptor complex by suppressing the expression of surrogate light chain VpreB and λ5 genes (238, 239). Indeed, it is well documented that IRF4 and IRF8 interact with Ets transcription factors PU.1 or SpiB on the EICEs found in the Ig κ 3 and λ enhancers to activate transcription (240, 241). Supporting evidence for the overlapping function of the two IRFs was shown when introduction of either IRF4 or IRF8 rescued the maturation arrest of Irf4−/− Irf8−/− B cells in vitro (239). The role of IRF8 in the germinal center program. Upon T cell–dependent antigen challenge, peripheral IgM+ B cells enter the GC of secondary lymphoid tissues, where they undergo afﬁnity maturation and

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isotype switching. Interestingly, IRF8 expression increases and IRF4 expression is suppressed in centroblasts within the dark zone (236, 242). IRF8 directly regulates the induction of two critical genes, AICDA and BCL6, during the GC reaction in both human and mouse (243). The AICDA gene encodes activation-induced cytidine deaminase (AID), which is required for both somatic hypermutation and class switch recombination, and the BCL6 gene encodes B cell lymphoma ¨ 6 (BCL6) protein, a Kruppel-type zinc ﬁnger transcriptional repressor that functions as a master regulator of the GC program. Although GCs can still form in Irf8−/− mice, Irf8−/− GC B cells show less organized morphology and reduced levels of Aicda and Bcl6 gene expression (243) (Figure 6). Regulation of germinal center reaction and plasma cell differentiation by IRF4. When centrocytes in the GC light zone differentiate into high-afﬁnity antibody-producing plasma cells, the expression of IRF8 declines, whereas the expression of IRF4 gradually increases (236, 244, 245). An earlier study has reported that Irf4−/− mice display a profound reduction in serum Ig, fail to produce antigenspeciﬁc antibodies, and do not generate GCs (137). A more detailed role for IRF4 in isotype switching and plasma cell differentiation was recently discovered by two groups (244, 246) (Figure 6). Using mice with conditional or conventional deletion of Irf4 alleles, these studies showed that class switch recombination and plasma cell differentiation from centrocytes or memory B cells require IRF4. Both studies also showed that IRF4 is indispensable for Aicda gene induction, illustrating the molecular basis for the failure of isotype switching in Irf4−/− B cells. Furthermore, one of the studies identiﬁed the Prdm1 gene as a direct target of IRF4 (244), although the other group found no regulation of Prdm1 by IRF4 (246). The Prdm1 gene encodes the zinc-ﬁnger transcriptional repressor Blimp-1, which is a master regulator

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Figure 6 Regulation of B cell and plasma cell differentiation by IRF4 and IRF8. Differentiation from pre-B to IgM+ B cell requires IRF4 or IRF8 (238). IRF8 positively regulates the germinal center (GC) program (243). IRF4 is essential for class switch recombination in the late stage of GC reaction and for plasma cell differentiation from centrocytes or memory B cells (244, 246).

of plasma cell differentiation (247). Although AID and Blimp-1 are key components of antagonistic developmental states (GC reaction and plasma cell differentiation, respectively), graded expression of IRF4 sequentially activates the two genes; during the ﬁnal stage of GC reaction, a relatively low level of IRF4 stimulates the expression of the Aicda gene to achieve class switch recombination, and then a higher concentration of IRF4 induces the Prdm1 gene that in turn represses the expression of Bcl6 and Aicda, thereby terminating the GC reaction and inducing plasma cell differentiation (244). The induction of Prdm1 by IRF4 is reminiscent of that by IRF8 in myeloid progenitor cells. However, the reason IRF8 does not induce Prdm1 in GC B cells may be because of the potent repression by Bcl6 in order to maintain the GC reaction program. Interestingly, IRF4 can interact with STAT6 and promote the expression of IL-4-inducible genes in B cells treated with CD40 and IL-4 (248). In addition to the above developmental defects, elder (10 to 15 weeks) Irf4−/− mice display a generalized lymphadenopathy because of an expansion of B and T cells in lymph nodes and spleen (but not thymus), suggesting that IRF4 is also important for the homeostasis of mature lymphocytes (137). www.annualreviews.org • The IRF Family Transcription Factors

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T Cells: Thymocyte Development and Th1/Th2 Differentiation Regulated by Multiple IRFs

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Development and/or function of CD8 + T cells regulated by IRF1, IRF2, and other IRFs. Irf1−/− mice display a profound reduction of mature CD4− CD8+ T cells in the thymus and peripheral lymphoid organs, suggesting a lineage-speciﬁc defect in thymocyte development (53) (Figure 7). Although Irf1−/− thymic stromal cells express decreased IRF2

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Figure 7 T cell differentiation regulated by IRF family members. In the thymus, IRF1 plays an essential role in CD8+ T cell development (53). Cytotoxic T lymphocyte (CTL) activation is positively regulated by IRF1, IRF4, and IRF8 (53, 137, 187). IRF2 is required for preventing hyperresponsiveness of CD8+ T cells to antigen stimulation, which is mediated by IRF2’s negative regulation of type I IFN signaling (252). Th1 differentiation requires IRF1, IRF2, and IRF8 (214, 215, 228, 233, 256). IRF1 is indispensable for CD4+ T cells to respond to the Th1 cytokine IL-12 (233) and is also required for the induction of genes encoding IL-12 in Ms and DCs (134, 233, 256). In addition, IRF1 is essential for the development of NK cells that produce IFN-γ, a M-stimulating cytokine (231, 232). IRF8 is critical for differentiation of Ms and several DC subsets and also for the induction of IL-12p40 in these cells (142, 175, 190, 214, 215). IRF2 is required for NK cell development and the induction of the gene encoding IL-12p40 (228, 234). In addition, IRF2 negatively regulates Th2 polarization in naive mice by limiting the number of basophils that secrete the Th2 cytokine, IL-4 (224). IRF4 is required for CD4+ T cells to differentiate into Th2 cells (261). 556

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levels of LMP2, TAP1, and MHC I (53, 249), the defect in CD8+ T cell development in Irf1−/− mice does not reside in the thymic environment and is instead due to thymocyteintrinsic defects in differentiation from immature T cells (TCRαβ− CD4+ CD8+ ) to mature CD8+ T cells (250). Further studies using MHC I–restricted TCR transgenic mice (H-Y and P14) reveal that IRF1 controls both the positive and negative selection of CD8+ thymocytes (250). Moreover, Irf1−/− thymocytes are defective in TCR-mediated signal transduction, and the induction of negative selection in TCR transgenic thymocytes from Irf1−/− mice require a 1000-fold higher level of the selecting peptide than that for wild-type mice. Thus, IRF1 may regulate the expression of gene(s) required for lineage commitment and selection of CD8+ T cells in developing thymocytes (250). The introduction of a Bcl2 transgene driven by the Eμ or lck promoter in Irf1−/− mice restores the development of CD8+ T cells (but not NK, NKT, or TCRγδ+ intestinal intraepithelial lymphocytes), suggesting that IRF1 may be required for survival signals involving Bcl2 to support CD8+ T cell development (251). Consistent with the impairment of CD8+ T cell in Irf1−/− mice, the cytotoxic T lymphocyte (CTL) response to LCMV-infected target cells is signiﬁcantly reduced in these mice. In contrast, Irf2−/− mice show normal CTL activity against LCMV-infected target cells, and some nonspeciﬁc cytotoxicity was detected (53). Upon LCMV infection, Irf4−/− mice show no CTL activity against target cells, whereas in Irf8−/− mice CTL responses after VSV and vaccinia virus infection revealed a three- to tenfold reduction of CTL activity (137, 187). Therefore, IRF1, IRF2, IRF4, and IRF8 all contribute to the regulation of CTL activity (Figure 7). The CD8+ T cell abnormality in naive Irf2−/− mice causes a spontaneous inﬂammatory skin disease resembling psoriasis (53, 252). Irf2−/− CD8+ T cells exhibited a hyperresponsiveness to antigen stimulation in vitro, accompanied by a notable upregulation

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of type I IFN–inducible gene expression. Furthermore, disease development and CD8+ T cell abnormality are suppressed by the introduction of nullizygosity to the genes that positively regulate the type I IFN signaling pathway (252). Thus, IRF2 represents a unique negative regulator that attenuates type I IFNinduced gene transcription and is necessary for balancing the beneﬁcial and harmful effects of type I IFN signaling in the immune system. IRF4 is expressed in mature T cells and is further induced by concanavalin A or CD3 cross-linking in mature T cells (253). In Irf4−/− mice, thymocyte development appears to take place normally, but T cell functions such as cytokine production, cytotoxic activity, and proliferation are abrogated. In addition, adult Irf4−/− mice display progressive lymphadenopathy (137). Two conﬂicting results have been reported with regard to IRF4 regulation of apoptosis in T cells. One study shows that ectopic expression of IRF4 in the Jurkat human T cell line results in a signiﬁcant enhancement of Fas-mediated apoptosis and that primary CD4+ T cells from Irf4−/− are defective in activation-induced cell death and in Fas receptor polarization (254). However, another study demonstrated that Irf4−/− CD4+ T cells are highly sensitive to apoptosis by TCR or Fas triggering (255). The latter study further shows that upon Leishmania major infection in Irf4−/− mice, the lesiondraining lymph nodes initially develop typical lymphadenopathy, but that this hyperplasia is followed by enhanced apoptosis and almost total loss of cellularity. Regulation of Th1/Th2 differentiation by IRF1, IRF2, IRF4, and IRF8. IRF1 is indispensable for the development of Th1-type immune responses, and its absence leads to the induction of Th2-type immune responses (233, 256). In fact, Irf1−/− mice are vulnerable to Listeria monocytogenes and Leishmania major, but resistant to Nippostrongylus brasiliensis (233, 256). The compromised Th1 differentiation of CD4+ T cells in Irf1−/− mice is due

to defects in multiple cell types (Figure 7). First, as described above, Irf1−/− Ms and DCs are defective in the induction of IL-12, a cytokine essential for Th1 differentiation, because of the impaired expression of genes encoding IL-12p40 and IL-12p35. Second, Irf1 is a target gene of IL-12 (257, 258), and IRF1 activates the Il12rb1 promoter, rendering Irf1−/− CD4+ T cells hyporesponsive to IL-12 (233, 259). Third, Irf1−/− mice lack NK cells, which produce IFN-γ to stimulate Ms to secrete IL-12 (see above). Unexpectedly, IL-12 production is also suppressed in Irf2−/− Ms (228, 234), and Irf2−/− mice are susceptible to Leishmania major infection owing to a defect in Th1 cell differentiation (234) (Figure 7). IRF2 apparently contributes to IL-12p40 gene expression in cooperation with IRF1 and other factors, rather than functioning as a transcriptional repressor. The defective NK cell differentiation present in Irf2−/− mice likely contributes to the impaired Th1 response (see above). In addition, excessive Th2 polarization in naive Irf2−/− mice is attributed to the expansion of basophils (see above) that produce the Th2 cytokine IL-4. Interestingly, IFN-γ represses the expression of IL-4 via IRF1 and IRF2 to promote Th1 and attenuate Th2 responses (260). IRF8 and IRF4 oppositely regulate Th1/Th2 differentiation (Figure 7). Irf8−/− mice are impaired in developing Th1 responses (214, 215), whereas Irf4−/− mice are defective in mounting Th2 responses (261–263). Although activated T cells express IRF8, Irf8−/− T cells show a capacity for normal Th1/Th2 differentiation when transplanted into Rag2−/− mice (213); thus, the defective Th1 response in Irf8−/− mice is attributed to the defects in Ms and DCs. IRF8 is required for the expression of genes encoding IL-12, as already discussed. In addition, CD8α+ DCs, whose development depends on IRF8, are a major producer of the Th1-promoting cytokine IL-12 and preferentially drive Th1 responses (264, 265). www.annualreviews.org • The IRF Family Transcription Factors

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Irf4−/− T cells, in contrast, are unable to differentiate into Th2 cells. IRF4 stimulates the expression of the Th2-inducing cytokine IL-4 via physical interaction with NFATc2 and/or NFATc1 (262, 266). Naive T helper cells from Irf4−/− mice are defective in the production of IL-4 and other Th2 cytokines. Upon IL-4 treatment in vitro, Irf4−/− CD4+ T cells proliferate poorly and fail to express GATA3, a transcription factor critical for Th2 development, suggesting that IRF4 has a T cell–intrinsic role (261, 263). IRF4 is also required for the development of CD4+ DCs, which may contribute to the defective Th2 responses seen in Irf4−/− mice because they are thought to stimulate Th2 responses (264, 265). Indeed, IRF8 and IRF4 inversely affect the Th1/Th2 balance by regulating DCs (173). Consistent with its role in promoting humoral immune responses, IRF4 is required for the somatic hypermutation and formation of antibody-secreting plasma cells, as described above. Thus, IRF8 and IRF4 participate in and are critical for the induction of cellular and humoral immune responses, respectively.

Regulation of Keratinocyte Development by IRF6 In humans, mutations in the IRF6 gene cause two related disorders, Van der Woude syndrome and popliteal pterygium syndrome. Recently, two groups have shown that mice with null or missense mutations in the Irf6 gene have abnormal skin, limb, and craniofacial development (267, 268). Furthermore, both studies reveal a critical role for IRF6 in keratinocyte development; Irf6 mutant keratinocytes are hyperproliferative and fail to terminally differentiate. Interestingly, mice deﬁcient for IKKα, a kinase involved in the phosphorylation of IRF7 via TLR-MyD88 signaling, display a similar skeletal phenotype as a secondary effect of abnormal keratinocyte differentiation (269), suggesting that IRF6 and IKKα may converge on the same pathway(s). The role of IRF6 in immune responses 558

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is unknown, but given that keratinocytes express functional TLRs and MyD88 and are able to kill pathogenic fungi and bacteria by producing antimicrobial substances (270), the participation of IRF6 in the innate immune function of keratinocytes is an interesting possibility.

REGULATION OF ONCOGENESIS Another critical facet of IRFs’ function in host defense is the regulation of oncogenesis. Indeed, as outlined below, many IRFs, such as IRF1, IRF3, IRF5, and IRF8, possess tumor suppressor gene activities.

IRF1 in Tumor Suppression Regulation of cell cycle and apoptosis by IRF1. The notion that IRFs participate in the regulation of oncogenesis ﬁrst arose from studies performed on IRF1. The expression of IRF1 is regulated throughout the cell cycle; Irf1 mRNA expression is markedly elevated in NIH3T3 cells subjected to serum starvation but rapidly declines upon seruminduced cell cycle progression (271). Irf1−/− MEFs are deﬁcient in their ability to undergo DNA damage–induced cell cycle arrest, a phenotype similar to that observed in MEFs lacking the tumor suppressor p53 (272). Importantly, transcriptional induction of the gene encoding the CDK inhibitor p21WAF1/CIP1 by gamma irradiation depends on both IRF1 and p53. In such DNA-damaged cells, IRF1 protein level was elevated via an ATM-dependent increase in both mRNA expression and protein half-life, so as to act on the p21 promoter region containing the IRF1- and p53-binding sites (272, 273). Thus, these two transcription factors cooperate in the DNA damage– induced cell cycle arrest by activating a common target gene. Other reports have also demonstrated the involvement of IRF1 in cell growth arrest (274–277). Apoptosis is one mechanism by which precancerous cells can be eliminated from the

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host. IRF1 is required for apoptosis induced by DNA damage or other stimuli. Wild-type MEFs, if introduced with an activated oncogene such as c-Ha-Ras, undergo apoptosis instead of arresting the cell cycle when treated with anticancer drugs or ionizing radiation. This apoptosis is a hallmark of tumor suppression and is dependent on both IRF1 and p53 (278). DNA damage–induced apoptosis in mitogenically activated mature T lymphocytes is dependent on IRF1 but independent of p53 (279, 280), whereas in thymocytes it is dependent on p53 but not on IRF1. Thus, IRF1 and p53 regulate DNA damage–induced apoptosis cooperatively and independently, depending on the type and differentiation stage of the cell. Interestingly, GAAP-1, a transcriptional activator of both IRF1 and p53, has a proapoptotic activity (281). In addition, numerous studies have shown that IRF1 is also essential for apoptosis induced by other stimuli. For example, IRF1 is required for IFN-γ-induced apoptosis in primary cultured hepatocytes (274) and ovarian cancer cells (282) and for IFN-γ-mediated enhancement of Fas/CD95-induced apoptosis (283). The target genes of IRF1 responsible for apoptotic responses have not been ﬁrmly identiﬁed but may include genes encoding Caspase 1 (280, 282, 284), Caspase 7 (283, 285), Caspase 8 (286), and TRAIL (TNFrelated apoptosis-inducing ligand) (287). Tumor suppressive activity of IRF1. Consistent with the observations described above, a tumor suppressor–like activity for IRF1 has been demonstrated in oncogenic transformation assays using Irf1−/− MEFs. The malignant transformation of normal MEFs, as assessed by formation of colonies in a soft agar and of tumors in nude mice, requires the activities of at least two oncogenes (288). However, the loss of IRF1 and the introduction of activated c-Ha-Ras is sufﬁcient to transform Irf1−/− MEFs (278). Moreover, conditions under which activated Ras inhibits cell growth of myeloid cells involve IRF1 and the induction of p21WAF1/CIP1 (289). Multiple

studies also show that ectopic expression of IRF1 can suppress the malignant properties of cancer cell lines and oncogene-transformed cell lines in vitro and in vivo (290–293). In the mouse, the loss of IRF1 alleles alone rarely induces tumor development (294). However, IRF1 deﬁciency dramatically exacerbates tumor predispositions caused by the expression of a c-Ha-Ras transgene or by nullizygosity of the p53-encoding gene, Trp53 (294). This accelerated tumor development may not be due to the abovementioned immunological disorders that resulted in the absence of IRF1 because in chimeric mice consisting of Irf1−/− Trp53−/− cells and Trp53−/− cells, generated by the aggregation of their respective embryos, most tumors originate from Irf1−/− Trp53−/− cells (294). In addition to an increased incidence, notable alterations in the relative proportion of the types of tumors observed in Irf1−/− Trp53−/− mice suggest that IRF1 is not hypostatic with respect to p53. Furthermore, Irf1−/− Trp53−/− MEFs are more sensitive to drug-induced mutagenesis and exhibit hyperactive proliferation compared with single-null cells. Thus, Irf1 is a tumor susceptibility gene the loss of which, in combination with other genetic alterations, signiﬁcantly increases the incidence of tumors. In attempting to clarify the mechanism underlying accelerated tumor susceptibility in the absence of IRF1, the lysyl oxidase (Lox) gene was identiﬁed as a target of IRF1 (295). Lox plays a critical role in the biogenesis of connective tissue matrices and is identical to the independently discovered Ras recision gene (rrg) implicated in the reversion of Ras-transformed NIH3T3 cells by preventing activation of NF-κB (296, 297). Ectopic expression of Lox partially suppresses the transformed phenotype of c-Ha-Ras-expressing Irf1−/− MEFs, suggesting that Lox may be one of the IRF1 target genes involved in mediating IRF1’s tumor suppressive activity (295). IRF1 and human cancers. In addition to the functional analyses described above, a number www.annualreviews.org • The IRF Family Transcription Factors

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of clinical studies have indicated that the loss of IRF1 may promote the development of some forms of human cancer, especially leukemia. The IRF1 gene maps to the chromosomal region 5q31.1 (298, 299), a region that frequently shows cytogenetic abnormalities in leukemia and preleukemic myelodysplastic syndrome (MDS) (300). It has been reported that among 13 patients with leukemia or MDS who exhibited cytogenetic abnormalities in this region, IRF1 was the only gene found to be consistently deleted or rearranged in either or both alleles (299). Moreover, 12 of 14 patients with MDS/AML associated with 5q abnormalities also had a loss of one allele of IRF1 (301). In addition to IRF1’s association with hematopoietic malignancies, the loss of one IRF1 allele is correlated with esophageal and gastric cancers (302, 303), and, in one out of four cases of gastric cancers examined, the deletion is accompanied by an inactivating point mutation in the other allele (304). In addition to the genetic alterations at the IRF1 gene per se, several mechanisms lead to the loss-of-function of IRF1. Nucleophosmin, a putative ribosome assembly factor often overexpressed in leukemic cells, binds to IRF1 and inhibits its function (305). Other studies have suggested that splicing aberrations in the IRF1 gene also account for the loss of IRF1 expression (306, 307). A mechanism for inactivation of IRF1 by human papilloma virus (HPV) 16-encoded E7 oncoprotein has also been reported (308). In addition, numerous reports indicate a decreased expression of IRF1 mRNA in several types of cancers, such as CML, breast cancer, endometrial cancer, and hepatocellular carcinoma (307, 309–312).

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Tumor Suppressive Function of IRF5 IRF5 has emerged as another IRF family member that possesses a tumor suppressor ability. Using Irf5−/− mice, a recent study demonstrated that IRF5 is required for DNA damage–induced apoptosis; c-HaRas-expressing Irf5−/− MEFs fail to undergo apoptosis efﬁciently in response to X-ray ir560

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radiation or anticancer drug treatment (65), and Irf5−/− MEFs undergo malignant transformation upon c-Ha-Ras expression as measured by formation of colonies in a soft agar and by the establishment of tumors in nude mice. However, IRF5 is not necessary for DNA damage–induced cell cycle arrest because Irf5−/− MEFs (without c-Ha-Ras) do stop cell growth in response to the same stimuli as efﬁciently as do wild-type MEFs. In addition, Irf5−/− MEFs are resistant to VSVinduced apoptosis, resulting in enhanced viral propagation despite the fact that the cells can produce normal levels of type I IFNs and IL-6 (65). Using Ifnar1−/− and Trp53−/− MEFs, it has been shown that Irf5 mRNA is induced upon viral infection through type I IFN signaling and upon DNA damage by p53 (65), the latter of which is consistent with a previous report indicating that the IRF5 gene is a direct target of p53 (313). IRF5 is then activated by an unknown mechanism(s) and translocates to the nucleus where it may regulate apoptosis-related gene(s) (65, 314). Because several p53 targets, such as the genes encoding Puma and Noxa, are induced normally in Irf5−/− MEFs, it is likely that IRF5 acts on an apoptotic pathway distinct from that for p53 (65). Indeed, overexpression of IRF5 inhibits in vitro and in vivo B cell lymphoma tumor growth in the absence of wild type p53 (315), and ectopic expression of IRF5 sensitizes p53-proﬁcient and p53-deﬁcient colon cancer cells to DNA damage–induced apoptosis (314). Interestingly, IRF5 mRNA expression may be suppressed in human leukemia cells (315). Further studies are required to clarify the transcriptional pathway by which IRF5 stimulates apoptosis.

IRF8 and IRF4 in the Regulation of Leukemia Several lines of evidence have indicated an antagonizing relationship between IRF8 and myeloid leukemias, especially CML. Human CML is caused by Bcr/Abl, the

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t(9; 22)-derived fusion oncoprotein with an aberrant kinase activity. As already discussed, Irf8−/− mice develop a CML-like syndrome (187). Consistent with this, IRF8 transcripts are absent in CML and AML patients, and several IRF8 target genes such as Bcl2 and Pml are downregulated in CML cells (205, 207). Moreover, therapeutic IFN-α for human CML induces IRF8 expression in vivo (197), and IRF8 expression correlates positively with pretreatment risk features and cytogenetic response to IFN-α in CML (196). In the laboratory, ectopic expression of IRF8 is able to override the mitogenic activity of Bcr/Abl by activating several genes that interfere with the c-Myc pathway, a downstream target of Bcr/Abl (204) and, in fact, ameliorate Bcr/Abl-mediated murine myeloid leukemia in vivo (316). These data indicate that the loss of IRF8 expression may be a major event leading to the development of human CML and that the restoration of IRF8 expression can antagonize the oncogenic activity of Bcr/Abl. IRF8 appears to exert its antileukemic activity not only by the direct control of cell growth, differentiation, and apoptosis (see above), but also by modulating antitumor immunity. Coexpression of IRF8 in a Bcr/Abltransformed pro-B cell line causes a CD8+ cytotoxic T cell response that prevents the establishment of leukemia in vivo (317). Indeed, it is well established that human CML cells are sensitive to T cell–mediated immunity. Because T cells are stimulated by APCs, IRF8’s ability to support the differentiation and function of Ms and DCs may also be relevant to antitumor immunity. In addition, given that IFN-α therapy is effective in human CML, it is interesting to note that IRF8 is required for the development of pDCs that produce high levels of type I IFNs and that IRF8 is a transcriptional activator of type I IFN genes (52). Interestingly, IRF4 transcript levels are also signiﬁcantly low in CML patients (318, 319), suggesting that the decreased expression of both IRF8 and IRF4, the two IRFs that cooperatively regulate DC and B cell develop-

ment, may be involved in the pathogenesis of CML. Investigators have suggested that IRF8 manifests antitumor activity even in nonhematopoietic tumors. For instance, IFN-γinduced IRF8 sensitizes human colon carcinoma cells to Fas-mediated apoptosis (320), and IRF8 expression is repressed by DNA methylation in human metastatic colon carcinoma cell lines and murine mammary carcinoma with lung metastasis in vivo (321).

Regulation of Apoptosis by IRF3 One host defense mechanism to prevent viral propagation is apoptosis of virus-infected cells. Virus-induced apoptosis may be mediated by activated IRF3 because, on the one hand, expression of a constitutively active mutant of IRF3 triggered apoptosis, while on the other hand dominant-negative mutants of IRF3 strongly inhibited Sendai virus– and NDV-induced apoptosis (322, 323). Interestingly, data suggest that IRF3-mediated apoptosis is independent of p53 and IFN (323). Instead, the gene encoding TRAIL, which is implicated to be involved in virus-induced apoptosis, has been identiﬁed as a potential mediator of IRF3-induced apoptosis because it was transcriptionally activated by ectopic expression of IRF3 (324). IRF3 does not appear to mediate virus-induced apoptosis against all viruses, as VSV-infected Irf3−/− MEFs efﬁciently undergo apoptosis as efﬁciently as infected wild-type cells do (65). Interestingly, Irf5−/− MEFs show a defect in VSV-induced apoptosis, suggesting that IRF5 functions in this viral response (65). IRF3 also participates in bacteriuminduced apoptosis. Certain bacteria induce M apoptosis by producing virulence factors that inhibit cell survival pathways such as the p38 or NF-κB pathways, suggesting the existence of a hidden proapoptotic pathway downstream of TLR signaling. A requirement for IRF3 has been shown when Irf3−/− Ms failed to undergo apoptosis induced by LPS in the presence of a p38 inhibitor www.annualreviews.org • The IRF Family Transcription Factors

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or upon Salmonella typhimurium infection (325). Finally, IRF3 may play a role in DNA damage–induced apoptosis. Upon being phosphorylated in response to DNA damaging agents, IRF3 translocates from the cytoplasm to the nucleus and activates transcription (323, 326). One study has provided evidence that DNA-dependent protein kinase (DNA-PK) may be responsible for the phosphorylation of IRF3 in response to DNA damage (327). Consistent with its role in DNA damage–induced apoptosis, IRF3 can inhibit the growth of cancer cell lines in vitro and in vivo, as shown by several overexpression studies (328, 329). It is also interesting that HPV 16’s E6 oncoprotein binds to and inhibits IRF3 transcriptional activity (330). Although further studies are required, these data suggest that IRF3 may function as an additional tumor suppressive IRF.

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IRF9 and Oncogenesis A critical link between type I IFNs and the p53 pathway was recently discovered when type I IFNs transcriptionally activated the tumor suppressor p53 gene through ISGF3; thus, Irf9−/− MEFs fail to upregulate p53 upon IFN-β stimulation (331). This p53 induction is functionally signiﬁcant because IFN-β suppressed oncogene-induced malignant cell transformation and enhanced DNA damage– induced apoptosis of cancer cells. Further, p53 is required for virus-induced apoptosis. These results indicate a link between type I IFNs and p53 in tumor suppression and antiviral immunity (331). Hence, as a component of ISGF3, IRF9 boosts the p53 pathway when cells are exposed to endogenously induced or exogenously administered type I IFNs. In addition, the Irf9 gene is directly activated by c-Myc, and a cell line lacking IRF9 expression has been shown to be more susceptible to the cytocidal action of anticancer drugs (332). These ﬁndings suggest an additional role of IRF9 in cell cycle regulation, 562

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although further work is required to clarify this point.

Oncogenic Potential of IRF2 and IRF4 In addition to its role as an IFN attenuator, the overexpression of IRF2 in NIH3T3 cells causes oncogenic transformation (271). Furthermore, a retroviral library genetic screen has identiﬁed IRF2 as an inhibitor of activated N-Ras-induced growth suppression in leukemic cells (333). Although the exact mechanism is unknown, IRF2 may exert its oncogenic function by interfering with IRF1 and/or other IRF family members that bind to the same ISREs (334). Indeed, the concomitant expression of IRF1 in NIH3T3 cells made to overexpress IRF2 revert these cells to a nontransformed phenotype (271). However, IRF2 can also activate gene transcription under certain conditions (335) and, in fact, stimulates the expression of genes involved in oncogenesis such as histone H4 (336, 337). The activity of IRF2 is regulated in a cell growth–dependent manner where IRF2 is acetylated and binds to the H4 promoter in proliferating cells only (338). Several experiments have suggested a connection between IRF4 expression and cancers. In human T cells, the expression of IRF4 mRNA is induced by human T cell leukemia virus-1 (HTLV-1) infection (339). Moreover, overexpression of the HTLV-1 oncoprotein Tax induces IRF4 mRNA expression in Jurkat T cells, and constitutive expression of IRF4 in these T cells results in reduced expression of the G2-M checkpoint gene encoding Cyclin B1 and DNA repair genes encoding Rad51, XRCC1, Yng1, RPA, and PCNA. Such transcriptional changes are strikingly similar to those that occur in HTLV-infected T cells (340, 341), suggesting a possible involvement of IRF4 in HTLV-1-induced leukemogenesis. Interestingly, in some patients with multiple myeloma and cell lines derived from this tumor, a chromosomal translocation t(6;14)(p25;q32) juxtaposes the Ig heavy chain

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locus to IRF4/MUM1 (multiple myeloma 1), resulting in the overexpression of IRF4 (342). IRF4 indeed possesses a transforming activity in vitro; exogenous expression of IRF4 in Rat-1 cells causes anchorage-independent growth (342). However, overexpression of IRF4 alone is not sufﬁcient for leukemogenesis in transgenic mice in lymphocytes (343), suggesting that additional factors are required for the oncogenic activity of IRF4 in vivo.

IMPLICATIONS AND PERSPECTIVES A prompt and controlled cellular response is central to host defense against pathogens and tumors. In this article, we reviewed how the IRF family of transcription factors directly impacts a number of aspects of host defense, from the development and function of a multitude of myeloid and lymphoid tissues to immune activation downstream of PRRs and danger signals. We also discussed how IRF family members exert control over the cell cycle and apoptosis and, thus, are involved in the oncogenesis of several cancers. Clearly, IRF family members do not function in these processes alone and are known to directly and indirectly cooperate and antagonize members of their own family, as well as members of other classes of transcription factors (for details, see Reference 10). Although beyond the main scope of this review and, hence, not touched upon here in more detail, the nature of intermolecular contact between IRFs and other transcription factors is an interesting area for future study. Indeed, it has already been shown that these interactions vary between essentially weak association caused by their independent binding to adjacent target DNA elements, as is known to occur between IRFs and NF-κB (60), to more direct interaction, such as is the case when IRF3 binds to the p65 subunit of NF-κB (also known as REL-A) to transactivate a set of NF-κB-dependent genes without binding to an ISRE (344).

The relationship between IRFs and NFκB is of particular interest because both are activated by a remarkably common set of stimuli, such as PAMPs and DNA damage. Both transcription factors play an essential role in immune cell development and function and, indeed, cooperatively regulate the expression of many cytokine genes. However, IRFs and NF-κB appear to exert opposite effects on cell growth and survival. In contrast to the tumor suppressive effects of IRFs, NF-κB acts as a potent prosurvival transcription factor and contributes to the development of tumors, including inﬂammation-linked cancers (345, 346). Precisely how these two transcription factor families cooperate and antagonize one another is an important question to be addressed. One evolutionary perspective on IRFs is important to mention. Because type I IFN and IRF genes are present in vertebrates, but not invertebrates, we surmise that they evolved after the divergence of these two branches. Recent studies have suggested that viral infection in invertebrates triggers an RNA-silencingbased antiviral response (also known as RNA interference or RNAi). However, presumably such a response was insufﬁcient to eradicate viruses in proto-vertebrates, thereby creating selective pressure under which the IRF-IFN system evolved. Interestingly, the presence of genes with some sequence similarities to vertebrate IRF and type I IFN receptor genes was reported by the recent genome-wide sequence analyses in the urochordate Ciona intestinalis, an organism that shares a common ancestor with vertebrates (347, 348). This is consistent with the idea that the IRF-IFN system arose at the boundary of early vertebrate evolution. Given that the IRF family is critical for two aspects of host defense—immunity against pathogens and tumor suppression—a more detailed understanding of how the IRF system’s signaling pathways are turned on and off could make the IRF family an attractive target not only for therapy for infectious diseases and immune disorders, but also in the multidisciplinary therapy of cancers. www.annualreviews.org • The IRF Family Transcription Factors

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DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

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We thank Drs. A. Takaoka and K. Honda for valuable discussions and advice. This work was supported by Kakenhi (Grant-in-Aid for Scientiﬁc Research) on Priority Areas “Integrative Research Toward the Conquest of Cancer” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. D.S. is a Japan Society for the Promotion of Science postdoctoral fellow.

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53. Reports the first gene-targeting study of IRFs.

61. Provides evidence for the involvement of IRF3 in type I IFN gene induction upon virus infection.

64. Provides definitive evidence that IRF7 is the main regulator of type I IFN gene induction.

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248. Gupta S, Jiang M, Anthony A, Pernis AB. 1999. Lineage-speciﬁc modulation of interleukin 4 signaling by interferon regulatory factor 4. J. Exp. Med. 190:1837–48 249. White LC, Wright KL, Felix NJ, Ruffner H, Reis LF, et al. 1996. Regulation of LMP2 and TAP1 genes by IRF-1 explains the paucity of CD8+ T cells in IRF-1−/− mice. Immunity 5:365–76 250. Penninger JM, Sirard C, Mittrucker HW, Chidgey A, Kozieradzki I, et al. 1997. The interferon regulatory transcription factor IRF-1 controls positive and negative selection of CD8+ thymocytes. Immunity 7:243–54 251. Ohteki T, Maki C, Koyasu S. 2001. Overexpression of Bcl-2 differentially restores development of thymus-derived CD4–8+ T cells and intestinal intraepithelial T cells in IFN-regulatory factor-1-deﬁcient mice. J. Immunol. 166:6509–13 252. Hida S, Ogasawara K, Sato K, Abe M, Takayanagi H, et al. 2000. CD8+ T cell-mediated skin disease in mice lacking IRF-2, the transcriptional attenuator of interferon-α/β signaling. Immunity 13:643–55 253. Matsuyama T, Grossman A, Mittrucker HW, Siderovski DP, Kiefer F, et al. 1995. Molecular cloning of LSIRF, a lymphoid-speciﬁc member of the interferon regulatory factor family that binds the interferon-stimulated response element (ISRE). Nucleic Acids Res. 23:2127–36 254. Fanzo JC, Hu CM, Jang SY, Pernis AB. 2003. Regulation of lymphocyte apoptosis by interferon regulatory factor 4 (IRF-4). J. Exp. Med. 197:303–14 255. Lohoff M, Mittrucker HW, Brustle A, Sommer F, Casper B, et al. 2004. Enhanced TCRinduced apoptosis in interferon regulatory factor 4-deﬁcient CD4+ Th cells. J. Exp. Med. 200:247–53 256. Lohoff M, Ferrick D, Mittrucker HW, Duncan GS, Bischof S, et al. 1997. Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo. Immunity 6:681– 89 257. Coccia EM, Passini N, Battistini A, Pini C, Sinigaglia F, Rogge L. 1999. Interleukin-12 induces expression of interferon regulatory factor-1 via signal transducer and activator of transcription-4 in human T helper type 1 cells. J. Biol. Chem. 274:6698–703 258. Galon J, Sudarshan C, Ito S, Finbloom D, O’Shea JJ. 1999. IL-12 induces IFN regulating factor-1 (IRF-1) gene expression in human NK and T cells. J. Immunol. 162:7256–62 259. Kano S, Sato K, Morishita Y, Vollstedt S, Kim S, et al. 2008. Contribution of the transcription factor IRF1 to the interferon-γ-interleukin-12 signaling axis and TH1 versus TH-17 differentiation of CD4+ T cells. Nat. Immunol. 9:34–41 260. Elser B, Lohoff M, Kock S, Giaisi M, Kirchhoff S, et al. 2002. IFN-γ represses IL-4 expression via IRF-1 and IRF-2. Immunity 17:703–12 261. Lohoff M, Mittrucker HW, Prechtl S, Bischof S, Sommer F, et al. 2002. Dysregulated T helper cell differentiation in the absence of interferon regulatory factor 4. Proc. Natl. Acad. Sci. USA 99:11808–12 262. Rengarajan J, Mowen KA, McBride KD, Smith ED, Singh H, Glimcher LH. 2002. Interferon regulatory factor 4 (IRF4) interacts with NFATc2 to modulate interleukin 4 gene expression. J. Exp. Med. 195:1003–12 263. Tominaga N, Ohkusu-Tsukada K, Udono H, Abe R, Matsuyama T, Yui K. 2003. Development of Th1 and not Th2 immune responses in mice lacking IFN-regulatory factor-4. Int. Immunol. 15:1–10 264. Maldonado-Lopez R, De Smedt T, Pajak B, Heirman C, Thielemans K, et al. 1999. Role of CD8α+ and CD8α− dendritic cells in the induction of primary immune responses in vivo. J. Leukoc. Biol. 66:242–46

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265. Pulendran B, Smith JL, Caspary G, Brasel K, Pettit D, et al. 1999. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc. Natl. Acad. Sci. USA 96:1036–41 266. Hu CM, Jang SY, Fanzo JC, Pernis AB. 2002. Modulation of T cell cytokine production by interferon regulatory factor-4. J. Biol. Chem. 277:49238–46 267. Ingraham CR, Kinoshita A, Kondo S, Yang B, Sajan S, et al. 2006. Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6). Nat. Genet. 38:1335–40 268. Richardson RJ, Dixon J, Malhotra S, Hardman MJ, Knowles L, et al. 2006. Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch. Nat. Genet. 38:1329–34 269. Sil AK, Maeda S, Sano Y, Roop DR, Karin M. 2004. IκB kinase-α acts in the epidermis to control skeletal and craniofacial morphogenesis. Nature 428:660–64 270. Pivarcsi A, Bodai L, Rethi B, Kenderessy-Szabo A, Koreck A, et al. 2003. Expression and function of Toll-like receptors 2 and 4 in human keratinocytes. Int. Immunol. 15:721–30 271. Harada H, Kitagawa M, Tanaka N, Yamamoto H, Harada K, et al. 1993. Anti-oncogenic and oncogenic potentials of interferon regulatory factors-1 and -2. Science 259:971–74 272. Tanaka N, Ishihara M, Lamphier MS, Nozawa H, Matsuyama T, et al. 1996. Cooperation of the tumour suppressors IRF-1 and p53 in response to DNA damage. Nature 382:816–18 273. Pamment J, Ramsay E, Kelleher M, Dornan D, Ball KL. 2002. Regulation of the IRF-1 tumour modiﬁer during the response to genotoxic stress involves an ATM-dependent signalling pathway. Oncogene 21:7776–85 274. Kano A, Haruyama T, Akaike T, Watanabe Y. 1999. IRF-1 is an essential mediator in IFN-γ-induced cell cycle arrest and apoptosis of primary cultured hepatocytes. Biochem. Biophys. Res. Commun. 257:672–77 275. Yim JH, Ro SH, Lowney JK, Wu SJ, Connett J, Doherty GM. 2003. The role of interferon regulatory factor-1 and interferon regulatory factor-2 in IFN-γ growth inhibition of human breast carcinoma cell lines. J. Interferon Cytokine Res. 23:501–11 276. Kirchhoff S, Schaper F, Hauser H. 1993. Interferon regulatory factor 1 (IRF-1) mediates cell growth inhibition by transactivation of downstream target genes. Nucleic Acids Res. 21:2881–89 277. Romeo G, Fiorucci G, Chiantore MV, Percario ZA, Vannucchi S, Affabris E. 2002. IRF-1 as a negative regulator of cell proliferation. J. Interferon Cytokine Res. 22:39–47 278. Tanaka N, Ishihara M, Kitagawa M, Harada H, Kimura T, et al. 1994. Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 77:829–39 279. Strasser A, Harris AW, Jacks T, Cory S. 1994. DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2. Cell 79:329–39 280. Tamura T, Ishihara M, Lamphier MS, Tanaka N, Oishi I, et al. 1995. An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes. Nature 376:596–99 281. Lallemand C, Palmieri M, Blanchard B, Meritet JF, Tovey MG. 2002. GAAP-1: a transcriptional activator of p53 and IRF-1 possesses proapoptotic activity. EMBO Rep. 3:153– 58 282. Kim EJ, Lee JM, Namkoong SE, Um SJ, Park JS. 2002. Interferon regulatory factor1 mediates interferon-γ-induced apoptosis in ovarian carcinoma cells. J. Cell. Biochem. 85:369–80 www.annualreviews.org • The IRF Family Transcription Factors

267, 268. Two papers that report that Irf6 null/mutant mice have abnormal skin, limb, and craniofacial development.

278. Provides for the first time definitive evidence of the tumor suppressive role of an IRF.

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349. Reports the discovery and cloning of the first IRF family member, IRF1.

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353. Levy DE, Kessler DS, Pine R, Darnell JE Jr. 1989. Cytoplasmic activation of ISGF3, the positive regulator of interferon-α-stimulated transcription, reconstituted in vitro. Genes Dev. 3:1362–71 354. Kimura T, Kadokawa Y, Harada H, Matsumoto M, Sato M, et al. 1996. Essential and nonredundant roles of p48 (ISGF3 γ) and IRF-1 in both type I and type II interferon responses, as revealed by gene targeting studies. Genes Cells 1:115–24

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:535-584. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

vi

Contents

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Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan1,3 and Ian Parker1,2 1

Department of Physiology and Biophysics, 2 Department of Neurobiology and Behavior, and 3 Center for Immunology, University of California, Irvine, California 92697; emails: [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:585–626

Key Words

First published online as a Review in Advance on January 2, 2008

T cell, B cell, dendritic cell, chemokine, antigen, motility

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.24.021605.090620 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0585$20.00

Abstract The immune system is the most diffuse cellular system in the body. Accordingly, long-range migration of cells and short-range communication by local chemical signaling and by cell-cell contacts are vital to the control of an immune response. Cellular homing and migration within lymphoid organs, antigen recognition, and cell signaling and activation are clearly vital during an immune response, but these events had not been directly observed in vivo until recently. Introduced to the ﬁeld of immunology in 2002, two-photon microscopy is the method of choice for visualizing living cells deep within native tissue environments, and it is now revealing an elegant cellular choreography that underlies the adaptive immune response to antigen challenge. We review cellular dynamics and molecular factors that contribute to basal motility of lymphocytes in the lymph node and cellular interactions leading to antigen capture and recognition, T cell activation, B cell activation, cytolytic effector function, and antibody production.

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INTRODUCTION

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The immune system is designed for detection of foreign antigens that have never been encountered, for ampliﬁcation of responses to previously encountered antigen, and for the delivery of coordinated effector responses ranging from mobilization of effector T cells, cytokine delivery, and antibody production. Each of these processes relies on cell-to-cell contact for initiation, followed by guided dispersal of effector cells and molecules to the appropriate location, while avoiding friendly ﬁre that could result in autoimmune disease. Lymphocytes are potentially dangerous cells, and numerous cellular and molecular mechanisms avoid inappropriate triggering, dampen initial responses, and amplify responses to second and subsequent encounters with antigen, while at the same time optimizing sensitivity to minute quantities of antigen. Within the past ﬁve years, since publication of the ﬁrst studies examining the motility of lymphocytes and thymocytes in native tissue environments (1–3), new imaging studies have appeared at an exponentially increasing rate. Two-photon microscopy has proved to be the method of choice for cellular immunoimaging, enabling visualization deep within intact organs and tissues while minimizing photodamage and bleaching. Efforts to map out the cellular choreography in vivo build upon a framework of in vitro studies of lymphocyte motility and response dynamics following contact with antigen-presenting cells (APCs). In this review, we ﬁrst discuss the technology of two-photon microscopy as applied to immunoimaging and then focus on its applications to live-cell imaging of immune cells in the lymph node under basal conditions and in response to antigenic challenge. Collectively, two-photon microscopy is revealing an elaborate and elegant choreography of cell motility, antigen capture, and cell-cell interactions in a variety of in vivo settings and holds great promise for understanding the dynamic response to infectious agents.

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TWO-PHOTON IMAGING METHODOLOGY Imaging the single-cell dynamics of the immune system within an intact environment requires the ability to look deep inside intact tissues and organisms with spatial and temporal resolution adequate to track cell morphology, motility, and signaling processes, all the while minimizing perturbation of the system under study. Optical microscopy employing ﬂuorescence techniques is highly suited for this purpose, permitting both labeling of speciﬁc cells, organelles, or proteins and functional readout of physiological events (4). However, conventional (single-photon) techniques such as wide-ﬁeld and confocal microscopy suffer severe disadvantages, principally because the short wavelengths required for ﬂuorescence excitation are subject to strong scattering in biological tissue and exacerbate phototoxicity. Nonlinear microscopy differs fundamentally from conventional techniques in that the elementary process involves near simultaneous interactions of two (or more) photons, so that the signal varies as the square (or higher power) of incident light intensity, rather than linearly. This nonlinear relationship leads to qualitatively new imaging modalities, of which ﬂuorescence excitation by two-photon absorption and second harmonic generation have proved most useful for immunoimaging. The essence of two-photon microscopy is that a ﬂuorophore is excited by the nearsimultaneous absorption of energy from two photons, each of which contributes one half of the energy required to induce ﬂuorescence. Because excitation then increases as the square of the incident light intensity, ﬂuorescence is essentially conﬁned to the focal spot formed by a microscope objective, thereby providing an inherent optical sectioning effect, analogous (though involving a completely different mechanism) to confocal microscopy. To achieve practicable ﬂuorescence signals, the photon density in the focal spot must be incredibly high, yet not so high as to damage the specimen. This is achieved by using

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femtosecond or picosecond pulsed lasers, which concentrate their output into brief bursts with enormous instantaneous power, yielding a two-photon advantage of ∼105 as compared with a continuous beam of the same average power. The continuing improvement of ultrafast pulsed lasers has been a key factor driving the adoption of two-photon microscopy by immunologists. In addition to its inherent optical sectioning effect, two-photon excitation has other major advantages for immunoimaging because the excitation wavelengths are roughly twice as long as would be used for conventional linear excitation by wideﬁeld or confocal microscopy. This use of long wavelength excitation is particularly advantageous for imaging deep into highly scattering biological tissues (5) because scattering decreases with increasing wavelength (6) and because absorption by hemoglobin and other proteins is minimized. Thus, the infrared wavelengths used for two-photon imaging enable a ﬁvefold or deeper tissue penetration than does confocal imaging employing visible wavelengths (2, 7–9) and cause negligible photodamage or photobleaching. Long-term imaging is thereby facilitated because bleaching and damage processes (induced by nonlinear processes) are largely conﬁned to those cells lying at the focal plane, whereas cells above and below experience only the innocuous infrared light. Nevertheless, the laser power must be kept below some sharp threshold value in order to maintain long-term viability of the preparation. Finally, the two-photon excitation spectra of most ﬂuorophores are appreciably broader than for one-photon excitation (10), so a single excitation wavelength can be used efﬁciently to excite multiple probes simultaneously with distinct emission wavelengths. Moreover, the ease of tuning of the latest generations of femtosecond lasers facilitates selection of a wavelength that balances excitation of different ﬂuorophores. In instances in which highly divergent excitation wavelengths are required, two independently tuned lasers may be used simultaneously (8).

In addition to multiphoton absorption, another nonlinear interaction that becomes prominent at very high light intensities is that of optical-harmonic generation, in which two (or more) photons are almost simultaneously scattered to generate a single photon with exactly twice (or higher multiples of ) the incoming energy (11). Second harmonic generation is produced by spatially ordered molecules and has proved especially useful for imaging ordered structural proteins such as collagen ﬁbers (12) and microtubules (13) by detecting emitted light through a bandpass ﬁlter of twice the wavelength of the excitation light without the need for ﬂuorescent labeling. Deep tissue imaging is currently limited by several factors, including the requirement for exponentially increasing excitation power at increasing depths to compensate for increasing scattering loss. Use of infrared-emitting dyes may be advantageous in reducing signal loss owing to scattering, and the advent of ever more powerful lasers extends the depth range but will be limited by thermal effects and excitation near the surface (5). Moreover, image quality (as well as brightness) degrades with transit through tissues with strongly varying refractive index but may be mitigated by correcting for wavefront distortions with deformable mirrors (14) or by applying depthdependent deconvolution algorithms to acquired images (15). Imaging yet deeper into thick tissues and organs will necessitate mechanical techniques, such as penetration or removal of overlying tissue, use of needle-like gradient index lenses to extend the range of otherwise bulky objectives (13), and development of miniaturized two-photon endoscopes (16, 17).

Fluorescent Labels and Probes The imaging techniques reviewed here rely primarily on the introduction of a ﬂuorescent probe or label into cells or structures of interest, although useful information may also be gained from intrinsic signals such as

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autoﬂuorescence and second harmonic generation. Extrinsic labeling may be accomplished in vitro using isolated cells that are then adoptively transferred into a recipient animal by introducing ﬂuorophores directly into the animal or organ being imaged (e.g., to stain vasculature) or by genetically engineering expression of ﬂuorescent proteins. The ﬁrst in situ imaging studies of lymphocytes and dendritic cells (DCs) employed cell tracker ﬂuorescent dyes, including CSFE (5,6-carboxyﬂuoresceine diacetate succinimidyl ester; green ﬂuorescence) and CMTMR (chloromethylbenzoyl aminotetramethylrhodamine; red) for staining of adoptively transferred cells, and this method remains a mainstay. Such dyes have been used for many years by immunologists for ﬂow cytometry and have the advantages of providing bright ﬂuorescence signals while being relatively benign. Adoptive transfer of labeled cells typically results in <1% of cells being labeled in dense tissue populated by a vast excess of unlabeled cells. Although this is a huge advantage for tracking single cells and visualizing their morphological characteristics, it is obviously important that the unseen cells may exert signiﬁcant effects on the labeled cells. The proportion of labeled cells can be adjusted to vary the density of labeled cells. Differently labeled cells can be coinjected and then distinguished by color. This approach has several advantages: It creates an internal control population for comparison; it allows labeling of a tissue landmark or compartment, such as the follicle, for orientation while imaging; and it allows comparison of cells by molecular perturbation with a drug treatment or a genetic deletion. Dyes are available with a wide range of emission spectra separated sufﬁciently to permit independent visualization of at least three cell populations separately labeled with, for example, blue-, green-, or red-emitting dyes, by use of appropriate dichroic mirrors and bandpass ﬁlters before three photomultiplier detectors. Moreover, using dyes such as CFSE, one can track the number of times that

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lymphocytes divide in vitro or in vivo by taking advantage of the fact that the original dye content is split equally into the daughter cells each time the cell divides. Thus, comparison of relative ﬂuorescence intensities among cells can be an indicator of how many times they have divided. However, this approach is complicated by the need to correct for variation in signal intensity with imaging depth into the tissue, and after a few cycles the remaining ﬂuorescence becomes too faint to detect so that it is not possible to visualize labeled lymphocytes for more than one or two days after antigen stimulation or to follow them into peripheral sites of effector function. Signiﬁcant disadvantages of in vitro dye labeling approaches arise because cells must ﬁrst be extracted from their normal environment in the donor animal before adoptive transfer into the recipient host. Expression of ﬂuorescent proteins by target cells has been the other main method for visualizing target cells. The original ﬂuorescent proteins derived from the Aequorea green ﬂuorescent protein (GFP) suffered from low brightness and closely overlapping emission spectra. Subsequent developments, including the identiﬁcation of a red ﬂuorescent protein from the coral Discosoma and extensive mutagenesis work, have now made available a wide range of ﬂuorescent proteins with peak emissions ranging from blue (475 nm) to red (610 nm), which have greatly improved properties in terms of brightness, lack of pH sensitivity, faster maturation, and lack of oligomerization (reviewed in 18, 19). The two-photon excitation maxima for ﬂuorescent proteins generally lie at longer wavelengths (approximately 900 nm) (20) than for organic dyes (approximately 780 nm). Simultaneous imaging of both types of label with a single femtosecond laser thus requires selection of an intermediate wavelength that provides an optimal compromise, whereas application of a dual, independently tuned laser system may offer appreciable advantage. The ﬂuorescence brightness obtained from a cell obviously depends on the level of protein

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expression and must be balanced against the possibility of cellular disruption with overexpression systems, particularly when the ﬂuorescent protein is tagged to some other protein of interest. There are several advantages to using expressed ﬂuorescent proteins over other extrinsic ﬂuorophores: (a) The label is not diluted by successive cell divisions, allowing tracking of cell progeny after clonal expansion. In a mouse with GFP under the control of a ubiquitous promoter such as β-actin or ubiquitin, T or B cells can be puriﬁed and adoptively transferred into recipients. For longer-term tracking, the recipient of choice would be one that has been tolerized by tissue-speciﬁc expression of GFP elsewhere, avoiding immune responses to GFP (21). (b) Fluorescent proteins can be expressed under the control of speciﬁc promoters to obtain selective expression of a particular cell type. For example, the CD11c promoter has been used to drive expression of YFP in DCs (22). The advantage of being able to visualize every cell of a particular cell type must be weighed against the potential difﬁculty of tracking individual cell behavior in densely populated tissue. An analogy may be made with Golgi staining of the nervous system, which reveals the morphological complexity of sparsely labeled individual neurons in the brain. (c) Speciﬁc proteins may be directly tagged with a ﬂuorescent protein to visualize in real time their subcellular expression and/or relocalization, rather than merely expressing free cytosolic ﬂuorescent protein as a cellular marker. (d ) Transgenic expression of ﬂuorescent proteins enables in situ labeling of cells, so that for many experiments the need for ex vivo manipulations is eliminated. (e) The percentage of a particular cell type that expresses a ﬂuorescent protein can be adjusted by making mixed bone marrow chimeras (23, 24), thus facilitating tracking of individual cell behavior. ( f ) Immune cells can be selectively ablated by irradiation in a mouse that ubiquitously expresses ﬂuorescent protein and then reconstituted from bone marrow of a nonﬂuorescent donor, allowing radiation-resistant

stromal and vascular cells to be visualized (25).

Analyzing Cell Motility and Interactions Multiphoton imaging yields 4-D (x, y, z, t) information of cell morphology and motility, but the very wealth of data (gigabytes) presents appreciable problems of visualization and analysis. The results are almost impossible to convey in static images on the printed page but are better appreciated as timelapse movies, usually presented as maximumintensity 2-D projections along the z-axis. Depth information is lost in this process (so it is impossible to discern whether cells touch or pass over one another), but it may be communicated by methods such as color-encoding the z-axis location of cells (26) or employing specialized 3-D visualization software. The instant replay of time-lapse videos is, however, only a ﬁrst step in immunoimaging, and deriving quantitative information by tracking cells is crucial (Figure 1). Quantitative tracking is most easily achieved by 2-D analysis after compression of image stacks, a procedure that circumvents errors that are due to the inherently lower z-axis resolution. 3-D tracking is essential in cases in which motility may be anisotropic in the z-axis. Cell tracks may be superimposed directly on the imaging ﬁeld (Figure 1a) or, if there are no reasons to suspect regional differences, by overlaying tracks from several cells after normalizing their starting coordinates to generate a ﬂower plot (Figure 1b). Various parameters (Table 1) can then be extracted from such tracking data to characterize cell motility and morphology (Figure 1c,d ). Instantaneous velocity is a simple and widely used parameter, but it is subject to errors. For example, jitter in measurements at brief time steps overestimates velocities and can give the appearance of motion in even stationary cells, whereas time steps longer than the persistence time for linear motion will tend to underestimate velocities.

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tion with time. Analogous to the diffusion coefﬁcient for Brownian motion, the slope of this relationship can be used to calculate a motility coefﬁcient. This has generally been done by plotting displacement versus square root of time (Figure 1e) (2), but a plot of displacement squared versus time is more appropriate when data are appreciably scattered. A

Early imaging studies (2) suggested that T cell motility in the lymph node approximates a random walk, and several approaches have subsequently been adopted to characterize whether motility is indeed random or is directed (e.g., along chemokine gradients). For a random-walk process, the mean displacement from origin increases as a square root func-

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Linear-directed motion

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Random walk

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Parameters used to characterize cell motility and migration

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Parameter

Description

Unit

Instantaneous velocity (v)

Velocity calculated as displacement/time during a single time step

μm/min

Mean velocity (V)

Mean velocity of a cell over several time steps (usually the entire imaging period)

μm/min

Contact time

Time for which a cell is in contact with a deﬁned cell or structure

sec: min

Shape index

Measure of cell polarization (long axis/short axis)

Path length

Cumulative distance traveled by a cell over a given time

μm

Displacement (D)

Straight-line distance of a cell from its starting point after any given time

μm

Motility coefﬁcient (M)

M = displacement2 /4t (for 2-D measurements) or displacement2 /6t (for 3-D measurements); analogous to the diffusion coefﬁcient for Brownian motion

μm2 /min

Chemotactic index

Displacement/path length for a given time interval (a measure of cell directionality)

Turning angle

The angle through which a cell deviates between successive time steps

degrees

Persistence time (length)

Time (length) for which a cell continues moving in a straight line before making an appreciable turn

min (μm)

practical difﬁculty with this method is that cells exit the imaging volume at longer times, resulting in increasing standard error as fewer cells remain and possible bias as the remaining cells are likely to have lower velocities. Deviations from linearity of the displacement versus square root time plot point to nonrandom processes (Figure 1e): (a) At intervals shorter than the persistence time, the relationship will

curve upward, as cells migrate without turning; (b) transition from a linear relationship to a plateau is consistent with migration conﬁned by physical or biological barriers; (c) a steeperthan-linear slope indicates directed motion (27). The motility coefﬁcient is a function of both the directionality of motion and the cell velocity, whereas the chemotactic index (displacement/path length) provides a measure of

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1 Basics of motility analysis. (a) Snapshot visualization of the 3-D locations of ﬂuorescently labeled cells throughout the imaging volume at a single time point. Tracks of three cells are superimposed. The cells are CFSE-labeled allogeneic CD8+ T cells (green), CMTMR-labeled allogeneic CD4+ T cells (red ), and CMF2HC-labeled syngeneic CD4+ T cells (blue) in the lymph node of B6D2F1 (H-2b/d) mice (Video 1a provided by Y. Yu). The total volume is (86 μm × 57 μm × 50 μm). (b) Flower plot representation of 2-D tracks of several cells, superimposed after normalizing their starting coordinates to the origin (adapted from Reference 26). (c) Schematic showing the track of a cell at ﬁve successive time points, t1 , t2 , etc. Instantaneous velocities are calculated from the net distance d traveled during each time interval t, and the turn angle θ is the angle through which the cell turns between time steps. The displacement D is the straight-line distance of the cell from its origin at any given time. Path length is given by the sum of d1 , d2 , d3 , etc. Persistence time (or length) is the time for which a cell continues to move without turning appreciably, as is illustrated for time points t2 –t4 . (d ) The instantaneous velocity of T cells ﬂuctuates in a characteristic manner over time, accompanied by changes in shape index (long axis/short axis) as the cell elongates while moving faster (adapted from Reference 2). (e) A plot of mean displacement from origin of multiple cells is expected to follow a straight line when plotted as a function of square root of time, if cell motility follows a random walk (adapted from Reference 26). The slope of this line can be used to derive a motility coefﬁcient M, analogous to the diffusion coefﬁcient for Brownian motion. Deviations from a straight-line relationship may be indicative of directed migration (upward curvature) or migration constrained by physical or biological barriers (plateau). ( f ) Chemotactic index. This measure, deﬁned as path length/displacement from origin, remains constant with a value of unity for linear-directed motion but decreases progressively with time for cells following a random walk. Video may be viewed separately by following the Supplemental Materials link on the Annual Reviews website at http://www.annualreviews.org. www.annualreviews.org • Cell Motility and Interaction Dynamics Imaged

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directionality that is independent of velocity but must be stated for some given time interval (Figure 1f ).

CELLULAR DYNAMICS WITHIN THE LYMPH NODE IN THE ABSENCE OF COGNATE ANTIGEN

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Since the pioneering work of Gowans and colleagues (28–30), the processes of lymphocyte homing and tissue localization have been investigated extensively, resulting in our current understanding of how lymphocytes distribute from the blood to lymphoid organs and to peripheral tissues of the body (31–34). In the following sections, we review how twophoton immunoimaging allows us to visualize the dynamics of lymphocytes within the black box of their native tissues. Although the individual cellular components had been investigated previously in vitro, cell behaviors and the dynamics of cellular interactions turn out to be quite different within the complex tissue environment. We ﬁrst consider the behavior of T and B cells following homing to lymph nodes under basal conditions without antigen priming.

Basal Motility of T and B Lymphocytes Early single-cell in vitro observation of living immune cells revealed their ability to crawl in an amoeboid manner on a variety of substrates (35–45). Yet many immunologists retained a view of resting lymphocytes as round, quiescent cells that somehow become more lively and energized in response to antigen. In contrast, the ﬁrst study to apply twophoton microscopy to the lymph node revealed rapid migration of T and B lymphocytes under basal conditions within a lymph node explant (2). T cells within the diffuse cortex migrate with about twice the average velocity of B cells in the follicle (10–12 μm/min versus 6 μm/min for 2-D velocities) and with a motility coefﬁcient ﬁve- to sixfold higher for 592

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T cells than B cells. T and B cells are highly polarized while crawling, sometimes to an extreme of being four to ﬁve times longer than they are wide, and they move in a stop-andgo manner, with pauses between bursts, and reach peak velocities up to 30 μm/min for T cells and 20 μm/min for B cells. Tracking single cells revealed that T cells in the diffuse cortex and B cells in the follicle apparently move in random directions in all three dimensions. Rather than moving en masse, as might be expected if their migration were predominantly inﬂuenced by pervasive interstitial chemokine gradients, T and B cell tracks meander away from any arbitrary starting point in a manner reminiscent of molecules diffusing in solution. As a simple test for a random walk, plotting cell displacement as a function of the square root of time reveals an approximately linear function, as it would be for diffusion. Of course, the cells are not moving by diffusion but are crawling in an amoeboid manner. Miller et al. (2) labeled the reticular ﬁber network by soaking lymph nodes in CMTMR and observed T cells interacting with the labeled scaffold elements as well as T cells crawling over and interacting with each other. Subsequently, it has been shown that T cells migrate along a scaffold of ﬁbroblastic reticular cells (FRCs) (25) that is associated with and extends beyond a network of resident DCs (22) near the cortical ridge adjacent to the B cell follicle. There was initial concern regarding whether the rapid and random motility of lymphocytes in the lymph node explant accurately reﬂects their true in vivo behavior. Perhaps the disruption of lymph ﬂow and blood circulation disturbed preexisting chemokine gradients that direct the migratory patterns of lymphocytes in the lymph node. Moreover, a contemporary report (3), using confocal microscopy of lymph node explants, described resting T cells as immotile. In retrospect, the primary reason for the latter difference is likely that T cells within the subcapsular sinus region of the lymph node are less motile than deeper cells (which are accessible to

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two-photon, but not confocal microscopy) because in this region they lack chemokine signaling that enhances motility (46–48). These issues provoked a follow-up investigation using two-photon microscopy to examine naive T cell motility in an intravital preparation (26), exposing the inguinal lymph node in the anesthetized mouse. In the absence of antigen, naive T cells were again seen moving at 10–12 μm/min following a random-walk pattern within the diffuse cortex. Homing events were also visualized, as labeled T cells, imaged at 30 Hz and moving 1000 times faster in blood than in the interstitium, suddenly arrested at speciﬁc sites in the high endothelial venule (HEV), extravasated, and began to crawl through the interstitium of the diffuse cortex. Together, these two studies by Miller et al. (2, 26) suggested an antigen-search strategy in which the purpose of rapid T cell migration is to locate and sample DCs for antigen within the relatively vast expanse of the diffuse cortex. Such a scanning mechanism could, in principle, solve the antigenic “needle in a haystack” conundrum of locating rare antigens without invoking speciﬁc attraction mechanisms. Moreover, the close agreement on quantitative measures of motility by several groups (22, 25, 26, 49, 50) conﬁrms the robust motility of lymphocytes observed originally in the lymph node explant preparation (2), validating the lymph node explant preparation for further investigation of lymphocyte motility. There is now general agreement that the predominant macroscopic behavior of T cells in the diffuse cortex and of B cells in the follicle is rapid migration along apparently randomly oriented trajectories.

Lymphoid Architecture, FRCs, and Stromal Cell Guidance Lymphoid organs are optimized for bringing together lymphocytes and antigen. T and B cells spend from 8 to 24 h in the lymph node interstitium before exiting by transiting across a lymphatic endothelium into a network of

sinuses in the medulla that drain to efferent lymphatic vessels. While in the lymph node, T and B cells localize differentially to occupy distinct niches (Figure 2a) within the diffuse cortex and follicles, respectively (51), guided by their differential expression of chemokine receptors (52). Although occupying distinct territory, T and B cells explore a very different landscape. Despite appearances from movies showing only ﬂuorescently labeled lymphocytes, these cells do not migrate in a void. Instead, they crawl among a complex landscape of closely packed cells, including a vast excess of unlabeled lymphocytes, DCs, and stromal cells. Therefore, many factors may inﬂuence cellular motility in the complex environment of the lymph node. The role of stromal cells in lymphocyte guidance has been clariﬁed by Bajenoff and colleagues (25), who conclude that FRCs serve as a primary guidance scaffold for T cells in the diffuse cortex (Figure 2b) and that follicular dendritic cells (FDCs) may fulﬁll the analogous function for B cells in the follicle. Bajenoff et al. (25) imaged in chimeric GFP mice that had been irradiated to destroy the endogenous ﬂuorescent cells of the immune system, which were then reconstituted with GFP− immune-precursor bone marrow cells. The resulting chimeras with an invisible immune system thus enabled intravital imaging of FRCs, FDCs, and vascular cells as GFP+ cells in the lymph node. Although these cellular elements were well known by histological examination, the overall effect for live-cell immunoimaging was as if a previously invisible but extensive highway system were suddenly illuminated. In the T cell zone, the scaffold consists of a 3-D chicken-wire network of 5-μm thin desmin+ ERTR-7+ cells with 20to 30-μm gaps between scaffold elements. Intravital imaging of red-labeled lymphocytes that were “salted in” as a random-sample survey revealed T cells crawling continuously along the FRC network, turning at branch points, and in rare instances jumping from one point on the scaffold to another, all the while making contact with scaffold elements

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b

Lymphoid follicles

B

B

B

T B

T T

HEV T

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c

Visualized T cells

Sessile dendritic cell in network

Medullary sinus

B

B Efferent lymphatic

Hilar region

Lymphatic endothelial barrier

T cell traversing the FRC corridor

Efferent /afferent blood vessels

Lymph node shutdown

Steady-state lymphocyte egress Transendothelial migration of lymphocytes into sinus

Lymphatic endothelial cell

S1P gradient

Unlabeled T cells

B

T

B

Subcapsular sinus

T

T

Collagen fibril

Fibroblastic reticular cells (FRC)

Afferent lymphatic

Agonist activity, receptor internalization, loss of S1P sensitivity

Rac Gi

S1P1 agonist

S1P1 receptor

S1P1 receptor internalization/ recirculation

Agonist activity, closing of endothelial gates

Egress stops, lymphatic sinuses clear

Lymphocyte LY MP HAT I C SIN U S

Figure 2 Structural and functional aspects of lymph node organization. (a) Macroscopic organization of lymph node regions, illustrating B cell follicles, T cell localization within the paracortex, and high endothelial venules (HEVs). (b) Schematic illustrating the motility of labeled T cells (orange) along and across the ﬁbroblastic reticular scaffolding, in the presence of DC and an excess of unlabeled T cells (yellow), only a small fraction of which are shown in one region. (c) Hypothesis for regulation of lymphocyte egress by sphingosine 1-phosphate (S1P), illustrated under conditions of steady-state egress (left) and lymphopenia (right). T cells are shown with S1P1 receptors either exposed or internalized in the presence of S1P. Junctions between the lymphatic endothelial cells close in the presence of an S1P1 agonist.

like children on a jungle gym. Bajenoff and colleagues (25) also provided examples of T cell homing behavior, which had previously been imaged only in the absence of cellular landmarks (26). T cells enter the lymph node across HEVs and follow exit ramps to encounter a network of FRCs and DCs (25). Recently homed B cells also follow the FRC network until they reach the follicle, but within 594

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the follicle they migrate in contact with relatively sessile FDCs. Simultaneous imaging of T cells with the stromal reticular network reinforces the importance of lymph node structural components that deliver antigen rapidly to the T cell zone and conﬁrms ideas developed by Anderson, Shaw, and others on the basis of static imaging of histological sections (53–56).

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FRCs and FDCs express the ligands ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule1), potentially providing an adhesive substrate for T and B cells, both of which express the corresponding integrins LFA-1 (lymphocyte function-associated antigen 1) and VLA-4 (very late activated antigen 4). The surfaces of FRCs and FDCs are decorated with differing chemokines bound to surface glycosaminoglycans. FRCs secrete the chemokines CCL19, CCL21, and CXCL12 and stain most strongly for surface-bound CCL21 in the paracortex (25, 57). CCL21 is a chemokine ligand of CCR7 on T cells. T cells have also been observed migrating directly in contact with resident DCs (22) that also express surface-bound CCL21 (58). The directionality of cells along a scaffold element is random, and thus movement along the scaffold is not guided by chemotaxis. In the next section, we review recent evidence strongly implicating haptotactic CCL21CCR7 signaling to generally augment basal T cell motility. FDCs bear surface-bound CXCL13, the chemokine ligand of CXCR5 expressed by B cells. By analogy with the role of CCR7 on T cells, CXCL13 on the surface of FDCs acting on CXCR5 receptors may thus exert a similar inﬂuence on B cell motility in the follicle. The ﬁdelity with which lymphocytes remain on the scaffold in the context of antigen challenge and molecular interventions will be intriguing to determine.

Signaling Pathways Regulating Basal Lymphocyte Motility In their default state in the absence of antigen, lymphocytes are highly polarized in the lymph node and move more rapidly than other cell types. On the one hand, although it is well established that chemokine receptor expression regulates lymphocyte homing and cellular localization within the lymph node (34, 52, 59–62), the predominant random-walk pattern of T and B cells within their respective

regions is inconsistent with what would be expected for chemotaxis, by which cells migrate collectively toward a source of chemokine. On the other hand, recent studies have clariﬁed the role of chemokines in enhancing overall lymphocyte motility (chemokinesis), particularly when bound to substrates (haptokinesis), the manner in which chemokines are presented on stromal cell surfaces in vivo (43, 63). Several recent studies have begun to unravel the molecular requirements for lymphocyte motility within the lymph node (Table 2) by dissecting the signaling pathway that leads from chemokine to receptor (G protein–coupled receptor, or GPCR) to G protein and, ultimately, to the cytoskeleton. Some studies have focused on lymphocyteextrinsic factors such as chemokines found in the lymph node environment, and others have focused on cell-intrinsic factors that include molecules or signaling pathways within the cell. G protein–coupled receptor. Chemokines bind to heterotrimeric GPCRs to trigger downstream changes in lymphocyte polarity and migration. In particular, pertussis toxin– sensitive Gαi isoforms play a key role in homing and localization, and the chemokine receptors expressed by T cells (e.g., CCR7 and CXCR4) couple through Gαi . Three studies now indicate that Gαi may play a major role in promoting basal T cell motility in the lymph node (47, 57, 64) by showing that pertussis toxin inhibition of Gαi results in a decreased T cell velocity and motility coefﬁcient (Table 2). Cells fail to migrate over long distances not only because they move more slowly, but also because they turn with broader angles. The Gαi subunit of heterotrimeric Gi exists in three mammalian isoforms. Genetic deletion of Gαi 2 results in a smaller effect in B cells (65) than in T cells (66). Chemokines and haptokinesis. Among the chemokine receptors that couple through pertussis toxin–sensitive Gi proteins, CCR7 has a substantive role in promoting T cell motility

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Molecular interventions and T cell motility in lymph node Homing and Egress Rates

Condition or treatment

Turning angle

Motility coefficient

Pertussis toxin

↓↓ homing

↓50%–60%

↑↑ (broader)

Gαi 2−/−

↓ homing

↓30%–50%

↑

CCR7−/−

↓ homing

↓20%–30%

↔a

↓50%–60%

CXCR4 antagonist or

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Velocity

CXCR4−/−

↓↓80%–90%

References 47, 57, 64 66

↔

47, 48, 64 57, 64

plt recipient mice

↔

↓20%–30%

↔

↓70%

48, 57, 64

p110γ−/−

↓homing

↔

↑

↓16%

71, 72, 76

p110δ−/−

↔

N/Ab

N/A

N/A

71

p85α−/−

N/A

↓12%

N/A

↓21%

70

p85β−/−

N/A

↓26%

N/A

↓56%

70

p85α/ p55α/ p50α/ p85β−/−

N/A

↓37%

N/A

↓78%

70

Wortmannin

↔

↓26%c

N/A

↓33%

70

↔c

57c

↔c DOCK2−/−

↓ homing and egress

↓40% paracortex, ↓60% medulla

↑↑

↓93%

72, 76

DOCK2/p110γ

↓↓ homing and egress

Similar to DOCK2−/−

↑↑

↓96%

72, 76

CD18−/− (lacking LFA-1)

↔

↓15%

↑

↓30%

63

ICAM1−/−

↓25%

63

antiα4 integrin

↓15%

63

MHC-II recipient after 7 days

↓70%

82

↓↓ egress

↔ in paracortex

47, 93

↓↓ egress

↔ paracortex

93

↔

↔ paracortex

84

FTY S1PR1

−/−

S1PR antagonist

↔ medulla ↓↓ egress

S1PR1 agonist

↔ paracortex, ↓↓ medulla

84

a

↔, no change. N/A, not applicable. c Different conclusions in cited references. b

in the lymph node. Four recent studies concur that CCR7−/− T cells show a 20%–30% reduction in T cell velocities (47, 48, 57, 64). CCL19 and CCL21, ligands for CCR7, both enhance motility of T cells when bound to substrates in vitro (43, 63). CCL21 is present in lymph nodes at much higher concentration than CCL19 (67) and is bound to the cell surface of both FRCs and DCs (57, 58). In recipient plt mice lacking CCL19 and CCL21, velocities of wild-type T cells are inhibited to a similar extent as in CCR7-deﬁcient T cells (48, 57, 64), and motility of wild-type T cells in plt recipients recovers to normal following 596

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i.v. injection of CCL19 (48). Together, these results indicate that CCR7 plays an important role in enhancing T cell motility within the lymph node, with residual motility of cells perhaps reﬂecting chemokinesis mediated by another chemokine. Inhibition of CXCR4, using a speciﬁc antagonist or by genetic deletion, has no effect in wild-type T cells (57, 64), but one report (57) observed a further diminution of motility in CCR7-deﬁcient T cells to a level comparable with inhibition by pertussis toxin. Thus, CCR7 appears to mediate an important chemokinetic effect on motility, with CXCR4 playing a possible secondary role.

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Integrins and adhesion molecules. Cell adhesion molecules, such as ICAM-1 on stromal cells and APCs and integrins on T cells, are also thought to regulate lymphocyte motility (68), whereas their role in supporting basal motility in the lymph node may be surprisingly limited. A recent paper (63), combining in vitro and in situ imaging of lymphocytes, elegantly reveals the interplay between CCR7, the integrins LFA-1 and VLA-4, and shear stress on lymphocyte motility. Shear stress induced by solution ﬂow over cultured cells rapidly causes arrest as a result of LFA-1 adhesion to ICAM-1 on the substrate (63). Such conditions are likely present in the vascular lumen, where integrins mediate the adhesive interaction that stops lymphocytes at sites of homing. Once inside the lymph nodes, however, shear stresses are absent, minimizing the adhesive function of LFA-1 interactions with ICAM-1. Consistent with this interpretation, Woolf et al. (63) further show that genetic deletion of LFA-1 in the donor T cell or ICAM-1 in the recipient lymph node has little effect on T cell motility, and blockade of VLA-4 using a blocking antibody has no additional effect. Phosphoinositide 3-kinase. Among members of the phosphoinositide 3-kinase (PI3K) family, two major classes, IA and IB, are important in cell motility (69). Class IA and IB PI3K regulatory subunits include ﬁve and two isoforms, respectively, that, together with distinct catalytic subunits, are expressed in combinations of heterodimers in a tissue-speciﬁc manner. Two groups explored the possible involvement of PI3K activity in modulating lymphocyte motility by employing wortmannin as an irreversible inhibitor of all PI3K isoforms, but they reported divergent results. In lymph node slices imaged by epiﬂuorescence microscopy, pretreatment with wortmannin had no effect on T cell motility (57). In contrast, recent work from our group, in collaboration with David Fruman (70), showed that wortmannin treatment resulted in a signiﬁcant decrease in T and B cell motility in lymph

node explants (Table 2). Although class IA and IB catalytic subunits (p110δ and p110γ, respectively) had previously been shown to be involved in lymphocyte chemotaxis and homing (71, 72), and p110γ is indispensable for neutrophil chemotaxis (73–75), p110γ deletion had only a minor effect on T cell motility and did not alter velocity or B cell motility (76). Among class IA regulatory isoforms, T cells lacking either p85α or p85β showed reduced velocities, with p85β deletion being more profound. Genetically targeting four class IA regulatory isoforms together produced an even more dramatic motility phenotype (Table 2), suggesting an additive effect (70). In B cells, regulatory isoforms may perform distinct functions, as p85α knockouts showed reduced velocity and displayed a striking, dendritic-like morphology, whereas p85β deletion had modest effects (70). Dedicator of cytokinesis 2 (DOCK2). Guanine nucleotide exchange factors such as DOCK2 are centrally poised to communicate between GPCRs and cytoskeletal elements, and DOCK2 in particular plays a key role in lymphocyte homing, chemotaxis, and motility (72, 77). In lymphocytes, DOCK2 is required for Rac activation and actin polymerization and thus results in migration defects of lymphocytes (78). Similar to effects of pertussis toxin treatment discussed above, DOCK2-deﬁcient T lymphocytes exhibit profound defects in lymphocyte motility (76) (Table 2), including a decrease in velocity and an even more severe decrease in motility coefﬁcient resulting from increased turning angle. Similar defects are observed in B cells from DOCK2−/− mice. Double deletion of DOCK2 together with p110γ does not produce further changes, suggesting downstream mediation of DOCK2, but the possible interplay between DOCK2 and class IA regulatory isoforms of PI3K has not been examined. Ca2+ ions and stop-and-go basal motility. The rise in cytosolic Ca2+ concentration triggered by T cell receptor (TCR) engagement

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leads to gene expression required for lymphocyte activation. However, even in the absence of cognate antigen, intermittent Ca2+ signals occur in T cells as they migrate. In two recent studies (57, 79), Ca2+ was monitored during basal locomotion of T cells in lymph nodes. Ca2+ spikes lasting less than 2 min and rising sharply to a peak of about 200 nM are associated with temporary slowing of adoptively transferred T cells loaded with the Ca2+ indicator indo-1 and imaged in lymph node explants (79). In lymph node slices, Ca2+ spiking occurs in about 25% of T cells loaded with fura-2 and with higher frequency in arrested cells (57). Ca2+ spiking may be triggered by contact with DCs in the absence of speciﬁc antigen, and such intermittent spiking may play a role in cell survival, as it is less often observed in the absence of MHC class II in vitro (80). Such Ca2+ spikes do not, however, correlate well with the ongoing stop-and-go behavior of motile T cells.

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MHC class II. Self-recognition mediated by contact with MHC class II is vital for T cell survival and proliferation in vivo (81). A recent study (82) showed that T cells deprived of contact with MHC class II in vivo by transferring OT-II T cells into MHC class II–deﬁcient recipients become progressively immotile and disabled in their in vivo proliferative response to antigen. After seven days, T cells are essentially immotile. Biochemical analysis of T cells from mice treated in vivo with anti–MHC class II antibody, which deprives CD4+ T cells of contact with self ligands, revealed a strong reduction in both active and total Rap1 and Rac1, but not in ras, suggesting a possible mechanism underlying the defect. The effects of MHC deprivation increase progressively, resulting in a diminished ability to proliferate from 30% after one day to complete inhibition within ﬁve days. The diminution in motility is likely not the sole cause of the decreased response to antigen but may contribute at later times, as motility is normal at intermediate times when activation responses had already declined substantially. 598

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Temperature and tissue oxygenation. Lymphocyte motility is extraordinarily sensitive to experimental conditions. By common observation, lymphocytes stop moving if the laser power is too high, if excessive pressure is placed upon the lymph node, if the temperature is too low or too high, or if the tissue is not oxygenated, both in vitro and in vivo. The potential interplay between temperature and tissue oxygenation has been examined in greater detail using the lymph node explant preparation (47).

Regional Variations of T Cell Motility in the Lymph Node Follicle edge. The trajectories of T and B cells at the edge of a follicle are decidedly nonrandom. There is a rather sharp divide of T cells outside and B cells inside the follicle, and cells migrating near the T-B boundary show relatively little mixing (83). Instead, most cells approach the edge and, without slowing, turn acutely and move back to join their compatriots. The occasional stray T cell that moves into the follicle and migrates among B cells maintains an average velocity typical of T cells in the diffuse cortex. Sharp segregation of T and B cells may be achieved simply by T and B cells remaining attached to their respective scaffold elements; Bajenoff et al. (25) show that most T cells straying into the B cell region still retain connection to FRCs. The important follicular edge, where helper T cells and B cells interact following antigen priming, is thus likely to be deﬁned by overlap of follicular reticular cells. Subcapsular sinus. T cells move with reduced velocity (35%–50%) and wider turning angles in the shallow subcapsular region of the lymph node (46–48, 66). These motility characteristics are similar to those of T cells in the paracortex in the absence of CCR7 or its ligands (Table 2). Indeed, the region of diminished motility in the subcapsular sinus correlates spatially with the absence of CCR7 ligands. Furthermore, subcutaneous injection

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of the CCR7 ligand, CCL19, greatly enhances motility and numbers of wild-type, but not of CCR7-deﬁcient, T cells in the subcapsular sinus (48). Medulla. T cells in the medullary region near the hilus of the lymph node move more slowly than in the paracortex, with a 2-D velocity of 6 μm/min (76, 84). CXCL12 is enriched in the medulla (57, 85), but this chemokine, the ligand for CXCR4, does not induce chemokinesis in vitro (64) and does not affect motility in the paracortex. Control of lymphocyte egress. Lymphocytes exit the lymph node by migrating from medullary cords across an endothelial barrier into efferent sinus vessels. A convergence of pharmacological (86, 87) and genetic evidence (88) indicates that sphingosine 1-phosphate (S1P1 ) receptors regulate lymphocyte egress. In particular, FTY720 in its in vivo–phosphorylated form and related compounds that bind with high afﬁnity and speciﬁcity to different Gi-coupled S1P receptors result in lymphopenia, as lymphocytes are trapped within secondary lymphoid organs (86, 87), and genetic targeting of S1P1 receptors prevents lymphocyte egress (88). S1P1 receptors are expressed on both lymphocytes and lymphatic endothelial cells, and there is continuing debate regarding the cellular targets and mechanisms regulating egress. Several recent reviews (52, 89, 90) discuss this topic. One school considers that lymphocytes migrate by chemotaxis toward a higher S1P concentration in the sinus lumen. The action of FTY720 is then explained by functional antagonism, whereby irreversible binding of FTY720 effectively removes S1P1 receptors from the lymphocyte surface and degrades them, rendering lymphocytes blind to an S1P gradient. An alternative (not necessarily mutually exclusive) hypothesis implicates stromal gating by lymphatic endothelial cells. In this view, S1P1 agonists engage endothelial receptors and close the gates through which lymphocytes migrate. Here, we focus on imaging

data showing that agonist activity is required for pharmacologically induced sequestration. Imaging of the egress step was ﬁrst achieved in explanted nodes by orienting medullary sinuses on the hilar side toward the microscope (76, 84), and subsequently extended to intravital imaging (91). In the presence of a reversible S1P1 receptor agonist that does not lead to receptor degradation (87, 92), medullary sinuses (visualized using a ﬂuorescently conjugated lectin to stain the endothelial lining) are empty, whereas rounded and immotile lymphocytes become logjammed in the adjacent medullary cords. Removal of S1P1 agonist, or addition of a molar excess of an S1P1 receptor agonist in the maintained presence of agonist, results in rapid resumption of motility, with lymphocytes often crossing into the sinus at speciﬁc portals (84). A further key point is that neither the S1P1 receptor agonism nor antagonism alters the robust motility of T cells in the paracortex of the node (Table 2), with the agonist action being restricted to the medullary region where basal motility is already lower (6 μm/min) than in the paracortex (10–12 μm/min). Any model of egress and the role of S1P receptors under basal conditions must account for the following results: (a) Transendothelial migration occurs at preferential sites (portals) in the lymphatic endothelium (84); (b) S1P1 receptor agonism induces actin polymerization in lymphocytes (76), potentially accounting for observed reductions in lymphocyte velocity in the medulla (84); (c) lymphocyte migration in the paracortex is unaffected by reversible S1P1 or receptor-degrading agonists or antagonist (84, 93); and (d ) reversible S1P1 agonists that induce lymphocyte sequestration produce a rapid and region-speciﬁc motility arrest that is reversible upon removal of the agonist or addition of a stoichiometric antagonist (84, 91). We propose a hybrid model (Figure 2c) in which both S1P1 receptors in lymphocytes and lymphatic endothelial cells play a role. Intrinsic S1P agonism inhibits lymphocyte motility in a region-speciﬁc manner, constrained to the medullary cords where

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CCR7-induced haptokinesis may be reduced. In the absence of agonist, the random motility of lymphocytes brings them to portals through which they transit into the lymphatic sinus system, where lymphatic ﬂow returns them to the blood. As a further checkpoint, the portals are gated by S1P1 agonist action.

Overview of Extrinsic Factors, Intrinsic Factors, and the Random Walk Annu. Rev. Immunol. 2008.26:585-626. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Within the past two years, the roles of stromal cells and chemokines in lymphocyte motility within the lymph node have been clariﬁed, and signiﬁcant progress has been made in deﬁning signaling molecules and pathways within the cell that are essential for motility (Table 2). Lymphocytes migrate along a stromal cell scaffold in which both FRCs and DCs display surface-bound CCL21. T cells, while crawling on FRCs that encapsulate the reticular conduits, make brief contacts with DCs to sample antigen along the way. T cell motility along the scaffold is enhanced by the chemokine CCL21 bound to the surface of FRCs. By haptokinesis, CCL21 binds to CCR7, which couples through the Gαi subunit of heterotrimeric Gi to enhance motility. When this function is disabled, T cells crawl with reduced velocity and broader turning angles, giving rise to highly localized cell tracks. In these instances, cell rounding of normally polarized T cells may reduce their ability to traverse adjacent elements of the scaffold, resulting in conﬁned motion and greatly reduced motility coefﬁcients. Class IA PI3K regulatory isoforms and DOCK2 mediate important effects on motility, but their links to chemokine receptors and downstream cytoskeletal effectors have not yet been fully deﬁned. A picture is thus emerging that the random walk of T and B cells in their respective compartments is guided and reinforced by haptokinetic signals obtained from stromal cells, and possibly also by DCs, and is mediated by chemokine receptor signaling through 600

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G proteins. To some extent, the stop-andgo program of motility may be intrinsic to lymphocytes because similar motile behavior occurs, albeit at lower velocity, even on a clean glass coverslip. Yet, there is no doubt that extrinsic factors enhance and guide motility in the lymph node environment. At the single-cell level, lymphocyte motility is not autonomous but rather is constrained by the scaffold that provides guidance cues as well as haptokinetic help via chemokines acting on GPCRs. From a systems perspective, however, the resultant motility patterns resemble swarm intelligence, by which the dynamic and seemingly autonomous motility of lymphocytes serves collectively to promote exploration of the interstitial lymphoid space and detect antigens. In addition, stromal guidance calls into question the role of chemokine gradients in guiding localization of T and B cells into separate compartments within the lymph node. If stromal cells are “rulers over randomness” (94), the exclusion of T lymphocytes from the follicle may depend on the distribution of FRCs, rather than on localized chemotaxis at the edge of the follicle. In this view, the issue of lymphocyte localization has been pushed back developmentally to the question of what limits the extent of FRCs and FDCs that form the respective scaffolds for T and B cells. B cells are more promiscuous in crawling both on FRCs soon after homing and among FDCs once inside the follicle. Further tests of the possible role of local chemokine gradients and stromal guidance at the edge of the follicle will rely on a more detailed analysis of cell behavior in this region and the ability to perturb local chemokine gradients. Figure 3 provides a visual representation, with videos available online, of basal motility in the lymph node in the absence of antigen.

Migration and Dynamics of DCs DCs initiate immune responses by capturing, processing, and presenting antigen. More than 60% of T cells in the lymph node are thought to be contacting a DC at any

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No specific antigen

a

B cell

T cell

VIDEO 3a

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MΦ

B

B

B

B

MΦ

FDC

B B

T Follicle

Diffuse cortex

T

T

T T Lymphatic endothelial barrier

Diffuse cortex Follicle

FDC

B

T T B

T

T T

B

B Follicle

DC VIDEO 3b

c T cell Lymphatic endothelium VIDEO 3c

Figure 3 Cell motility and interactions in the absence of antigen. T and B cells are depicted migrating randomly in diffuse cortex and follicles, respectively. A recently homed B cell is on its way toward the follicle. Macrophages and FDCs within the follicle are devoid of speciﬁc antigen. A T cell is shown interacting transiently with a resident DC, while another T cell crosses the lymphatic endothelium to gain access to the efferent lymphatic vessel. Shown on right are frames from Videos 3a, 3b, and 3c, respectively: (a) Montage of T cell and B cell dynamics, imaged separately in their respective regions (2); (b) T cells interacting transiently with a DC (96) (panel on the left is a “true” color maximum intensity projection along the z-axis and that on the right shows the same sequence after color depth encoding); (c) T cells migrating across the lymphatic endothelium (endothelial cells are stained with red ﬂuorescent lectin) (84). Videos may be viewed separately by following the Supplemental Materials link on the Annual Reviews website at http://www.annualreviews.org.

time (95). Steady-state and freshly migrated Langerhans-derived DCs populate the entire diffuse cortex of the lymph node, whereas newly immigrated dermal DCs localize to the cortical ridge, adjacent to reticular conduits for efﬁcient antigen capture and also near

b

Depth encoded

B

T

Dendritic cell

T

T

B

T cell

HEVs to favor encounters with newly homed T cells. Resident DCs are in intimate contact with FRCs that surround the reticular conduits, and both cell types bear surfacebound CCL21 (25, 58). Networks of CD11c+ steady-state DCs have been visualized using

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two-photon microscopy (22). Resident DCs are sessile, but long and active dendritic processes actively probe passing motile T cells. Freshly migrated DCs carry antigen from the periphery and dynamically traverse the diffuse cortex of the draining lymph nodes, often approaching the cortical ridge, and continually scan T cells with actively probing dendrites (49, 96). FDCs are not of hematopoietic lineage. These radiation-resistant stromal cells populate B cell follicles and have a high capacity to capture and retain antigen in the form of immune complexes (97–99). Imaging of FDCs by Bajenoff et al. (25) shows them to be stationary but displaying irregular and highly motile strands. While migrating in the follicle, B cells extensively contact the cell body and strands of FDCs. FDCs have also been imaged in germinal centers (21).

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Natural Killer Cells: Stationary or Motile? Although sparse in lymph nodes (<0.5% of total cell count), natural killer (NK) cells serve distinct roles in both innate and adaptive immunity (100) and are mobilized during infection and transplantation to produce cytokines and directly kill virus-infected or foreign cells. They are strategically poised to inﬂuence antigen-speciﬁc responses during parasite infection, tumor rejection, and transplantation (101, 102). Two studies have now imaged NK cells in lymph nodes (103, 104). Bajenoff and colleagues (103) isolated NK cells by magnetic bead selection using CD49b, an α2 integrin with afﬁnity for collagen. Imaged in the paracortex, these positively selected NK cells are scarcely motile, averaging less than 3 μm/min (in contrast to highly motile T cells moving three- to fourfold faster in the same region), and form prolonged contacts with both resident and newly arrived DCs. During Leishmania infection, NK cells are recruited from the blood, resulting in a tenfold increase in NK cell numbers in the lymph node, and localize primarily under and between B cell follicles. Here, a substan602

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tial fraction of interferon (IFN)-γ-producing cells (80%) are CD49b+ NK cells in close proximity to DCs and CD4+ T cells undergoing activation. Again, the NK cells responding to Leishmania infection and interacting closely with DCs are immotile. Localized IFN-γ secretion by NK cells in close proximity to resident DC networks could alter DC maturation and promote a Th1 polarized response (100). A very different view of NK cell motility in the lymph node emerged from a study utilizing NK cells from RAG−/− donors in order to avoid the need for positive selection (104). These untouched NK cells localize to the paracortex, consistent with the previous study (103), but they are highly motile (6– 7 μm/min) along randomly oriented trajectories (104). To resolve the apparent discrepancy in cell motility with the earlier report, Garrod et al. (104) showed that in vitro cross-linking of CD49b in the positive selection procedure renders the RAG−/− NK cells immotile following subsequent transfer and homing into lymph nodes. Positively selected NK cells have increased adherence to collagen, and immotile NK cells are found in the lymph node attached to collagen ﬁbers, whereas unmanipulated NK cells migrate rapidly along collagen ﬁbers. These ﬁndings sound a cautionary note about potential effects of positive selection on cell motility and, more generally, on other cellular responses studied following cell isolation. It will be of interest to track NK cell behavior in a more collagen-rich environment such as the skin, where these cells are mobilized in psoriasis or transplant.

CELL DYNAMICS AND INTERACTIONS DURING ANTIGEN ACQUISITION AND RECOGNITION Dramatic changes in cellular choreography accompany the initiation of an immune response to a speciﬁc antigen. Although the immune response usually begins and ends in the periphery at sites of infection or immunization, the lymph node functions as a crucial

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ﬁlter to capture, concentrate, and deliver antigens and as a site where cells interact to make decisions about whether to secrete cytokines, proliferate, and differentiate. It is now clear that very different mechanisms are employed

to deliver antigens to T cells than are used for antigen capture by B cells. Moreover, a very different cellular choreography takes place for T and B cells when they are activated (Figure 4). Focusing primarily on recent

Antigen: two waves

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Figure 4 Cellular choreography with antigen. B cells move by chemotaxis to the follicle edge, after picking up antigen from macrophages and from FDCs. A T cell/B cell conjugate pair is shown at the edge of a follicle, together with T cells clustering around a dendritic cell bearing cognate antigen. T cells proliferate at this time, and egress is blocked. Shown on right are frames from the following videos: (a) B cell chemotaxis (146); (b) T cell/B cell motile pairs (146); (c) T cell/DC clustering (84, 117); (d ) stereo movie of T cell proliferation (26); (e) egress block and subsequent recovery following removal of S1P1 agonist at lymphatic barrier. Video Links 4a–4e may be viewed separately by following the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org. www.annualreviews.org • Cell Motility and Interaction Dynamics Imaged

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Antigen Acquisition

of immunization is limited, and local capture by DCs followed by migration into the lymph node will dominate. The chemical composition of the adjuvant can also polarize a subsequent response by triggering different TLRs on DCs to promote subsequent Th1 or Th2 differentiation (109, 110). Different TLR agonists are known to differentially regulate the production of DC-derived IL-12, thus skewing Th1 or Th2 responses (109). Soluble antigen can use both routes, arriving passively within minutes or being conveyed as pMHC by tissue-derived DCs.

Antigen delivery to and capture by DCs. Pioneering static imaging studies showed that the delivery of soluble antigen from the periphery via afferent lymphatic vessels into the lymph node occurs in two successive waves (105). Soluble antigen arrives ﬁrst, conveyed rapidly within reticular conduits (106), and is then taken up, processed, and displayed as peptide-MHC (pMHC) by resident DCs within 30 min (105). A second wave of antigen, borne by dermal-derived DCs from the skin, subsequently arrives a few hours later. Live cell imaging in the skin shows epidermal CD11c+ Langerhans cells (langerin+ ) and deeper, immature dermal DCs (107) as being entirely immotile cells with stationary dendrites under basal conditions. After triggering by inﬂammatory cytokines and by activation of Toll-like receptors (TLRs), Langerhans cells and dermal DCs migrate to the lymph node via afferent lymphatic vessels in a process that requires upregulation of CCR7 by DCs and CCL21 by lymphatic endothelial cells (108); this process has not yet been visualized. The maturation of DCs and their cellular journey in afferent lymphatic vessels impose a delay of several hours, and CFSE-labeled migratory DCs are ﬁrst visualized following their arrival in the lymph node more than 4 h after subcutaneous injection, together with adjuvant-containing cytokines to induce maturation (96). Their numbers increase during the next day and then decline after two days in the lymph node. In an adjuvant depot, diffusion of soluble antigen away from the site

Antigen capture and delivery to B cells, and by B cells to FDCs. Antigen enters the lymph node in differing forms and by various routes. A recent study by Pape et al. (111) focuses attention on the rapid response capabilities of follicular B cells to capture soluble antigen entering the follicle. Following subcutaneous injection, soluble antigen arrives in the lymph node follicle by diffusion across small gaps in the ﬂoor of the subcapsular sinus directly to nearby follicular B cells, even faster (within a minute) than it is transmitted by reticular conduits into the T cell area. Such rapid delivery of soluble antigen from the subcapsular sinus to the interior of the follicle was not seen previously (55, 105, 106), perhaps because soluble antigen was washed away during ﬁxation. The subsequent spread of antigen into the follicle interior proceeds more rapidly than B cells can migrate, indicative of passive diffusion. Within 10 min, all the antigen-speciﬁc B cells in the follicle have taken up antigen, and by 4 h, the antigen is processed and presented bound to MHC by B cells with greater efﬁciency (more antigen acquired) than by DCs in the paracortex. DCs do not play a role in the acquisition of soluble antigen by B cells. To sustain the response to antigen, mechanisms for storage, sustained release, and delivery are also in place at the boundary of the subcapsular sinus and within the follicle. The subcapsular boundary functions as a sieve, restricting passive diffusion of larger forms of

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cellular immunoimaging studies, we follow in sequence the diverse routes by which antigen enters the lymph node; the cellular interactions between T cells and antigen-presenting DCs (close encounter of the ﬁrst kind); and then the B cell side of the story as B cells become activated, interact with helper T cells (close encounter of the second kind), and differentiate in germinal centers to become antibody-secreting plasma cells.

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antigen, such as that contained in immune complexes or bacterial particles. Two recent live-cell imaging studies (23, 112) show that antigen-trapping cells in the subcapsular sinus capture and retain surface-bound antigen. Phan et al. (23) implicate the macrophages that span the border of the subcapsular sinus, showing them in the process of capturing ﬂuorescently labeled immune complexes in the sinus lumen and handing off immune complexes to migrating B cells just inside the follicle. Unlike many other macrophages, those here are not highly phagocytic and do not rapidly degrade the antigen. Instead, they acquire and hold immune complexes on the luminal side of the subcapsular sinus and somehow convey them to the follicular face, where the cargo is displayed attached to long processes. Nonspeciﬁc B cells slow during contact with macrophages, pick up immune complexes, and then depart at the normal velocity of randomly migrating follicular B cells (2). Phan et al. (23) further show that the departing B cells display distinctive dendritic processes reminiscent of germinal center B cells and carry away noncognate immune complexes as cargo attached to the trailing uropod while migrating toward the follicle interior, where immune complexes are off-loaded to FDCs. Antigen recognition is not involved in this transport process, which relies on complement receptors CR1 and CR2 in the B cell for initial capture of immune complexes (but not for the acquisition of dendritic morphology). FDCs have a high capacity to retain immune complexes bound to high-afﬁnity FcγRIIB and complement receptors, so that B cells are relieved of the antigen burden by the time they leave the follicle. If cognate antigen is present in the immune complexes or is attached to a particle, the B cells pause longer near the macrophages as B cell antigen receptors (BCR) become engaged (23, 112), allowing them to pick up more antigen cargo. BCR engagement in turn triggers relocalization of the antigen-engaged B cell toward the T cell area. Antigen-speciﬁc B cells may also

encounter, recognize, and pick up antigen carried by nonspeciﬁc B cells that recently visited the macrophages at the subcapsular sinus (23). Collectively, these mechanisms will preserve antigen load within the follicle and allow latearriving B cells to participate in the antibody response. A third mode of antigen capture by B cells takes place outside the follicle near the sites of homing. There, a DC that has acquired antigen arriving in the reticular conduit system not only processes and presents the antigen as pMHC recognized by T cells, but also displays whole antigen bound to the cell surface in a form that is recognizable by B cells. Qi et al. (113) show by intravital immunoimaging that B cells survey the DCs immediately after homing. If speciﬁc antigen is present, the B cells stall and capture some of the surface antigen from the DC. The fate of these B cells, having engaged antigen in a most unusual locale, is to remain extrafollicular, where they can interact with helper T cells.

Activation of T Lymphocytes Antigen presentation: scanning the T cell repertoire. In the diffuse cortex, FRCs surround collagen ﬁbers and a basement membrane, forming a reticular conduit for antigen (53–55, 106). The cortical ridge region just outside the follicle includes resident DCs near sites of lymphocyte extravasation across HEVs (56). In this region, T cells sample resident DCs for the presence of cognate peptide antigen bound to MHC. The second wave of antigen that arrives from the periphery carried into the paracortex by maturing dermalderived DCs hours after antigen injection may have differing functional consequences. Live-cell imaging reveals recently immigrated DCs to be highly active cells that localize to the cortical ridge close to HEVs. While migrating with an average velocity of 2–6 μm/min (3, 49, 96, 114), DCs rapidly extend and retract dynamic dendritic processes, thereby greatly expanding their scanning range to >60 μm from the cell soma

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(possibly longer with nanotubes), and enabling DCs to contact many more T cells than if they were stationary. The effective swept volume covered by motile dendrites is about threefold larger than if DCs were rounded (96). In the absence of antigen, individual DCs make contact with up to 5000 T cells per hour (96, 114), an astonishing rate of cellular interaction that is promoted by dynamic DC dendrites and robust T cell motility. Contacts occur primarily on DC dendrites, rather than on the cell body, with a surface contact area on the order of 10 μm2 (96), comparable to that needed to trigger a T cell Ca2+ signal through presentation of TCR ligand on beads (115). Interactions between T cells and DCs appear predominantly to involve random encounters, as T cells approach DCs without changing course and migrate away after brief contact at the same velocity as their arrival (96). Such randomly initiated, shortduration encounters followed by rapid departure avoid unwanted competition for DC surface space by irrelevant T cells that would accompany nonspeciﬁc chemoattraction. The measured frequency of interaction suggests that any given DC can probe hundreds of motile T cells within a few minutes. Miller et al. (96) thus proposed a stochastic mechanism of antigen scanning that allows T cells to survey DCs within the diffuse cortex by randomly initiated contacts and initiate activation responses within 6 h, provided that ∼100 antigen-bearing DCs are present. However, in addition to random motility, we have also noted striking instances in which several T cells simultaneously migrate from different positions toward a particular DC (M.D. Cahalan, S. Wei, and M. Matheu, unpublished observation), raising the possibility of intermittent chemokine-mediated guidance—for example by puffs of soluble CCL19 emitted from DCs, consistent with a proposed role of this chemokine in scanning behavior (116).

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Stages of T cell–DC interaction during the first day of an immune response. In contrast to the brief contacts between T cells and 606

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DCs in the absence of antigen, early reports described T cells forming long-lived (hours) contacts with adoptively transferred, peptidepulsed DCs, with ten or more T cells clustered on a single DC (3, 114). Covisualization of T cells and DCs at different times during the ﬁrst day of in vivo antigen priming subsequently dissected distinct stages of interaction leading to cell proliferation (49, 117). These stages may differ somewhat between CD4+ and CD8+ T cells. During the initial stage, both CD4+ (117) and CD8+ T cells (49) make transient, serial contacts with several DCs, encounters that are sufﬁcient to trigger upregulation of the activation marker CD69. This ﬁrst stage is shorter for CD4+ T cells (2– 4 h) (117) than for CD8+ T cells (8 h) (49). Both long and transient CD4+ T cell/DC encounters were seen during the ﬁrst 2 h in our original study (96), but long-duration encounters were predominant in subsequent investigations by Shakhar et al. (50) and from our group in a study that incorporated Ca2+ imaging (79), possibly reﬂecting a greater quantity of antigen delivery in these later studies. After progression to the second stage, T cells are observed in dense clusters, with several T cells usually maintaining continuous contact with a single DC for >90 min (the usual limit for live-cell observation by two-photon microscopy). Although generally stable, clusters also exhibit dynamic changes, with DCs ripping T cells away from nearby DCs (96), suggesting one type of competition that could occur if a second wave of DCs arrived. This second, cluster stage is accompanied by IL2 production, indicating activation of NFAT (nuclear factor of activated T cells)-dependent downstream signaling leading to gene expression. From 16–24 h after initially encountering antigen, enlarged T cells dissociate from DC clusters and begin to swarm in looping patterns around DCs in a localized region. During this third stage, T cells again form transient contacts with additional DCs and later begin to proliferate. Motile T cell blasts that had previously separated from DCs round up and divide within 20–30 min. The daughter

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T cells are immediately motile with 2-D velocities of about 10 μm/min, and within ﬁve days T cells have divided up to eight times. T cell Ca2+ signal during antigen recognition. Progressing beyond an initial phenomenological description of cell behavior in the lymph node, the next challenge is to understand how cell interactions and signaling cascades within the cell are integrated to promote various immunological outcomes. Within a few seconds of TCR engagement, Ca2+ signaling is initiated by IP3 -induced Ca2+ release from the endoplasmic reticulum (ER) and is subsequently sustained by Ca2+ inﬂux through store-operated Ca2+ releaseactivated Ca2+ (CRAC) channels. CRAC channels are formed by Orai1 multimers activated by STIM1; these STIM1 multimers function dually as the ER Ca2+ sensor and as the activator of CRAC channel activity (118– 120). Ca2+ signals must exceed 500 nM for at least 1–2 h to drive gene expression via the calcineurin-dependent NFAT transcription factor pathway (121–123). The Ca2+ signal may be oscillatory, spike-like, or sustained in individual cells. The frequency and pattern of Ca2+ signaling are important for activating various transcription factor pathways in a differential manner (124, 125). Although in vitro studies have revealed much about the molecular mechanism and patterns of T cell Ca2+ signaling that lead to downstream transcriptional activation, an important goal remains to monitor signaling in primary T cells within the physiological context of antigen presentation inside the lymph node. A recent paper (79) describes Ca2+ signaling in lymph node T cells under basal conditions, during nonspeciﬁc inﬂammatory conditions, and during antigen-speciﬁc responses. Under basal conditions, T cells migrate rapidly and have low cytosolic Ca2+ , showing only infrequent, small-amplitude Ca2+ spike activity associated with T cell slowing. Inﬂammation by itself reduces motility and nearly eliminates intermittent Ca2+ spiking without affecting average Ca2+ lev-

els. Antigen priming reduces velocities further and evokes strong Ca2+ signaling activity (79), evident as irregular transients to >500 nM. Cells with the strongest Ca2+ signals are associated with the lowest velocities. Those T cells observed in direct contact with labeled DCs have more pronounced Ca2+ signals, but the initiation and termination of DC contacts are not clearly correlated with any change in Ca2+ signaling. T cell Ca2+ signaling continues even after detachment from all visible DCs. These results deﬁne the pattern of the Ca2+ signal in naive T cells during antigen presentation under physiological conditions as consisting of persistent and irregular spike activity. Using confocal microscopy and a nonratiometric Ca2+ indicator, Skokos et al. (126) detected Ca2+ signals associated with T cell slowing in freshly homed T cells interacting with DC that had taken up DEC-205conjugated high-potency peptide under tolerizing conditions. Lower-potency peptides failed to elicit Ca2+ signaling, T cell arrest, and downstream proliferative responses, even though they elicited upregulation of CD69. Together, these ﬁrst two Ca2+ imaging studies (79, 126) in lymph node emphasize the involvement of Ca2+ signaling in producing T cell arrest, regardless of T cell priming or tolerization, and herald a new phase of imaging that incorporates intracellular signaling responses together with tracking cell behavior in relation to functional outcomes. Stop signal. Calcium signaling during antigen recognition inhibits T lymphocyte motility in vitro (38, 127, 128). T cell motility in vitro is also inhibited by elevated Ca2+ when proximal TCR signaling is bypassed by the use of Ca2+ ionophores or by treatment with thapsigargin. Immunoimaging of T cells, B cells, and thymocytes indicates that the Ca2+ stop signal hypothesis also applies in vivo. Intermittent Ca2+ signaling in the absence of antigen is sufﬁcient to reduce the velocity of T cell migration (57, 79). Furthermore, T cells

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and B cells produce Ca2+ signals and slow at sites of interaction with antigen-bearing DCs (79, 113). The most rigorous test was performed by Bhakta et al. (129), who showed that Ca2+ signaling is both necessary and sufﬁcient for thymocytes to stop crawling in the thymic environment. Importantly, by changing the concentrations of Ca2+ and K+ ions in the medium, Bhakta et al. were able to restore the motility of arrested thymocytes by reducing Ca2+ inﬂux. Using thapsigargin treatment, they showed that moderate Ca2+ levels stop cells reversibly, proving sufﬁciency. Lowering Ca2+ levels also inhibits Ca2+ signaling in a selecting environment and results in the resumption of motility, proving necessity. Like T cells interacting with APCs, the Ca2+ dependence of thymocyte motility prolongs interactions with stromal cells and promotes sustained Ca2+ signaling that in turn alters gene expression critical for deleting autoreactive thymocytes. Further validation of the Ca2+ stop signal hypothesis was obtained in recent work by Skokos et al. (126), who showed in the lymph node that, even under noninﬂammatory conditions, T cells decelerate and produce Ca2+ signals when responding to YFP-expressing DCs primed with the most potent in a series of altered peptide ligands. Together, these studies support the conclusion that Ca2+ signaling both in vitro and in vivo promotes the stability of T cell–APC interactions, an essential prerequisite for stabilizing the immunological synapse and downstream effector function. A potentially important new wrinkle in the stop signal hypothesis has been raised by Schneider et al. (130), who report that CTLA4 (cytotoxic lymphocyte antigen-4) increases basal T cell motility and overrides the TCRinduced stop signal required for stable conjugate formation between T cells and APC. CTLA-4 is well known as an inhibitory coreceptor on T cells that is induced upon activation and opposes costimulation by APC proteins CD80 and CD86 delivered to CD28 on the T cell. After separating CTLA-4+ and CTLA-4− cells among a preactivated pool of

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antigen-speciﬁc T cells, the authors distinguished their motility and response to subsequent antigen challenge. In the lymph node, CTLA-4+ cells do not arrest during antigen challenge, suggesting that CTLA-4 expression prevents cell arrest during contact with antigen-presenting DCs. On the basis solely of differences in T cell behavior, one may conclude that the differences in cell arrest between these preactivated populations are due to a lack of initial antigen recognition by CTLA-4+ cells and to a corresponding suppression of the Ca2+ signal to below what is needed for cell arrest (131).

Antigen Quality, Quantity, and Timing The initial descriptions of T cell–DC interactions (3, 49, 96, 114) have been extended in several studies that examined the consequences of varying conditions that alter T cell/DC dynamics and functional outcomes. When one considers the demonstrated effects of costimulatory signaling; antigen quality, quantity, and persistence; competition between cells for antigen; and timing of T cell and DC arrival to the lymph node, the potential complexities are enormous. Late T cell/DC encounters may provide a rationale for prolonging the stay inside a lymph node. During the days-long period of egress block (lymph node shutdown) during an antigen response, T cells may encounter DCs again and again to integrate signals delivered by successive waves of DCs migrating from the periphery. How these signals are integrated remains to be seen, but antigen persistence during the ﬁnal days in the lymph node can also modulate T cell differentiation programs. Experiments tracking CD4+ T cell proliferation revealed that the presence of antigen throughout their expansion phase is essential for optimal activation (132). Furthermore, T cells induce antigen loss from the surface of DCs, resulting in high-afﬁnity T cells outcompeting lowerafﬁnity T cells during a response to antigenic challenge (133).

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Tolerance and Priming. Three studies imaged T cell–DC interactions under conditions that produced either priming or tolerizing immune responses (50, 134, 135). In a priming response, initiated usually by antigen in combination with an adjuvant containing TLR ligands such as lipopolysaccharide (LPS) to promote DC maturation, the subsequent exposure to antigen induces a more rapid and robust functional response, whereas a tolerizing regimen using antigen alone results in a diminished functional response. Hugues et al. (134) and Shakhar et al. (50) used DEC-205 (to facilitate antigen uptake by steady-state DCs) conjugated to ovalbumin in the presence or absence of LPS or anti-CD40 (mimicking DC costimulation) to induce priming or tolerance. Hugues et al. (134) found that in the tolerizing regimen, CD8+ T cells slow but do not arrest to the same degree during stage II as during priming. Differing from these results on CD8+ T cells, Shakhar et al. (50) found that CD4+ T cells arrest equally well in response to priming and tolerizing regimens but become more motile sooner during tolerization (at 18 h) compared with priming (at 24 h). Comparing oral priming with oral tolerance induction, Zinselmeyer et al. (135) found only minor differences in CD4+ T cell motility and clustering behavior, with larger and more stable clusters seen in the priming regimen. Common themes among all three studies are that priming and tolerizing routines do not lead to markedly different T cell behaviors, but that priming is associated with more prolonged and/or more stable cluster formation during stage II. Such subtle differences in T cell–DC interactions could inﬂuence or reﬂect costimulatory signals delivered during the interaction. The quality and context of pMHC presentation also matter. Skokos et al. (126) compared the dynamics and functional outcomes of steady-state DCs presenting high-, medium-, or low-potency pMHC complexes varying by only a single amino acid substitution in the peptide antigen conjugated with DEC-205 under tolerizing conditions.

All three peptide ligands induced CD69 upregulation and anergy, but only high-potency MHC complexes resulted in Ca2+ signaling, T cell arrest, and stable T cell–DC interactions that subsequently led to the strongest proliferative response and cytokine production. These latter responses, but not CD69 upregulation, were inhibitable by cyclosporin A. However, CD69 upregulation induced by low- and medium-potency pMHC was shown to be sufﬁcient to produce T cell anergy that is not inhibited by cyclosporin A, suggesting that anergy can be induced by Ca2+ independent events that are activated during hit-and-run contacts with DCs. Together, these results emphasize the importance of stable T cell–DC interactions, during which effective Ca2+ signaling can take place, for proliferative responses and cytokine production in vivo.

Timing of DC arrival: sustaining the response. During the third stage of antigen presentation, CD4+ T cells resume their motility and swarm away from their clusters while making contact with additional DCs (117). The consequences of a subsequent late encounter with an additional wave of DCs have been evaluated by Celli et al. (136), who sent in reinforcements, i.e., antigen-bearing DCs, during an already developing immune response. Swarming T cell blasts found and reengaged this fresh set of recent DC immigrants, sometimes again forming long-lasting contacts. Activated T cells that reengaged fresh antigen-bearing DCs did not proliferate to a greater extent but were more likely to express the high-afﬁnity IL-2 receptor CD25 and to secrete IFN-γ. As this cytokine secretes directionally into the immunological synapse (137), it may exert a localized effect on the DC. These results on CD4+ cell behavior differ from ﬁndings in CD8+ cells rechallenged with antigen; in the latter case, CD8+ cells did not reengage in long-lasting contacts following antigen delivered as a second wave at 20 h (134).

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Timing of T cell arrival: competition and antigen persistence. Using static methods and by blocking homing to inhibit the arrival of T cells, Catron et al. (138) compared outcomes for resident and late-arriving T cells that entered the lymph node after a speciﬁc antigen response was already underway. Although this was not imaged, the late arrivers engaged DCs that presumably would have already accumulated T cell clusters. Tracking the outcome of residents and late arrivers separately showed that competition results in a weaker proliferative response among the late arrivers and predestines them to become central memory T cells. The resident T cells that beneﬁt from the full antigen load have a greater tendency to become fully differentiated effector cells. Garcia and colleagues (139) extended the theme of competition by sending in labeled T cells either alone or accompanied by a tenfold excess of identical but unlabeled cells. With low numbers of cognate T cells, competition for antigen-presenting DCs is reduced and the speciﬁc T cells form stable interactions with antigen-pulsed DCs for a longer period. At higher densities, competition among antigenspeciﬁc T cells for space on the DC surface is not limiting because the frequency of transient interactions with DCs increases when more cognate T cells are transferred. On a per cell basis, however, CD69 is less efﬁciently induced. As a direct test of whether the quantity of antigen is the limiting factor, i.v. injection of the peptide antigen restores long-duration late contacts 2 days after the initial exposure of antigen. Thus, competition for the quantity of antigen plays an important role in determining the stability of T cell/DC contacts and the duration of the second stage of stable T cell/DC clustering. The results emphasize that the initial phase of T cell expansion is limited by the quantity of antigen present. Furthermore, they imply that under the normal condition of low cognate T cell frequency, longer-duration conjugate T cell–DC interactions will form. As T cell expansion continues, competition for residual antigen by

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T cells becomes self-limiting. The latearriving T cells that have a greater likelihood of becoming central memory cells (138) may do so because they endure competition for antigen by early arrivers which are more likely to become fully differentiated effectors. Another form of negative feedback involves late-arriving cytolytic effector cells returning to the draining lymph node from the periphery. CD8+ effector cells (CCR7− Lselectin− ) return by targeted homing specifically to draining lymph nodes in a CXCR3dependent manner. Once inside the lymph node, these effectors were visualized making prolonged contacts with, and killing, antigen-bearing DCs, thereby providing negative feedback by eliminating speciﬁc antigenpresenting DCs (140).

CD4+ T Cell/CD8+ T Cell/DC Ternary Clusters During an immune response to whole antigen, both CD4+ and CD8+ T cells often become activated by contact with differing pMHC complexes on DCs. The activation of CD4+ cells augments CD8+ cell clonal expansion and differentiation to become memory and cytolytic effector CD8+ cells (141). The initial recognition of antigen by either CD4+ or CD8+ T cells may be understood in the sense of a stochastic mechanism by which T cells make numerous random contacts with DCs, but the problem is compounded by the need to get all three cell types together. To investigate mechanisms of help provided by CD4+ cells for the CD8+ response, two groups examined the dynamics of CD4+ and CD8+ T cells interacting with DCs during responses that engage both T cell subsets. Each group imaged ternary clusters with both CD4+ and CD8+ cells in direct contact with antigen-bearing DCs (142, 143). Clustering of CD8+ cells to DCs is enhanced by the presence of CD4+ cells that recognize their cognate peptide on the same DC. Notably, this tripartite interaction is diminished by several molecular interventions: blocking

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antibodies directed against either CCL3 or CCL4 (chemokines that are produced by T cells and DCs following their engagement); CCR5 genetic deletion in CD8+ T cells; and DCs lacking CD40. The assembly of the ternary cluster enhances high-afﬁnity IL-2 receptor expression and promotes generation of CD8+ memory T cells (142, 143). A variation on this theme was shown recently by the demonstration that help to naive CD8+ cells can be provided by other CD8+ cells through preferential recruitment of polyclonal CD8+ T cells to speciﬁc CD8+ T cells responding to DCs (144). As with help delivered by CD4+ T cells, the tendency for increased clustering of nonspeciﬁc polyclonal CD8+ cells requires upregulation of CCR5, induced in CD8+ T cells by inﬂammation without the need for speciﬁc antigen stimulation. Such a mechanism would encourage greater vigilance in surveying the repertoire during an ongoing immune response. A key question is whether CD8+ cells are preferentially recruited or retained (or both) by the presence of CD4+ (or CD8+ ) cells in contact with DCs. Castellino et al. (143) present evidence suggesting that CD8+ cells are recruited to clusters through chemotaxis, by analyzing the hit rate of CD8+ cells making contact with DC clusters. Within 25 μm of a DC, the wild-type cells show a pronounced directional bias toward the DC, and they approach clusters more frequently and with greater velocity than do CCR5-knockout cells. Although a caveat remains that guidance may involve ﬁne DC processes (nanotubes) below the limit of optical resolution, the results of this exciting paper suggest a scenario in which preferential recruitment of CD8+ cells is enabled by upregulation of the chemokine receptor CCR5 on CD8+ cells following antigen stimulation in the inﬂammatory environment, leading to chemotaxis in response to secretion of CCL3 and CCL4 by CD4+ /DC clusters. By favoring prolonged contacts between CD8+ T cells and DCs, the larger and longer-lasting resulting clusters would thus promote CD8+ cells to ex-

press high-afﬁnity IL-2 receptors and to become memory CD8+ cells.

Dynamics of Target Cell Recognition and Lysis by Cytotoxic T Lymphocytes and NK Cells Activated CD8+ T lymphocytes and NK cells both possess the ability to recognize and kill target cells by triggered exocytosis of cytolytic granules. Two studies describe the dynamics of target cell lysis within the lymph node environment. In a beautiful, yet chilling, description of cellular genocide, Mempel and colleagues (145) imaged recognition and lysis of antigen-engaged B cells by in vivo primed CD8+ cytotoxic T lymphocytes (CTL). Freshly homed and motile B cells are sampled but ignored after a short-duration encounter by CTLs when the B cell suspect lacks speciﬁc antigen. CTLs latch onto genuine B cell targets in prolonged interactions only when these surrogate targets have previously taken up the speciﬁc antigen recognized by the CTL. Then, CTL cells are taken for a ride by the B cell, with the B cells leading the way in motile conjugate pairs analagous to helper T cells interacting with activated B cells (146). Motility arrest provides the ﬁrst sign of the cellular murder that is taking place. After ∼10 min, the monogamous CTL/B pair stop abruptly, and 5–10 min afterward the dually labeled B cells bleed soluble CMTMR from the cytoplasm while nuclear staining remains intact. At this point, the CTL generally detaches from its victim and migrates away in search of another target. The B cell victims are observed blebbing, leaving fragments of the corpses. These cell fragments are later cleaned up by presumptive macrophages that carry ﬂuorescent B cell body parts within granules. In a further fascinating part of this study, when antigen-speciﬁc regulatory T cells (Tregs) are present, CTLs recognize their targets and pair off in monogamous and highly motile conjugate pairs, but the targets are not killed, and the CTL/B cell conjugate pairs continue to migrate together until

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the CTL eventually detach, leaving the target unscathed. In contrast to CD8+ T lymphocytes, NK cells need no initial activation and priming step to recognize and kill their target. Target recognition is not antigen speciﬁc, although in both cases the lethal hit can be delivered by exocytosis of cytolytic granules or by ligandinduced death. NK cells are restrained from killing through engagement of inhibitory receptors that recognize self–MHC class I. Failure to recognize self–MHC I in combination with activating signals unleashes the lethal potential of NK cells, and their cytotoxic granule contents are released following contact. NK cells reject metastatic cells and bone marrow transplants from MHC-mismatched donors by this mechanism. In the lymph node, NK cells actively patrol the territory near B cell follicles, migrating at 6–7 μm/min (104). Garrod et al. (104) set up allogeneic foreign B cells as potential NK cell targets along with cotransferred syngeneic B cells. Both syngeneic and allogeneic cells migrate at 5– 7 μm/min, and NK cells sample both in a series of short contacts. When allogeneic MHC-mismatched B cells are recognized, NK interactions are prolonged and the B cell targets stop moving. Sometimes several NK cells swarmed a common B cell target. Shortly after dissociation of cognate NK cells, allogeneic B cell blebbing and fragmentation occurred.

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Regulatory T Cells Regulatory T cells (Tregs) modulate numerous types of immune responses and protect against autoimmunity by inhibiting activation responses during antigen priming through mechanisms that may require direct contact between Tregs and DCs and/or bystander effects mediated by cytokines (147–151). Three papers have applied immunoimaging techniques to investigate these processes in different settings of speciﬁc T cell priming by introducing Tregs as puriﬁed CD25+ cell populations [Foxp3 GFP cells (151) would 612

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be an attractive alternative but unfortunately have low ﬂuorescence]. In a model of type 1 diabetes, CD4+ T (Th) cells speciﬁc for islet autoantigens are highly motile and migrate randomly in nondraining inguinal lymph node but swarm and arrest in pancreatic lymph nodes of prediabetic NOD (nonobese diabetic) animals (152). Antigenspeciﬁc Tregs behave similarly in both environments. In CD28−/− NOD hosts with fewer Tregs, autoimmune progression is more rapid, and the static clusters of Th cells are predominant. When cotransferred together with CD25+ Tregs, little change is evident, but Tregs transferred ahead of time inhibit swarming and cluster formation of diabetogenic Th cells. Imaging both cells together shows that the presence of Tregs prevents swarming and arrest of Th cells, but without apparent direct interactions. In pancreatic lymph node, where DCs were visible because they had taken up GFP released by dying β cells, dynamic but stable contacts between Th cells and DCs, as well as swarming activity, were observed, reminiscent of T cell/DC dynamics during CD4 T cell priming (117). Tregs also swarm and engage in long-lasting interactions with DCs, but their presence inhibits stable contacts of Th cells with DCs. These results point strongly to indirect actions of Tregs on Th cells, possibly mediated by DCs. Could it be that Tregs somehow reduce the quantity of speciﬁc antigen on DCs, or do they produce factors that inhibit antigen recognition by Th cells? Without visualizing Tregs directly, Tadokoro et al. (153) showed that the presence of Tregs inhibits stable contacts of autoreactive myelin basic protein-speciﬁc T cells with autoantigen presented in the lymph node following an immunization by injected myelin basic protein. The presence of Tregs in both systems favors robust, apparently normal motility of CD4+ T cells, despite the presence of autoantigens, by inhibition of antigen recognition and the stop signal subsequent to interaction with autoantigen-presenting DCs.

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A somewhat different picture of Treg action was obtained by Mempel and colleagues (145) while imaging the ability of CD8+ CTLs to interact with antigen-presenting target cells. Instead of altering the initial recognition and stable attachment to an APC, Tregs do not inhibit CTL recognition and stable interactions with B cell targets. Instead, Tregs profoundly inhibit the ability of CD8+ CTLs to release cytolytic granules and to lyse the targets (145). Despite being fully armed, potentially dangerous, and in contact with a target that would otherwise be destroyed, CTLs do not deliver the lethal hit. Tregs themselves were imaged in this study but did not interact directly with CD8+ effectors. The ability of Tregs to suppress CD8+ cytolytic function depends on TGF-β production. These results (145) indicate that Tregs can prevent the ﬁnal cytolytic step of CTL-target interaction but do not interfere with all the previous steps, including the initial priming and subsequent recognition and long-lasting conjugate interaction of CTLs with their targets. A picture thus emerges from these imaging studies that Tregs can exert effects both on initial recognition with APCs and on subsequent activation events.

Activation of B Lymphocytes B cells exhibit great ﬂexibility to capture and respond to antigen in varying contexts. B cells at the follicle edge acquire, sample, and respond to antigen diffusing in or held by macrophages and FDCs. B cells in the follicle interior sample FDCs for antigen captured and displayed on their surface, activate, and then migrate to the edge of the T cell area. In contrast, newly homed B cells sample, respond to, and capture whole antigen displayed on the surface of DCs in the paracortex. T cell help delivered to B cells by direct cell contact is required for B cell immunoglobulin class switching and hypermutation in germinal centers to become high-afﬁnity antibody-producing plasma cells. Live-cell immunoimaging is now illuminating the

underlying choreography of each of these events. B cell–DC interaction. A recent paper presents a fascinating scenario for newly homed B cells on their way to the follicle to interact with cognate antigen-bearing DCs in the paracortex. When speciﬁc antigen corresponding to the BCR is present, B cell contact with DC results in B cell Ca2+ signaling, cell arrest, and antigen uptake by newly homed B cells. Qi et al. (113) show that B cells emerging from HEVs make contact with DCs immediately after homing. Soluble antigen, delivered through reticular conduits, is taken up by the DC and presented, in a form recognized by the BCR (i.e., without complete processing to small peptide fragments bound to MHC), to B cells on their way to the follicle. Ca2+ signaling is rapidly initiated, and the B cells fail to localize to the follicle but linger in direct contact with DCs while upregulating surface expression of the chemokine receptor CCR7 and CD86, the costimulatory ligand for CD28 on T cells. This hit-and-run robbery of antigen provides an opportunity for a localized m´enage a` trois, consisting of DCs, T cells, and B cells interacting together locally in the cortical ridge. Thus, DCs may provide a platform for localized T cell–B cell interaction that would potentially accelerate the delivery of T cell help to activating B cells. B cells seeking help by chemotaxis. B cells that are already in the follicle respond to the arrival of antigen in a very different manner. In the absence of antigen, B cells migrate inside the follicle at 6–7 μm/min and follow random trajectories (2). In order for B cells to receive T cell help during antigen challenge, they must make direct contact with CD4+ T cells by migrating to the edge of the follicle (follicular exclusion) after detecting the presence of antigen with their BCRs. Studies by Okada et al. (154) reveal that this is achieved by directed migration of antigen-engaged B cells, rather than by trapping of cells that randomly arrive at the follicle edge (146).

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Antigen induces a rapid upregulation of CCR7 expression that may permit the cells to detect and then migrate directionally along a CCL21 gradient (visualized by immunostaining) projecting from the T cell zone ∼150 μm into the follicle. Initially, antigenengaged B cells move at a reduced rate (4 μm/min) but then later migrate more rapidly, after 1 day, at 8–9 μm/min. Recent studies have conﬁrmed the initial decline in B cell velocity upon antigen engagement, ﬁrst seen by Okada et al. (146), by visualizing B cells following subcutaneous injection of antigen-coated small particles (112) or antigen-containing immune complexes (23). Okada et al. (146) then went on to show that upon reaching the CCL21 gradient by random migration, the B cells dither but then make consistent progress along more linear paths toward the T zone (a B-line, so to speak). Further supporting this interpretation, Okada et al. (154) showed that CCR7−/− antigen-speciﬁc B cells fail to relocate, as do CCR7+/+ in plt recipients that lack CCL19 and CCL21, consistent with conclusions from static imaging of ﬁxed tissue (51). The directed migration of B cells may be the ﬁrst example in which chemotaxis (or more properly haptotaxis) of lymphocytes has been clearly visualized in the lymph node.

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T cells delivering help: B cell/T cell waltz at the follicle edge. B cells that encounter activated T cells at the edge of the follicle form stable, motile conjugate pairs and migrate as partners, waltzing at the follicle edge with the characteristic velocity of an activated B cell (146). The B cell always leads, dragging the T cell behind as a rounded partner, sharing the dance ﬂoor with activated solitary T and B cells. Although generally stable (with contacts often lasting >45 min), conjugate pairs also undergo partner exchange, with single B or T cells cutting in on a motile pair. Activated solitary T and B cells that have not yet paired off migrate in the same region with conjugate pairs, the T cells more rapidly at ∼12 μm/min. During partner exchange, con614

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tact between two Bs and one T or between one B and two Ts (threesomes) rarely lasts more than a minute or two before the new T/B pair moves away (146), resulting in serial monogamy. Rare long-lasting threesomes do not migrate effectively. T cell help to B cells promotes differentiation of B cells to antibody-producing plasma cells. But what purpose might be served by the motility of cognate T/B pairs and by partner exchange? As B cells present antigen to T cells, the resultant T cell Ca2+ signaling will inhibit T cell motility, permitting coordinated motion to be led by the B cell. Polarized cytokine secretion by helper T cells in contact with B cells was discovered nearly 20 years ago (155), and more recently Huse and colleagues (137) showed that T cells can secrete certain cytokines locally toward the B cell, while releasing other cytokines and chemokines multidirectionally from the distal side of a T cell engaged with a B cell partner. Thus, polarized cytokine production in a motile conjugate pair may allow the T cell to secrete IL-2 and IFN-γ locally into the immunological synapse for a private conversation with the B cell, while at the same time broadcasting IL-4, tumor necrosis factor, and the chemokine CCL3 into the surrounding tissue. The motility of the conjugate pair would allow for more extensive distribution of inﬂammatory cytokines and chemokines than if the cells were immotile. Moreover, their motility may permit the conjugate pair to seek and colonize new territory, with B cells possibly carrying T cells into the follicle, and T cell/B cell partner exchange may optimize T/B cooperation during a mixed antigen response, thereby reducing the likelihood of ineffective T cell help being delivered to the B cell. Germinal center dynamics. Germinal centers are sites of organization for B cells and FDCs, and perhaps also T cells, to interact during a developing humoral immune response. In germinal centers, the humoral immune response becomes ﬁne-tuned to produce high-afﬁnity antibodies generated by

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hypermutation of the variable regions of the antibody locus and by selection of cells. It is thought that competition among newly arising clones underlies a survival advantage for those clones that produce the highest afﬁnity BCR (99). Based on histological examination of ﬁxed tissue, germinal centers are subdivided into dark zones close to the T cell region and more distal light zones. B cell blasts undergoing cell division predominate in the dark zone, and the light zone contains a network of FDCs with extensive ﬁne processes that bind immune complexes. Three recent imaging studies have begun to examine cellular dynamics in germinal centers. All three groups imaged highly motile B cells within the light and dark zones (21, 156, 157). B cells exhibit an elongated, dendritic morphology and migrate similarly at 6 μm/min within both regions. Two populations of B cells, including motile cells that moved along straighter paths than naive B cells and relatively stationary B cells, were distinguished in one study (21). Clusters of relatively stationary B cells make contact with FDCs, sometimes through long membrane tethers. All three groups demonstrated bidirectional migration between light and dark zones, but detailed cell tracking in favorably situated germinal centers showed surprisingly little crossing of the border between light and dark zones in two of the studies (21, 156). Furthermore, episodes of B cell division and death were seen in both light and dark zones (21). Apoptotic B cells release blebs and larger cell fragments including nuclei; both are taken up by macrophages. B cell blebs are also acquired by motile T cells that are present, mainly in the light zone, and that are moving with higher velocity than B cells. T cells are seen to engage B cells only in short-duration contacts, suggesting limited delivery of help at this stage. One group saw frequent visits to the germinal center by follicular B cells (157); another saw B cells leaving toward the T cell area, but not toward the follicle mantle (21). These recent imaging studies are causing reevaluation of a classical model for afﬁnity

maturation (158) to account for B cell proliferation and death in both light and dark zones and to consider the implications of a low incidence of interzonal migration (21, 99, 156). In a strict interpretation of the cyclic reentry model, somatic hypermutation during cell division in the dark zone and selection events in the light zone would require migration from the light zone to the dark zone and back to the light zone, necessitating double crossing of the zonal boundary (once in each direction) during each cell cycle. The relative infrequency of such border crossings and the corresponding primarily intrazonal pattern of migration may favor a model of intrazonal germinal center development in which B cells need not migrate to and fro in order to be selected (21). A revised cyclic reentry model would allow recombination and division in both subcompartments and considers competition for germinal center T cells to promote selection of favorable rearrangements (99). Direct tracking of antigen, along with longerterm and wider-ﬁeld imaging, may help to distinguish these models or to reveal aspects of both in differing circumstances.

IMMUNOIMAGING APPLIED TO OTHER ORGANS: A BRIEF OVERVIEW In addition to enabling the dynamics of lymphocyte motility and cellular interactions to be imaged within lymph nodes, two-photon immunoimaging is being applied in several other sites. Page constraints prohibit a detailed account of these results, but two-photon imaging has been effectively performed in spleen (159–162), thymus (1, 24, 129), bone marrow (163–165), skin (166; D. Sen, M. Matheu, M.D. Cahalan, and I. Parker, unpublished observations), gut (167, 168), the central nervous system (169, 170), and tumors (171–174). Highlights particularly relevant to themes developed in this review include the mixture of randomly oriented and highly directional paths of developing thymocytes during positive selection in the thymus (24);

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gesticulation of dynamic DC dendrites into the gut lumen (168); extrusion of protoplatelets from megakaryocytes into blood vessels (165); random migration and arrest of encephalitogenic T cells invading the spinal cord and recognizing myelin autoantigens (170); and the random migration and stable target interactions of tumor-inﬁltrating lymphocytes (173) and cytotoxic T lymphocytes (171) in the tumor microenvironment. Random walks, chemokine receptors, antigen acquisition, cell recognition events, and stop signals no doubt apply in many of these settings.

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SUMMARY AND FUTURE DIRECTIONS The advent of two-photon microscopy allows us to visualize, in real-time and deep within intact organs and tissues, the ongoing cellular drama of the immune system under basal conditions and during responses to antigen. Although previous static imaging approaches had provided great insights, they may be likened to trying to understand the rules of baseball using only still photographs. Dynamic, multidimensional imaging is now helping us comprehend the cellular rules of the game inside the lymph node and other organs. The ﬁeld has evolved beyond initial phenomenological descriptions, and quantitative analyses together with computer modeling and simulation are beginning to make sense of the apparently chaotic activity within lymph nodes as lymphocytes continually migrate amid the waving dendrites of DCs. At a macroscopic scale, random-walk motility prevails, constituting a swarm intelligence of autonomous cellular elements that enables efﬁcient and highly sensitive scanning for rare antigens. However, at a microscopic level, the individual cell motions are clearly directed both by chemokine gradients and by structural elements, as, for example, with the antigen-induced march of the B cells toward the T cell area, the migration of CD8+ cells toward DC/T cell clusters, and 616

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the regulation of lymphocyte egress from the node. At least nine types of cellular close encounters have now been visualized by live-cell immunoimaging in the lymph node, providing fresh insight on modes of cell communication during basal migration, antigen capture, antigen recognition, cell activation, differentiation, and death. (a) Under basal conditions, T cells migrate along FRCs that provide haptokinetic signaling to enhance their velocity. (b) In their ongoing search for antigen, T cells scan dynamic DC dendrites in short-lived, randomly initiated encounters. (c) After emerging from the HEV into the paracortex, newly homed B cells scan the DC surface for whole antigen. (d ) Inside the follicle, B cells migrate in direct contact with FDCs, exchanging antigen back and forth. (e) B cells scan subcapsular macrophages and other B cells, capture immune complexes and particulate antigen, and convey the cargo to other sites. ( f ) Upon encountering speciﬁc antigen, both T and B cells undergo Ca2+ signaling, arrest, and upregulation of gene expression, which lead to directed migration, cytokine secretion, and cell proliferation. (g) After migrating to the follicle edge, B cells pair with T cells and waltz while receiving their help. (h) Foreign or tumor cells are killed by activated CD8+ cytolytic T cells. (i ) Foreign cells are killed by NK cells. The application of live-cell in vivo imaging to the immune system has just begun, and we can anticipate that technological advances in microscopy and design of improved morphological and functional probes will provide new opportunities. Purely descriptive studies are being supplanted by a new functional immunoimaging approach that takes advantage of targeted gene deletion or overexpression and the application of selective pharmacological agents or antibodies to pinpoint molecular targets. Particularly important developments will be to apply immunoimaging to visualize host/pathogen dynamics and to elucidate and reﬁne the actions of therapeutic agents at the level of cellular dynamics. Ultimately,

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these methods may be applied to diagnose and treat human disease as well as continue

to illuminate cellular and molecular mechanisms in the immune response.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

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ACKNOWLEDGMENTS The authors thank members of our joint laboratory group—Melanie Matheu, Debasish Sen, Ying Yu, and Kym Garrod—for their comments on the manuscript, and M. Matheu and Y. Yu for assistance in preparing ﬁgures. In addition, we wish to thank Mark Miller (Washington University) and David Fruman (University of California, Irvine) for their careful reading and helpful suggestions. This work is supported by grants from the National Institutes of Health (GM41514 to M.D.C. and GM-48071 to I.P.).

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Annual Review of Immunology

Annu. Rev. Immunol. 2008.26:585-626. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:585-626. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno Trudeau Institute, Saranac Lake, New York 12983; email: [email protected]

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Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

lymphotoxin, homeostatic chemokine, lymphoid tissue inducer

This article’s doi: 10.1146/annurev.immunol.26.021607.090257

Abstract

c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0627$20.00

Secondary lymphoid organs develop during embryogenesis or in the ﬁrst few weeks after birth according to a highly coordinated series of interactions between newly emerging hematopoietic cells and immature mesenchymal or stromal cells. These interactions are orchestrated by homeostatic chemokines, cytokines, and growth factors that attract hematopoietic cells to sites of future lymphoid organ development and promote their survival and differentiation. In turn, lymphotoxin-expressing hematopoietic cells trigger the differentiation of stromal and endothelial cells that make up the scaffolding of secondary lymphoid organs. Lymphotoxin signaling also maintains the expression of adhesion molecules and chemokines that govern the ultimate structure and function of secondary lymphoid organs. Here we describe the current paradigm of secondary lymphoid organ development and discuss the subtle differences in the timing, molecular interactions, and cell types involved in the development of each secondary lymphoid organ.

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INTRODUCTION ILF: isolated lymphoid follicle NALT: nasal associated lymphoid tissue LTi: lymphoid tissue inducer

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LTin: lymphoid tissue initiator

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Primary immune responses are initiated in secondary lymphoid organs, including spleen, regional lymph nodes, Peyer’s patches, isolated lymphoid follicles (ILFs), tonsils, and nasal associated lymphoid tissue (NALT). These tissues are situated throughout the body at strategic sites where antigens from pathogens are most likely to be encountered. Regional lymph nodes are found along lymphatic vessels that collect antigen and antigenpresenting cells from nonlymphoid organs, whereas mucosal lymphoid organs, such as Peyer’s patches, ILFs, tonsils, and NALT, lack afferent lymphatics and acquire antigen directly across the mucosal epithelium. The spleen is also a secondary lymphoid organ and has evolved a unique structure to sample blood-borne antigens. All these secondary lymphoid organs have specialized architecture and microenvironments that promote the controlled interactions of immune cells in order to elicit a rapid and appropriate immune response to infectious agents (reviewed in 1–3). Most infectious agents gain entry to the body via the skin or mucosal epithelium, which comprise an extremely large surface area. Moreover, many infectious agents target nonlymphoid tissues and organs to complete their life cycle. Because the frequency of naive T or B lymphocytes speciﬁc for particular epitopes is in the order of 1 in 105 –106 , it would be very difﬁcult for these few cells to monitor every inch of each peripheral tissue and organ for foreign antigens and pathogens. This problem is elegantly solved by secondary lymphoid organs, which recruit naive lymphocytes from the blood and attract activated, antigen-bearing antigen-presenting cells from regional tissues. In essence, lymphocytes recirculate from lymph node to lymph node and allow antigen to be delivered to them. As a result, the process of immune surveillance is much more efﬁcient, and adaptive immune responses to infectious agents are initiated more quickly (reviewed in 1, 4, 5).

Randall

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Secondary lymphoid organs develop during embryogenesis or in the early postnatal period. This process occurs independently of antigen or pathogen recognition at predetermined sites throughout the body as a result of complex interactions between various hematopoietic, mesenchymal, and endothelial cells. Many, but by no means all, of the cellular and molecular interactions that are involved in secondary lymphoid organ development are understood, and some of these mechanisms are also involved in the maintenance of secondary lymphoid organ architecture in adults as well as the function of these organs during immune responses (previously reviewed in 6–11). Moreover, similar mechanisms govern the formation of tertiary lymphoid tissues, which develop in adults at sites of persistent infection or chronic inﬂammation. Because the formation and function of tertiary lymphoid organs has been recently reviewed (12, 13), this review focuses exclusively on the preprogrammed organogenesis of secondary lymphoid organs.

EARLY CELLULAR INTERACTIONS IN PEYER’S PATCH DEVELOPMENT Many of the cellular interactions that initiate secondary lymphoid organ development are well characterized, particularly with regard to Peyer’s patch development (11). Hematopoietic cells derived from precursors in the fetal liver ﬁrst begin to colonize the gut between days E12.5 and E15.5 (14, 15). Although these cells are evenly distributed throughout the gut on day E15.5, they rapidly form clusters at sites of Peyer’s patch development (16). This is Step 1, as shown in Figure 1. These hematopoietic cells include a population of CD4+ CD3− IL-7Rα+ c-kit+ lymphoid tissue inducer (LTi) cells as well as a population of CD4− CD3− IL-7Rα− ckit+ CD11b+ CD11c+ lymphoid tissue initiator (LTin) cells (16, 17). Both the LTin and LTi cells cluster together with nonhematopoietic VCAM-1+ ICAM-1+ lymphoid tissue

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ARTN

LTin

Step 1

LTo

Rapid migration

LTαβ

TRANCE /IL-7 /chemokines

LTi

Step 2

LTo

Clustering of LTin, LTi, and LTo cells

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LTαβ /others?

TRANCE /IL-7 /chemokines

Step 3 LTi

Lymphocyte-independent initiation of follicle formation

LTo LTαβ /others?

IL-7 /chemokines

NK/T

Step 4

Stroma

Entry of lymphocytes and B/T segregation

LTαβ /others?

Chemokines

B

Step 5

FDC

Mature B cell follicle formation

TNF/LTαβ

Figure 1 General scheme of secondary lymphoid organ development. As noted in text, the details in the developmental pathways of Peyer’s patches and individual lymph nodes are often slightly different. Therefore, this scheme incorporates elements of both Peyer’s patch and lymph node development and outlines major steps that are likely to be in common in these pathways. Importantly, this scheme implies several events that are purely speculative. For example, it is unknown whether LTo cells are the direct precursors of mature stromal cells and follicular dendritic cells (FDCs). It is also unclear whether LTi cells and LTin cells are maintained in mature secondary lymphoid organs in adults. [Abbreviations: LTαβ, lymphotoxin-αβ; NK/T, natural killer and/or T cell; ARTN, artemin; TRANCE, TNF-related activation-induced cytokine; TNF, tumor necrosis factor.]

organizer (LTo) cells on the antimesenteric side of the small intestine and form the primitive anlagen of the Peyer’s patch (15, 16). The available evidence suggests that both LTin and LTi cells are important for the initial

stages of Peyer’s patch development. For example, mice depleted of CD11c+ cells during embryogenesis develop fewer Peyer’s patches, and mice lacking the tyrosine kinase receptor RET, which is expressed by LTin cells,

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completely lack Peyer’s patch anlagen (16). Interestingly, Ret−/− mice have normal numbers of LTin and LTi cells, although these cells do not aggregate nor do they trigger the differentiation of VCAM-1+ ICAM-1+ LTo cells (16). One of the ligands for RET, ARTN (artemin), is produced by nonhematopoietic VCAM-1+ cells—probably LTo cells—and promotes the clustering of LTin and LTi cells at sites of Peyer’s patch development. In fact, the ectopic application of ARTN to embryonic gut tissue leads to the clustering of both LTin and LTi cells, the local appearance of VCAM-1+ cells, and the formation of an ectopic Peyer’s patch anlagen (16). LTi cells are also important for secondary lymphoid organ development, as mice deﬁcient for the transcription factor Id2 (18) and mice deﬁcient for the nuclear hormone receptor RORγ (19) fail to develop IL7Rα+ CD4+ CD3− LTi cells and consequently lack lymph nodes and Peyer’s patches (18, 19). Furthermore, the adoptive transfer of puriﬁed LTi cells from normal mice into neonatal CXCR5−/− recipients promotes the development of Peyer’s patches (20), and the adoptive transfer of IL-7Rα+ CD4+ CD3− cells into neonatal Id2−/− mice induces the development of NALT (21). Mice lacking the transcription factor Ikaros also fail to develop lymph nodes (22, 23), a fact that is attributed to the loss of LTi cells. However, given the defects in the dendritic cell (DC) lineage in Ikaros mutant mice (24), it is possible that the development of LTin cells is also compromised in these animals and may contribute to the loss of lymphoid organs. Additional evidence for the involvement of LTi cells in lymphoid organ development comes from transgenic mice that overexpress IL-7 (25). These mice have abnormally high numbers of LTi cells and develop a much higher number of lymph nodes and Peyer’s patches than normal mice (25). However, IL-7 by itself is not sufﬁcient to drive the development of these lymphoid organs because IL-7 transgenic mice crossed to Rorc−/− mice

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fail to produce LTi cells and do not develop any lymph nodes or Peyer’s patches (25). Thus, IL-7Rα+ CD4+ CD3− cells appear to be essential for lymphoid organ development. Despite the essential roles that LTin and LTi cells play in secondary lymphoid organ development, the lineage relationship between these cell types remains obscure. LTin cells seem to have some characteristics of DCs, including CD11c, CD11b, and MHC class II expression. However, they lack DEC205 and instead express NK1.1 and Gr1 (16). Importantly, they lack IL-7Rα, which is important for the expansion of LTi precursors and for the expression of lymphotoxinαβ (LTαβ) on LTi cells, particularly at mucosal sites. On the other hand, LTi cells are not easily categorized into a particular lineage. They arise from common lymphoid progenitors in the fetal liver (26) but lack any features of B or T lymphocytes. Under the appropriate conditions, they mature into NKlike cells and CD11c+ DCs (27), suggesting that they may give rise to CD11c+ LTin cells. However, it is currently unclear whether the development of LTin cells also requires RORγ or Id2; such a ﬁnding would provide better evidence that the two populations are related.

EARLY EVENTS IN LYMPH NODE DEVELOPMENT The early stages of lymph node development are slightly different that those of Peyer’s patch development. Unlike Peyer’s patches, lymph nodes are encapsulated by lymphatic endothelium, and their development occurs concurrently with the process of lymphatic vascularization. As ﬁrst shown by ink injections and microdissection, lymph sacs form from endothelial cells, which bud from the veins during early development (28, 29). These primitive lymph sacs then form buds that branch and form the lymphatic network. This process is controlled by the transcription

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factor Prox1, which is expressed exclusively by lymphatic endothelial cells (30) and is essential for the formation of lymphatic vessels (31, 32). In fact, the early lymph node anlagen is bordered by a mix of endothelial cells that have characteristics of both lymphatic and blood vascular endothelial cells (33). Analogous to what happens in developing Peyer’s patches, CD4+ CD3− IL7Rα+ LTi cells aggregate near endothelial cells at sites of future lymph node development and trigger the differentiation of VCAM-1+ ICAM-1+ LTo cells to form the primitive lymph node anlagen (34). Although CD11c+ cells are also observed in developing lymph node anlagen (34), it is currently unclear whether these cells are the equivalent of CD11c+ LTin cells or whether RET/ARTN interactions are also important for the development of lymph nodes.

DIFFERENTIAL ROLE OF TRANCE AND IL-7 IN PEYER’S PATCH AND LYMPH NODE FORMATION The differences in Peyer’s patch and lymph node organogenesis are also illustrated by the differential roles of the TNF family member TRANCE and the cytokine IL-7 in these processes (34, 35). TRANCE (TNF-related activation-induced cytokine, also known as OPGL/ODF/RANKL/TNFSF11), its receptor, RANK (receptor activator of NF-κB, also known as TRANCE-R/ODFR/OFE), and a critical component of the TRANCE signaling pathway, TRAF6 (TNF receptorassociated factor 6), are all required for the formation of lymph nodes, but not of Peyer’s patches or NALT (35–38). Although Trance−/− mice do develop some cervical lymph nodes, those that form are small and poorly populated (35, 36). Moreover, Trance−/− mice have relatively few LTi cells, and those that are present fail to properly induce the differentiation of mesenchymal cells (35). TRANCE is expressed by mesenchy-

mal LTo cells in sites of lymph node development and functions to promote the differentiation and survival of LTi cells and to upregulate LTαβ on their surface (33, 34). In turn, the ligation of the LTβR on local mesenchymal cells triggers their differentiation and promotes the expression of CCL19, CCL21, and CXCL13 as well as VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) (34, 39). This is Step 2 as shown in Figure1. These chemokines and adhesion molecules promote LTi clustering and continue to recruit newly emerging lymphocytes to the developing lymph nodes. Therefore, in the absence of TRANCE, VCAM-1+ ICAM-1+ cells fail to appear in the lymph node anlagen and lymph nodes fail to complete their program of development. Conversely, IL-7Rα, IL-2Rγ, and JAK3 are required for the organogenesis of Peyer’s patches, but not lymph nodes (14, 15). This was initially surprising because the LTi cells in developing lymph nodes express IL-7Rα just as the LTi cells in developing Peyer’s patches (34). In fact, LTi cells in developing lymph nodes are capable of responding to IL-7, as the ectopic application of IL-7 to developing Traf6−/− embryos (which cannot signal through TRANCE) promotes the development of lymph nodes (34). Like TRANCE, IL-7 upregulates LTαβ on the surface of LTi cells (40), which leads to the expression of chemokines and adhesion molecules by LTo cells. Other cytokines that signal through the IL-2Rγ chain, such as IL-4, are also capable of triggering LTαβ expression on LTi cells. However, there is no evidence that these cytokines are involved in secondary lymphoid organ development, probably because these cytokines are not expressed in the developing embryo. Thus, TRANCE and IL-7 perform similar functions, and the reason that TRANCE is required for lymph node development and IL-7R is required for Peyer’s patch development is due to differential expression of these cytokines in lymph nodes and Peyer’s patches.

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Lymph node anlagen: the unstructured collection of hematopoietic and mesenchymal cells that initially forms at sites of secondary lymphoid organ development

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Although these data would suggest that the role for IL-7 in lymph node genesis is minimal, other data demonstrate that mice doubly deﬁcient in CXCL13 and IL-7Rα completely fail to form lymph nodes (40), including mesenteric lymph nodes, which are normally present in Cxcl13−/− mice (40, 41). Moreover, the transgenic overexpression of IL-7 promotes the development of extra lymph nodes as a result of much higher numbers of LTi cells (25). Thus, IL-7 probably also plays a role in the expansion or development of LTi cells from common lymphoid progenitors and is also a critical inducer of LTαβ expression in some locations. The counterparts to LTi cells in the development of secondary lymphoid organs are LTo cells, which are mesenchymal in origin and form the stromal cell matrix of the developing lymphoid organ (14, 33, 39, 42). Just as there seem to be multiple types of hematopoietic cells that trigger lymphoid organ development, there are also multiple types of mesenchymal LTo cells, with different expression patterns of adhesion molecules, cytokines, and receptors (43). In fact, the VCAM-1+ ICAM-1+ LTo population can be divided into VCAM-1int ICAM1int and VCAM-1hi ICAM-1hi populations (33). Whereas both of these populations are present in peripheral and mesenteric lymph nodes, the frequency of VCAM-1int ICAM1int cells is dramatically lower in peripheral lymph nodes (33). Differences between these populations are also found in developing Peyer’s patches and mesenteric lymph nodes. In fact, the LTo cells found at sites of mesenteric lymph node and Peyer’s patch development express very different gene programs (43). For example, LTo cells at sites of mesenteric lymph node development express much higher levels of TGF-β and stem cell factor (the ligand for c-kit), whereas LTo cells at sites of Peyer’s patch development express higher levels of IL-6, TRANCE, CXCL1, CCL7, and CCL11 (43). These differences in gene expression may explain why

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various lymphoid organs have different requirements for cytokines and growth factors, such as the requirement for IL-7 in Peyer’s patch development and the requirement for TRANCE in lymph node development. However, it remains an open question whether all secondary lymphoid organs have slightly different populations of LTo cells and whether the differences in these populations are intrinsic or are caused by the local environment.

LYMPHOTOXIN SIGNALING AND SECONDARY LYMPHOID ORGAN DEVELOPMENT One of the key events in the development of secondary lymphoid organs is lymphotoxin signaling, which promotes the differentiation of mesenchymal LTo cells and leads to the expression of various chemokines and adhesion molecules that are necessary for secondary lymphoid organ development. The essential role of lymphotoxin is illustrated by Lta−/− mice, which lack Peyer’s patches and most lymph nodes, except for rudimentary mesenteric lymph nodes that occasionally appear in some mice (44, 45). This was initially somewhat surprising because LTα is a soluble cytokine that is structurally and functionally related to TNF (46). In fact, LTα binds to both TNFR1 and TNFR2, suggesting that the biological functions of LTα and TNF should be very similar (47). However, Tnf −/− mice have normal numbers of lymph nodes, albeit with some structural alterations (48, 49). Moreover, Tnfr1−/− and Tnfr2−/− mice also have normal numbers of lymph nodes (48, 50). However, there are some strains of Tnf−/− and Tnfr1−/− mice that completely lack Peyer’s patches (50a, 50b), suggesting that the type of genetic disruption at either the Tnf or the Tnfr1 locus may affect the sensitivity of Peyer’s patch development to the loss of TNF. The discrepancy between the activities of LTα and TNF was resolved by the observation that LTα also forms heterotrimers with

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LTβ, which is expressed as a transmembrane protein (51, 52). The LTαβ heterotrimer is expressed on the cell surface of lymphocytes (53) as well as on LTi and LTin cells (16) and binds to the LTβR. Because the organogenesis of secondary lymphoid organs is disrupted in Ltb−/− mice as well as in Ltbr−/− mice (54, 55), the surface form of LTαβ must be primarily responsible for triggering secondary lymphoid organ development. However, Ltb−/− mice still retain cervical and mesenteric lymph nodes (54), whereas Lta−/− mice lack these organs (44). Therefore, the soluble form of LTα must cooperate with surface LTαβ to contribute to the development of some lymph nodes. Furthermore, because all lymph nodes and Peyer’s patches are absent from Ltbr−/− mice (55), it is likely that another TNF family member, such as LIGHT (56, 57), acts through LTβR to facilitate the development of cervical and mesenteric lymph nodes. In fact, TNF, LTα, LTβ, and LIGHT all participate in secondary lymphoid organ development to some degree. For instance, mice treated in utero with soluble LTβR fused with the Fc portion of immunoglobulin (LTβR-Ig) retain mesenteric and cervical lymph nodes (58, 59), whereas mice treated in utero with both LTβR-Ig and TNFR1Ig do not develop these organs (60). Moreover, mice treated with LTβR-Ig and blocking antibodies against TNF also lack all lymph nodes and Peyer’s patches (60), indicating that TNF signaling through TNFR1 plays some role in mesenteric lymph node development. As discussed above, LIGHT also contributes to mesenteric lymph node development. Although both Light−/− and Ltb−/− mice have mesenteric lymph nodes (57), a portion of Light-Ltb−/− mice lack mesenteric lymph nodes (57), suggesting that LIGHT and LTβ cooperate to induce mesenteric lymph node formation. Furthermore, because all Ltbr−/− mice lack mesenteric lymph nodes (55), these results also suggest that there may be another ligand for the LTβR that facil-

itates mesenteric lymph node development. Thus, although LTαβ signaling through the LTβR is the major pathway through which secondary lymphoid organ development is triggered, other TNF family members also contribute to lymph node development. Despite the essential nature of interactions between LTαβ-expressing LTi cells and LTβR-expressing LTo cells, this interaction alone is not sufﬁcient to promote secondary lymphoid organ development. For example, although the administration of an agonistic anti-LTβR antibody in utero promotes lymph node development in Lta−/− mice, the administration of this same antibody in Rorc−/− mice, which lack LTi cells, does not promote lymph node development (19). Furthermore, the administration of anti-LTβR to Cxcl13−/− mice also fails to promote lymph node development. These data demonstrate that LTi cells or possibly LTin cells provide additional signals to mesenchymal or stromal cells that are necessary for secondary lymphoid organ development. The identiﬁcation of these additional signals is key to our future understanding of secondary lymphoid organ development. Most TNFR family members are coupled to the NF-κB signaling pathway (61), and, not surprisingly, molecules important in this signaling pathway are involved in LTβR signaling and lymphoid organ development. In particular, triggering of the LTβR induces the canonical pathway of NF-κB activation that involves IKKβ and IKKγ/NEMO and leads to the phosphorylation of IκB and the translocation of p50/RelA or p52/RelA complexes to the nucleus (62). In turn, these complexes control the expression of inﬂammatory proteins, including VCAM-1, CCL4, and CXCL1 (62). This pathway also promotes an increased production of the NF-κB2/p100 precursor (62). However, LTβR signaling also induces a second pathway that leads to the sequential activation of NIK and IKKα and leads to the processing of p100 to p52 (62, 63). In association with RelB, p52 translocates to the nucleus

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Homeostatic chemokines: chemokines that are constitutively expressed in secondary lymphoid organs and that control the steady-state recruitment and positioning of lymphocytes and DCs; their expression is often regulated by lymphotoxin Table 1

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and activates transcription of molecules involved in secondary lymphoid organ development and homeostasis (62). These molecules include the homeostatic chemokines CCL19, CCL21, and CXCL13. Both pathways appear to be important in lymphoid organ development because mice homozygous for a natural mutation in NIK (alymphoplasia or aly/aly mice) (64, 65) as well as Tnfr1-Rela−/− mice lack lymph nodes and Peyer’s patches (66). Furthermore, RelB is essential for Peyer’s patch organogenesis and aspects of splenic architecture (67–69). These results partly explain why LTβR and NIK signaling pathways are essential for lymphoid organ development and also explain why signaling through

TNFR1 can facilitate lymph node development under some circumstances. (A list of mutations that impair secondary lymphoid organ development is provided in Table 1.)

LYMPHOTOXIN SIGNALING ELICITS CHEMOKINE AND ADHESION MOLECULE EXPRESSION BY STROMAL AND ENDOTHELIAL CELLS As mentioned above, interactions between LTαβ-expressing LTi and LTin cells promote the differentiation of LTβR-expressing mesenchymal and endothelial cells (7, 11, 34). As part of this differentiation pathway, the

Mutations that impair secondary lymphoid organ development mLNsa

pLNsb

PPsc

NALT

±d

−

−

±e

21, 44, 45, 110

stroma

±

−

−

±

21, 54, 110

stroma

−

−

−

±

21, 55, 110

LTβR

stroma

+

+

+

NDf

57

Light/Ltb−/−

LTβR

stroma

−

−

−

ND

57

Aly/aly

LTβR

stroma

−

−

−

±

21, 65, 110

Il7ra−/−

IL-7R

LTi

+

+

−

±

15, 21, 34, 110

Il2rg/rag−/−

IL-7R

LTi

+

+

−

−

86

Jak3−/−

IL-7R

LTi

+

+

−

ND

15, 34

Trance−/−

TRANCE-R

LTi

−

−

+

+

35, 37, 110

Rank−/−

TRANCE-R

LTi

−

−

+

ND

36

Traf6−/−

TRANCE-R

LTi

−

−

+

ND

38

Rorc−/−

LTi

−

−

−

+

10, 19, 110

Id2−/−

LTi

−

−

−

−

18, 21

Ikaros−/−

LTi

−

−

−

ND

23

Mutation

Signaling pathway

Cells affected

Lta−/−

LTβR

stroma

Ltb−/−

LTβR

Ltbr−/−

LTβR

Light−/−

References

Cxcl13−/−

CXCR5

LTi/B

−

−

−

±

40, 41

Cxcr5−/−

CXCR5

LTi/B

−

−

−

ND

74

Plt/plt

CCR7

LTi/B/T

+

+

+

+

40, 65

Ccr7−/−

CCR7

LTi/B/T

+

+

+

ND

129

Cxcl13/Ccl19/21−/−

CXCR5/CCR7

LTi/B/T

−

−

−

±

40

Ret−/−

RET

LTin

ND

ND

−

ND

16

Graf 51/51

RET

LTin

ND

ND

±

ND

16

a

mesenteric lymph nodes. peripheral lymph nodes. c Peyer’s patches. d small mLNs developed in a few mice. e small, disorganized NALT developed. f ND, Not determined. b

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mesenchymal cells express adhesion molecules, such as VCAM-1, ICAM-1, and MAdCAM (mucosal addressin cell adhesion molecule-1), as well as homeostatic chemokines, including CXCL13, CCL19, and CCL21. LTi cells and probably LTin cells express chemokine receptors, such as CXCR5, the receptor for CXCL13, and CCR7, the receptor for CCL19 and CCL21 (40). LTi cells also express the integrins α4β7 and α4β1 (27, 70), which bind to the vascular adhesion molecules MAdCAM and VCAM-1, respectively. Homeostatic chemokines, particularly CXCL13, are expressed in Peyer’s patch anlagen during development (39). This low-level expression attracts CXCR5-bearing IL-7Rα+ CD4+ CD3− cells to the Peyer’s patch anlagen and activates α4β1 integrin in order to facilitate adhesion to the local stroma (20). Once IL-7Rα+ CD4+ CD3− cells have arrived at the future sites of Peyer’s patch genesis, they provide surface LTαβ (27), which induces the expression of higher levels of chemokines as well as adhesion molecules on the mesenchymal cells (39, 71, 72). These newly expressed molecules then recruit lymphocytes, which provide a sustained source of surface lymphotoxin and induce the differentiation of local stromal cells (73). The chemokine receptors and integrins expressed by LTin cells are currently unknown. However, because LTin cells cluster with LTi cells in the developing Peyer’s patch at the same time, we can safely assume that they express similar chemokine receptors and adhesion molecules. Homeostatic chemokines do more than simply attract LTi cells to sites of secondary lymphoid organ development. Like IL-7 and TRANCE, they promote the expression of LTαβ on the surface of LTi cells and lymphocytes (40, 41). For example, CXCL13 promotes the expression of LTαβ on the surface of LTi cells as well as on mature B cells (41), whereas CCL21 and CCL19 promote the expression of LTαβ on LTi cells as well as on mature CD4 T cells (40). In fact, the expression of homeostatic chemokines and

lymphotoxin is codependent in a positivefeedback loop (41) in which lymphotoxin signaling triggers the expression of homeostatic chemokines by mesenchymal/stromal cells and chemokine signaling maintains expression of LTαβ on the surface of LTi cells and lymphocytes. The essential role of homeostatic chemokines in secondary lymphoid organ development is demonstrated by Cxcl13−/− mice, which fail to develop Peyer’s patches and most lymph nodes (41). Cxcr5−/− mice also lack Peyer’s patches and most lymph nodes (74). Although mice with a natural mutation (paucity of lymph node T cells, plt) that disrupts CCL19 and CCL21 generally form a full complement of secondary lymphoid organs (75–78), the lymph nodes and Peyer’s patches that do develop in plt/plt mice are small and poorly organized (75–77). In part, this is due to the reduced capacity of these organs to recruit mature lymphocytes from the blood and activated antigen-presenting cells from regional tissues (75–77, 79–81). However, CCL19 and CCL21 also play a role in the development of secondary lymphoid organs, as shown by mice triply deﬁcient in CXCL13, CCL19, and CCL21, which exhibit a more frequent absence of facial and cervical lymph nodes than mice lacking CXCL13 alone (40). The loss of these additional lymph nodes is attributed to poor recruitment of LTi cells as well as to the failure of LTi cells to upregulate LTαβ and to promote the differentiation of local mesenchymal cells (40).

PROGRAMMED COLONIZATION AND DEVELOPMENT OF FOLLICULAR STRUCTURES IN DEVELOPING LYMPHOID ORGANS Although IL-7Rα+ CD4+ CD3− LTi cells and VCAM+ ICAM+ mesenchymal cells are initially arranged in homogenous clusters, they begin to segregate into follicular structures around day E18 (82). CD11c+ cells also

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segregate into follicular structures in the developing Peyer’s patches at this time (82), although it is not clear whether these cells are CD11c+ LTin cells or whether they are CD11c+ DCs that have differentiated in situ from LTi cells (27, 70). Regardless, the initial follicular formation requires LTαβ and occurs independently of B and T cells (82). Similar events are thought to take place during lymph node development (34). This is Step 3 in Figure 1. The last steps in the development of fully mature secondary lymphoid organs involve the recruitment of lymphocytes, the segregation of B and T cell areas, and the formation of mature B cell follicles. In mouse lymph nodes, this process is delayed until day 3 after birth (83) owing to the sequential expression of MAdCAM and PNAd on high endothelial venules (HEVs) (83, 84). Prior to day 3, MAdCAM-1 is the only vascular addressin expressed on HEVs (83). As a result, cells entering developing lymph nodes during this time must express α4β7, the receptor for MAdCAM. Cells that use this pathway include CD4+ CD3− LTi cells and γδ T cells (27, 70, 84). However, around day 3, MAdCAM expression is downregulated on the HEVs of lymph nodes and PNAd expression is upregulated, allowing the entry of mature naive B and T cells (83). This developmental switch is controlled in part by the de novo expression of CD34 and GlyCAM1 (83), which are substrates for the sulfation reaction that generates the PNAd epitope. After B and T cells start arriving in the developing lymph node, they begin to take over the role of CD4+ CD3− LTi cells by expressing LTαβ and maintaining the differentiation and survival of LTβR-expressing mesenchymal and stromal cells. When B cells ﬁrst arrive in the developing lymph node, they do not respond to CXCL13 and lack surface LTαβ expression (85). At this time, CXCL13 expression is still dependent on LTαβ expressed by CD4+ CD3− LTi cells (85). The lymph node becomes more organized upon the entry of T cells, when B cells begin to

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segregate in the outer cortex. Interestingly, the initial segregation of T and B cells occurs independently of CXCL13. This is Step 4 in Figure 1. However, from day 4 on, the architectural changes are dependent on CXCL13 and CXCL13-responsive B cells. B cells also express LTαβ at this time (85), suggesting that LTαβ-expressing B cells play a major role in maintaining the architecture of secondary lymphoid organs. Consistent with the idea that LTαβ-expressing lymphocytes take over the role of LTαβ-expressing LTi cells, mice that lack NK, B, and T cells develop the initial lymph node anlagen, which persists for a while after birth before dissipating (86). Interestingly, the adoptive transfer of IL-7Rα+ T cells or NK cells, but not B cells, restores the ﬁnal stages of lymph node development (86), suggesting that these cells have the ability to replace LTi cells as the source of LTαβ at this later stage. Although it is not totally understood why adoptively transferred NK and T cells would be more efﬁcient at promoting lymph node maturation than B cells, the authors of this study speculate that IL-7-driven homeostatic expansion of NK and T cells may play a role (86).

ROLE OF LYMPHOTOXIN IN THE MAINTENANCE OF LYMPHOID ARCHITECTURE Lymphotoxin and TNF expression are also required for the maintenance of lymphoid architecture in adults as well as for the appropriate homing and segregation of B and T cells within secondary lymphoid organs (44, 45). For example, the spleens of Lta−/− mice lack organized B and T cell areas (44, 45), marginal zones (45, 87), and germinal centers (87, 88). Moreover, follicular dendritic cells (FDCs) fail to develop in the absence of lymphotoxin or TNF signaling (73, 89, 90). As in the developmental stages of secondary lymphoid organs, the expression of lymphotoxin and homeostatic chemokines are codependent in a positive-feedback loop in which lymphotoxin promotes the differentiation and

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survival of the stromal cells and DCs that express CXCL13, CCL21, and CCL19 (72), whereas chemokine receptor signaling controls the steady-state recruitment and positioning of naive B and T cells and maintains LTαβ expression on B and T cells (41, 72). Most evidence suggests that the expression of LTαβ and TNF on mature B cells is essential for the maintenance of CXCL13 expression and FDCs in the B cell follicle (73, 91). However, mice in which LTβ is genetically deleted speciﬁcally in the B cell lineage have normal levels of chemokines, FDCs, and B cell follicles in the lymph nodes (92, 93). In this case, LTαβ expression by T cells is sufﬁcient to maintain these structures. Together, these data suggest that LTαβ expression on both B and T cells is important for the proper maintenance of lymphoid architecture in adults. In addition to maintaining B cell follicles and segregating B and T cells areas, lymphotoxin signaling is also important for maintaining DC numbers via homeostatic proliferation (94) and for the activation and maturation of DCs during immune responses (95). The development of gp38-expressing stromal cells in the spleen is also dependent on lymphotoxin-expressing B cells (96). Furthermore, the continued expression of the vascular addressins, PNAd and MAdCAM, on HEVs is dependent on lymphotoxin signaling (60, 97, 98). Interestingly, soluble LTα and membrane-bound LTαβ are responsible for different aspects of lymphoid architecture. For example, the defects in B and T cell separation are less severe in the spleens of Ltb−/− mice (54) than they are in the spleens of Lta−/− mice (45) and Ltbr−/− mice (55), suggesting that LTα signaling through TNFR1 as well as LTαβ signaling through LTβR are important for lymphocyte organization. In contrast, the presence of DCs in the spleen and the expression of MAdCAM and PNAd on HEVs and in marginal zone sinuses are controlled by surface LTαβ and the LTβR but not by TNF, LTα, or TNFR1 (59, 94). Finally, the differentiation of FDCs requires

TNF, LTα, and LTβ (49, 73, 90, 99). Thus, various aspects of lymphoid architecture are controlled by the overlapping, yet distinct, activities of soluble LTα, membrane LTαβ, and TNF.

SECONDARY LYMPHOID ORGANOGENESIS DEVELOPMENT IS STRICTLY CONFINED TO A DEVELOPMENTAL WINDOW The development of conventional secondary lymphoid organs occurs during a temporal window in embryogenesis that varies depending on the particular lymphoid organ (58). For example, mesenteric lymph nodes develop ﬁrst (around day E9–E10), followed by brachial (day E13), axillary (day E15), inguinal (day E16), and popliteal lymph nodes (day E17) (58). Mucosal lymphoid organs, such as Peyer’s patches and NALT, appear to develop last, as disruption of lymphotoxin signaling just before birth blocks Peyer’s patch formation (58), whereas the adoptive transfer of LTi cells can restore the development of NALT in neonatal Id2−/− mice (21) and the development of Peyer’s patches in neonatal Cxcr5−/− mice (20). In fact, there is a strict developmental window within which the organogenesis of lymph nodes and Peyer’s patches must occur (58). Once this window has passed, the development of these organs cannot take place, regardless of whether lymphotoxin signaling is restored. There also seems to be a developmental window that restricts the formation of lymphoid tissues at ectopic sites, as demonstrated by the intradermal transfer of cells from dissociated lymph nodes to recipient mice (100). When given to adults, the transferred cells form disorganized clumps. However, when given to neonates, the transferred cells organize into follicular structures with segregated T cell zones and B cell follicles that contain FDCs (100). In contrast, there does not seem to be a ﬁxed developmental window for the formation of ILFs. Although ILF development is dependent on

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Developmental window: a deﬁned period in embryogenesis during which most secondary lymphoid organ development occurs. If certain developmental steps are not completed, these organs’ ability to develop is permanently disabled

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lymphotoxin signaling (101, 102), these structures can be restored in adult Lta−/− mice by reconstitution with normal hematopoietic cells (102). Although it is unclear why these particular tissues lack a ﬁxed developmental window, we do know that RORγ-expressing CD4+ CD3− LTi-like cells are found in the cryptopatches of adults (10). Thus, the developmental window for each lymphoid organ may be restricted by the availability of LTi cells in that location. Interestingly, there is even a developmental window that restricts the formation of some splenic structures, such as the gp38expressing stromal cells in the T cell areas of the spleen (96). The development of these stromal cells requires LTαβ-expressing B cells during the neonatal period (96). As a result, gp38+ stromal cells cannot be restored in the spleens of B cell–deﬁcient or Lta−/− adult mice by reconstitution with normal bone marrow (96). However, most of the other defects in splenic architecture, including the differentiation of FDCs, expression of homeostatic chemokines, formation of the marginal zone, and the development of germinal centers after immunization, can be reversed upon reconstitution of Lta−/− mice with normal bone marrow (73, 87, 90, 99).

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A ROLE FOR CD4+ CD3− LTI CELLS AFTER LYMPHOID ORGANOGENESIS One of the most intriguing questions in lymphoid organogenesis is whether the sole purpose of CD4+ CD3− LTi cells is to promote secondary lymphoid organ development during a narrow temporal window in fetal and neonatal development or whether these cells are also present and functional in adults (103). Although LTi cells are easily located with relatively high frequency within the early anlagen of developing Peyer’s patches and lymph nodes (14, 34), they are difﬁcult to locate in adults. In fact, the presence of LTi cells in adults remains a controversial point (104). However, RORγ+ LTi-like cells can be ob638

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served within the cryptopatches of the adult gut (105), where they are postulated to promote the development of ILFs in response to microbial stimulation (106). CD4+ CD3− LTi cells are also proposed to regulate Th2 responses in the spleen (107). CD4+ CD3− cells in adult spleens appear to have a program of gene expression similar to that in CD4+ CD3− cells found at sites of embryonic lymph node development. For example, CD4+ CD3− cells in adults express high levels of LTαβ and TNF (104). Moreover, the adoptive transfer of normal CD4+ CD3− cells from either embryonic or adult tissues into Lta−/− mice leads to the segregation of B and T cell areas and the expression of CCL21 (108). However, unlike embryonic LTi cells, adult CD4+ CD3− cells express high levels of OX40 ligand and CD30 ligand (109). Therefore, the precise lineage relationship between embryonic LTi cells and adult CD4+ CD3− cells remains unclear. To make matters even more complicated, there is evidence that LTi cells in embryo are not a single homogenous population. For example, the development of NALT occurs in the absence of RORγ (110), whereas it is completely dependent on Id2 (21). Because both RORγ and Id2 are thought to be essential for the differentiation of LTi cells (18, 19, 111) and because normal LTi cells restore NALT development in Id2−/− mice (21), these data suggest that there must be some populations of LTi cells that develop independently of RORγ. These different populations of LTi cells may express different patterns of chemokine receptors that preferentially attract them to particular sites of secondary lymphoid organ development. This corresponds with the idea that there are also different populations of mesenchymal LTo cells at sites of Peyer’s patch, mesenteric, and peripheral lymph node development that express different arrays of chemokines (43). Thus, the CD4+ CD3− LTi-like cells found in the spleens and cryptopatches of adults may simply be a few examples of the multiple types of these intriguing cells.

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EXCEPTIONS TO THE PARADIGM OF SECONDARY LYMPHOID ORGAN DEVELOPMENT Although Peyer’s patch organogenesis serves as an instructive model for the organogenesis of all lymphoid organs (11), the molecular requirements for the development of Peyer’s patches and other lymphoid organs are not identical. This is illustrated by the differential requirement of lymph nodes and Peyer’s patches for TRANCE and IL-7 (34, 35). Although these two cytokines seem to share a similar purpose in secondary lymphoid organ development (34), their differential expression at sites of Peyer’s patch and lymph node development leads to their differential importance in the development of these organs. In contrast, the development of some mucosal lymphoid tissues differs much more substantially from that of Peyer’s patches. For example, although NALT and Peyer’s patches share an architectural similarity, the development of these organs is very different. The organogenesis of NALT occurs during the ﬁrst week or two after birth (21, 112), which is signiﬁcantly later than that of Peyer’s patches. As in Peyer’s patch development, CD4+ CD3− LTi cells are among the ﬁrst to appear in the developing NALT anlagen (21, 113). On the one hand, NALT development does not require RORγ-dependent LTi cells (110). On the other hand, it does require Id2-dependent LTi cells (21). In fact, the organogenesis of NALT can be restored in Id2−/− mice by the adoptive transfer of normal LTi cells to neonatal recipients (21). Most importantly, unlike the development of lymph nodes and Peyer’s patches, the development of NALT is not dependent on the lymphotoxin signaling pathway (21, 110). In fact, NALT is present in Lta−/− , Ltb−/− , Tnf-Lta−/− , Ltbr−/− , and Tnfr1−/− (21, 110). Moreover, NALT develops normally in mice treated in utero with both soluble LTβR and soluble TNFR1 (21), even though these mice do not develop any lymph nodes or Peyer’s patches. These data

argue that none of the ligands that bind to either the LTβR or the TNFR1, including the LTα homotrimer, the LTαβ heterotrimer, TNF, or LIGHT, are essential for NALT development. Furthermore, mice with mutations in the NF-κB signaling pathway, including aly/aly, Nfkb1−/− , Nfkb2−/− , and Relb−/− mice, all have NALT to some degree (114). Together, these data argue that the central feature in the paradigm of secondary lymphoid organ development—the interaction between LTαβ-expressing LTi cells and LTβRexpressing mesenchymal LTo cells—is not a part of the NALT developmental program. Although it is not clear why NALT development requires IL-7Rα+ CD4+ CD3− cells in Id2−/− mice but not in Rorc−/− mice, the difference may reﬂect the activities of additional cell types that facilitate NALT development. Because Id2−/− mice lack NK cells as well as multiple populations of DCs (18, 115, 116) and have defects in the CD8 T cell lineage (117), these cells may play important, although currently undeﬁned, roles in NALT development in the absence of RORγ-dependent LTi cells. This would be consistent with other data showing that NK cells participate in the stabilization of lymph nodes during development (86). However, if CD4+ CD3− cells are a central component in the NALT developmental pathway, even in the absence of lymphotoxin signaling, then what signals might these cells provide during NALT organogenesis? It is clear from other studies that additional signals from LTi cells do play a role in lymph node development because agonistic antibodies to the LTβR do not promote lymph node development in Rorc−/− mice (19). Deﬁning these interactions will be key to our future understanding of secondary lymphoid organ development. The discrepancy between Rorc−/− and Id2−/− mice in the relative importance of CD4+ CD3− cells in NALT development may also reﬂect diversity in the LTi population— with some LTi cells being formed independently of RORγ. Consistent with this idea,

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the LTi cells found in NALT express very low levels of CCR7 and CXCR5 relative to their levels on LTi cells from Peyer’s patches (113). Moreover, NALT develops in the absence of CXCL13, CCL19, and CCL21 (113, 118), and normal numbers of LTi cells migrate to the NALT of Cxcl13−/− or plt/plt mice just after birth (113). These data demonstrate that conventional LTi cells are probably not involved in NALT development and further suggest that additional, as yet undeﬁned chemokines attract LTi cells to the NALT anlagen. One of these chemokines might be CCL20, which is normally expressed in the dome epithelium of NALT in a lymphotoxin-dependent manner (118). If CCL20 is required for NALT formation, possibly by recruiting CCR6-expressing LTi cells, then NALT development might be more analogous to the development of ILFs, which are highly dependent on CCR6 (119). Despite the fact that the initial stages of NALT development occur independently of the lymphotoxin signaling pathway, the structure of NALT is severely compromised in Lta−/− , Tnf-Lta−/− , and Ltbr−/− mice (110, 118). In fact, the B and T cells are not segregated into separate areas, FDCs and BP3+ stromal cells do not develop, HEVs cannot be found, and the normal array of homeostatic chemokines are not expressed in the NALT of Lta−/− mice (110, 118). However, the architectural defects in the NALT of adult Lta−/− mice can be repaired by the restoration of normal lymphotoxin-expressing hematopoietic cells (110). Again, this is dramatically different than other lymphoid organs, which cannot be reconstituted in Lta−/− mice, even after the transfer of normal bone marrow into irradiated recipients (120). Thus, the structural defects in the NALT of Lta−/− mice are primarily due to the failure of lymphotoxin to promote the differentiation of stromal and endothelial cells and to the loss of homeostatic chemokine expression. The development of ILFs is also different than that of Peyer’s patches, despite the similarity of their locations and presumably their

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functions in the small intestine. For example, the formation of ILFs is clearly dependent on lymphotoxin signaling, as ILFs are absent in Lta−/− and aly/aly mice (101, 102, 121). However, like NALT, ILFs are completely restored in Lta−/− mice after reconstitution with normal bone marrow (102, 122). Moreover, even though the treatment of mice in utero with LTβR-Ig or anti-IL-7R blocks Peyer’s patch development, it does not prevent the development of ILFs (101, 122). These data suggest that lymphotoxin signaling is important for the maintenance of ILFs after embryonic development rather than for the developmental formation of ILFs. This is probably analogous to the role of lymphotoxin in the formation of NALT, in which lymphotoxin is essential for the expression of chemokines and the differentiation of stromal and endothelial cells (118). An alternative explanation is that lymphotoxin is required for ILF development but that there is no temporal window in development during which ILF formation must occur. As it is in NALT, CCL20 is highly expressed in the dome epithelium of ILFs and seems to attract CCR6+ DCs as well as B and T cells to the dome region (119, 123). The central importance of CCR6/CCL20 interactions in the development of ILFs is demonstrated in Ccr6−/− mice, in which ILFs fail to develop properly (119). Like other homeostatic chemokines, CCL20 expression is dependent on LTα (118), suggesting that the lymphotoxin-induced expression of CCL20 may be the essential step in ILF formation. Although this might imply that LTi cells responsible for ILF formation must express CCR6 (124), it reﬂects instead a requirement for CCR6 on B cells (119). Given that LTαβ expression on B cells and not T cells is also required for the maturation of ILFs (125), one could speculate that there is a positive-feedback loop between CCL20 expression in the dome epithelium of ILFs and LTαβ expression on B cells. Interestingly, B cells in ILFs do not have to be antigen speciﬁc to mediate their effects. In fact, normal ILFs develop in mice in which all B

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cells express a transgenic BCR speciﬁc for lysozyme (125). Despite the central role of LTαβ+ CCR6+ B cells in ILF formation, ILF development is compromised in Rorc−/− mice (106), suggesting that LTi cells are important for ILF formation. However, it is unclear at this time whether LTi cells are attracted to sites of ILF development via CCL20 or other homeostatic chemokines, like CXCL13, which is also highly expressed in mature ILFs. Another unusual feature of ILF development, unlike that of any other secondary lymphoid organ, is that it is linked to microbial exposure (102, 125–127). For example, ILF formation is barely detectable in germ-free mice (102), and those ILFs that do develop have only small clusters of c-kit+ cells and essentially lack T cell zones or B cell follicles. However, other investigators ﬁnd that ILFs (also known as solitary intestinal lymphoid tissue, or SILT) are present in normal numbers in germ-free mice but are very small and immature and lack identiﬁable B cell follicles (126, 127). Importantly, ILFs return to normal upon restoration of intestinal ﬂora (102). These data argue that the location and number of ILFs is determined developmentally and cannot be altered by inﬂammation or infection. However, the ﬁnal maturation and organization of ILFs depends on microbial stimuli and can even be enhanced by infection with pathogenic bacteria (128). Together, these data demonstrate that the de-

velopment of ILFs has some characteristics in common with the development of ectopic lymphoid follicles but have other characteristics of secondary lymphoid organs.

CONCLUDING REMARKS Our understanding of the programmed development of secondary lymphoid organs has progressed remarkably over the past decade. We now appreciate that interactions between newly emerging populations of LTi and LTin with immature mesenchymal LTo cells are instrumental in this process and that these interactions are orchestrated by a variety of homeostatic chemokines, cytokines, and growth factors. In particular, the lymphotoxin signaling pathway is centrally important for the initial differentiation of mesenchymal LTo cells and for the maintenance of architectural elements that comprise the scaffolding of secondary lymphoid organs. However, it is now apparent that the developmental pathways governing each secondary lymphoid organ are often subtly and sometimes dramatically different. These differences are reﬂected in the variety of cytokines and chemokines that are required for the development of each organ and in the various types of LTi cells and LTo cells that are present in each site. Understanding these differences and determining how they impact the ultimate structure and function of each secondary lymphoid organ will be the challenge for the next decade.

SUMMARY POINTS 1. The development of secondary lymphoid organs is initiated during embryogenesis by interactions between hematopoietic cells (CD4+ CD3− IL-7Rα+ α4β7+ LTi cells and CD4− CD3− IL-7Rα− CD11c+ LTin cells) that arise in the fetal liver and mesenchymal cells that are located at sites of future lymphoid organ development. 2. The development of LTi cells from precursors in the fetal liver and their local differentiation into lymphotoxin-αβ-expressing cells requires the activities of cytokines, such as TRANCE or IL-7. IL-7 seems to be most important for the differentiation of LTi cells at mucosal sites, such as the Peyer’s patches, whereas TRANCE is most important at sites of peripheral lymph node development.

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3. LTi cells are attracted to sites of lymphoid organ development by homeostatic chemokines, including CXCL13, CCL19, and CCL21. Like TRANCE and IL-7, these chemokines also maintain surface lymphotoxin-αβ expression on LTi cells. 4. The lymphotoxin signaling pathway plays a central role in the development of secondary lymphoid organs owing to its ability to trigger mesenchymal cell differentiation, elicit homeostatic chemokine expression, and promote the differentiation of HEVs, stromal cells, and DCs. 5. Signaling through the lymphotoxin-βR initiates both the canonical and alternative NF-κB signaling pathways. Annu. Rev. Immunol. 2008.26:627-650. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

6. The vascular addressins, MAdCAM and PNAd, are sequentially expressed during lymph node development so that LTi cells and mature lymphocytes are recruited in a temporally ordered fashion. 7. CD4+ CD3− cells can be found in adults and seem to have a function in facilitating follicular T cell responses. However, the lineage relationship between embryonic CD4+ CD3− LTi cells and those found in adults remains uncertain. 8. The developmental pathways for peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches differ in their requirements for chemokines, cytokines, and growth factors. However, the pathways that govern the development of ILFs and NALT are dramatically different from those that govern conventional lymph nodes and Peyer’s patches.

FUTURE ISSUES 1. What is the relationship between CD4+ CD3− IL-7Rα+ LTi cells and CD4− CD3− IL7Rα− CD11c+ LTin cells? 2. Does each secondary lymphoid organ use slightly different populations of LTi and LTo cells for their development, and does this differential utilization of cell types explain why each lymphoid organ uses subtly different combinations of chemokines, growth factors, and cytokines for their development? 3. Other than the lymphotoxin signaling pathway, what pathways are used by LTi cells to trigger the differentiation of mesenchymal cells and initiate secondary lymphoid organ development? 4. Why is the development of most secondary lymphoid organs (lymph nodes, Peyer’s patches, and NALT) restricted to embryogenesis, when the development of ILFs can occur in adults? 5. Why does the development of NALT occur in the absence of lymphotoxin signaling and what pathways substitute for lymphotoxin signaling in this process?

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review. 642

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ACKNOWLEDGMENTS This work was supported by the Trudeau Institute, by NIH grants HL69409 and AI072689, and by the Sandler Program for Asthma Research.

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104. Kim MY, Toellner KM, White A, McConnell FM, Gaspal FM, et al. 2006. Neonatal and adult CD4+ CD3− cells share similar gene expression proﬁle, and neonatal cells up-regulate OX40 ligand in response to TL1A (TNFSF15). J. Immunol. 177:3074–81 105. Eberl G, Littman DR. 2004. Thymic origin of intestinal αβ T cells revealed by fate mapping of RORγt+ cells. Science 305:248–51 106. Ivanov II, Diehl GE, Littman DR. 2006. Lymphoid tissue inducer cells in intestinal immunity. Curr. Top. Microbiol. Immunol. 308:59–82 107. Kim MY, Gaspal FM, Wiggett HE, McConnell FM, Gulbranson-Judge A, et al. 2003. CD4+ CD3− accessory cells costimulate primed CD4 T cells through OX40 and CD30 at sites where T cells collaborate with B cells. Immunity 18:643–54 108. Kim MY, McConnell FM, Gaspal FM, White A, Glanville SH, et al. 2007. Function of CD4+ CD3− cells in relation to B- and T-zone stroma in spleen. Blood 109:1602–10 109. Kim MY, Anderson G, White A, Jenkinson E, Arlt W, et al. 2005. OX40 ligand and CD30 ligand are expressed on adult but not neonatal CD4+ CD3− inducer cells: evidence that IL-7 signals regulate CD30 ligand but not OX40 ligand expression. J. Immunol. 174:6686–91 110. Harmsen A, Kusser K, Hartson L, Tighe M, Sunshine MJ, et al. 2002. Organogenesis of nasal associated lymphoid tissue (NALT) occurs independently of lymphotoxin-α (LTα) and retinoic acid receptor-related orphan receptor-γ, but the organization of NALT is LTα-dependent. J. Immunol. 168:986–90 111. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. 2004. An essential function for the nuclear receptor RORγ(t) in the generation of fetal lymphoid tissue inducer cells. Nat. Immunol. 5:64–73 112. Ying X, Chan K, Shenoy P, Hill M, Ruddle NH. 2005. Lymphotoxin plays a crucial role in the development and function of nasal-associated lymphoid tissue through regulation of chemokines and peripheral node addressin. Am. J. Pathol. 166:135–46 113. Fukuyama S, Nagatake T, Kim DY, Takamura K, Park EJ, et al. 2006. Cutting edge: uniqueness of lymphoid chemokine requirement for the initiation and maturation of nasopharynx-associated lymphoid tissue organogenesis. J. Immunol. 177:4276–80 114. Weih F, Caamano J. 2003. Regulation of secondary lymphoid organ development by the nuclear factor-κB signal transduction pathway. Immunol. Rev. 195:91–105 115. Ikawa T, Fujimoto S, Kawamoto H, Katsura Y, Yokota Y. 2001. Commitment to natural killer cells requires the helix-loop-helix inhibitor Id2. Proc. Natl. Acad. Sci. USA 98:5164– 69 116. Hacker C, Kirsch RD, Ju XS, Hieronymus T, Gust TC, et al. 2003. Transcriptional proﬁling identiﬁes Id2 function in dendritic cell development. Nat. Immunol. 4:380–86 117. Kusunoki T, Sugai M, Katakai T, Omatsu Y, Iyoda T, et al. 2003. TH2 dominance and defective development of a CD8+ dendritic cell subset in Id2-deﬁcient mice. J. Allergy Clin. Immunol. 111:136–42 118. Rangel-Moreno J, Moyron-Quiroz J, Kusser K, Hartson L, Nakano H, Randall TD. 2005. Role of CXC chemokine ligand 13, CC chemokine ligand (CCL) 19, and CCL21 in the organization and function of nasal-associated lymphoid tissue. J. Immunol. 175:4904– 13 119. McDonald KG, McDonough JS, Wang C, Kucharzik T, Williams IR, Newberry RD. 2007. CC chemokine receptor 6 expression by B lymphocytes is essential for the development of isolated lymphoid follicles. Am. J. Pathol. 170:1229–40 120. Moyron-Quiroz JE, Rangel-Moreno J, Kusser K, Hartson L, Sprague F, et al. 2004. Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity. Nat. Med. 10:927–34 www.annualreviews.org • Secondary Lymphoid Organ Development

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121. Taylor RT, Patel SR, Lin E, Butler BR, Lake JG, et al. 2007. Lymphotoxin-independent expression of TNF-related activation-induced cytokine by stromal cells in cryptopatches, isolated lymphoid follicles, and Peyer’s patches. J. Immunol. 178:5659–67 122. Kweon MN, Yamamoto M, Rennert PD, Park EJ, Lee AY, et al. 2005. Prenatal blockage of lymphotoxin β receptor and TNF receptor p55 signaling cascade resulted in the acceleration of tissue genesis for isolated lymphoid follicles in the large intestine. J. Immunol. 174:4365–72 123. Wang C, McDonald KG, McDonough JS, Newberry RD. 2006. Murine isolated lymphoid follicles contain follicular B lymphocytes with a mucosal phenotype. Am. J. Physiol. Gastrointest. Liver Physiol. 291:G595–604 124. Lugering A, Kucharzik T. 2006. Induction of intestinal lymphoid tissue: the role of cryptopatches. Ann. N. Y. Acad. Sci. 1072:210–17 125. McDonald KG, McDonough JS, Newberry RD. 2005. Adaptive immune responses are dispensable for isolated lymphoid follicle formation: antigen-naive, lymphotoxinsufﬁcient B lymphocytes drive the formation of mature isolated lymphoid follicles. J. Immunol. 174:5720–28 126. Pabst O, Herbrand H, Friedrichsen M, Velaga S, Dorsch M, et al. 2006. Adaptation of solitary intestinal lymphoid tissue in response to microbiota and chemokine receptor CCR7 signaling. J. Immunol. 177:6824–32 127. Pabst O, Herbrand H, Worbs T, Friedrichsen M, Yan S, et al. 2005. Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes. Eur. J. Immunol. 35:98–107 128. Halle S, Bumann D, Herbrand H, Willer Y, Dahne S, et al. 2007. Solitary intestinal lymphoid tissue provides a productive port of entry for Salmonella enterica serovar Typhimurium. Infect Immun. 75:1577–85 129. Forster R, Schubel A, Breitfeld D, Kremmer E, Renner-Muller I, et al. 1999. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23–33

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:627-650. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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Immunity to Citrullinated Proteins in Rheumatoid Arthritis 1,2 ¨ Lars Klareskog,1 Johan Ronnelid, Karin Lundberg,3 Leonid Padyukov,1 and Lars Alfredsson4 1

Rheumatology Unit, Department of Medicine, Karolinska Institutet/Karolinska University Hospital, SE-171 76, Stockholm, Sweden

2

Clinical Immunology Unit, Uppsala University/Uppsala University Hospital, SE-751 85, Uppsala, Sweden

3

Kennedy Institute of Rheumatology, Imperial College London, W6 8LH, London, United Kingdom

4

Institute of Environmental Medicine, Karolinska Institutet, SE-171 77, Stockholm, Sweden; email: [email protected], [email protected], [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:651–75

Key Words

First published online as a Review in Advance on January 2, 2008

genes, environment, autoimmunity, smoking, HLA-DR

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090244 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0651$20.00

Abstract Antibodies to citrullinated proteins (ACPA), i.e., to peptides posttranslationally modiﬁed by the conversion of arginine to citrulline, are speciﬁc serological markers for rheumatoid arthritis (RA). Studies on anticitrulline immunity, summarized in this review, demonstrate that the criterion-based syndrome RA should be subdivided into at least two distinct subsets (ACPA-positive and ACPA-negative disease). A new etiological model is proposed for ACPA-positive RA, built on MHC class II–dependent activation of adaptive immunity. Fundamentals of this model include the following: (a) ACPA antedate onset of arthritis; (b) ACPA may aggravate arthritis in rodents; (c) ACPA are triggered in the context of genes that confer susceptibility to RA (HLA-DRB1 SE) and by environmental agents triggering RA (smoking or bacterial stimuli); (d ) ACPA may complex with citrullinated proteins present in target tissue as part of a multistep process for arthritis development. The model provides a new basis for molecular studies on the pathogenesis of ACPA-positive arthritis.

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BACKGROUND RA: rheumatoid arthritis RF: rheumatoid factors MHC: major histocompatibility complex

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ACPA: antibodies to citrullinated protein antigens CII: collagen type II

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Rheumatoid arthritis (RA) is a disease deﬁned by criteria (1) that have been useful in harmonizing clinical trials and clinical practice but that are not based on what is now known about its etiology or pathogenesis. It follows that any studies on the molecular pathogenesis of arthritis as deﬁned by these criteria should consider the possibility that the ﬁndings are relevant only for a subset of RA. RA is often denoted an “autoimmune” disease, largely based on the presence of rheumatoid factors (RF) (2), one of the seven classiﬁcation criteria. However, the presence of RF is not speciﬁc for RA but is rather a general consequence of immune activation in the context of immune complex formation (3, 4); no experimental studies have demonstrated any proarthritogenic effects of RF. However, other data favor a role for adaptive immunity and possibly for autoimmune reactions in the disease. Such features are the genetic linkage to MHC class II genes (5, 6), the pattern of immune cells and MHC class II molecules in inﬂamed synovium (7), and the presence of activated T and B cells in the joint (7–9). Taken together, these observations underscore a pathogenetic role for MHC class II–dependent immune activation in RA (7, 10). The nature of such speciﬁc immune reactions has, however, been surprisingly difﬁcult to deﬁne. In recent years, advances in research and therapy within the ﬁeld of cytokine regulation and cytokine-directed therapy have largely dominated the research ﬁeld of RA (11–14), illustrating how therapeutic progress is possible even though the role of adaptive immunity in this disease is not fully understood. In parallel with this progress, there has also been a major development in the ﬁeld of immunity focused on antibody reactivity to proteins modiﬁed by citrullination, i.e., an enzyme-mediated posttranslational modiﬁcation of peptidylarginine to peptidylcitrulline. Notably, epidemiological and genetic studies of RA in relation to the anticitrulline immunity have redeﬁned RA phenotypes, demonstrating major differences Klareskog et al.

in genetic and environmental risk factors, and thus probably in molecular pathogenesis too, between RA patients with and without the presence of antibodies to citrullinated proteins (ACPA). The implication for immunological studies of RA is that meaningful molecular studies on RA, particularly when dealing with adaptive immunity, should no longer be performed in patients with “RA” but rather in subsets of patients, grouped according to serology as well as to genetic, environmental, and clinical determinants. A short summary of studies from many groups over the past 10 years tells us that (a) antibodies to citrullinated proteins can be found in approximately 60% of RA patients (15–19). These antibodies are highly speciﬁc for RA, i.e., they exist in around 2% of normal populations (15) and are also quite rare in other inﬂammatory conditions (15). (b) The occurrence of ACPA is seen several years before onset of disease (20–23), and very few patients with RA develop ACPA after onset of their symptoms (24, 25). (c) The occurrence of ACPA is closely linked to the presence of MHC class II alleles that predispose for RA (26, 27); most notably, the association of HLA-DRB1 alleles is seen exclusively for the ACPA-positive subset of disease but not for the ACPA-negative variant (26, 27). (d ) Immunity toward citrullinated self proteins contributes to arthritis in rodents. The two experiments supporting this conclusion demonstrated that transfer of monoclonal antibodies to citrullinated ﬁbrinogen can enhance arthritis development in mice at the same time as tolerization against citrullinated antigens diminishes arthritis severity (28), and that immunization with the citrullinated self antigen type II collagen (CII) leads to a more severe arthritis than immunization with the same noncitrullinated protein (29). These studies have renewed interest in the ﬁeld of adaptive immunity in RA by focusing on a deﬁned subset of the disease. This review summarizes this research and suggests potential directions for research into the etiology of selected RA subpopulations. By dissecting the

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syndrome now called RA, we might be able to devise immunotherapies speciﬁcally adapted to individual arthritis subpopulations.

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CITRULLINATION, A POSTTRANSLATIONAL PROTEIN MODIFICATION OF LARGELY UNKNOWN SIGNIFICANCE Deimination of the charged peptidyl arginine to the neutral peptidylcitrulline is an enzymatic process in mammals mediated by a series of enzymes denoted peptidyl arginine deiminases (PADs) (30, 31). The activity of these enzymes is dependent on high concentrations of calcium, and deimination can occur intracellularly in conjunction with apoptosis (32) as well as extracellularly given high enough Ca2+ concentrations (33). In addition, certain proteins present in the stratum corneum of outer epidermis (34) and in the CNS in conjunction with astrocytes appear to be constitutively citrullinated (35, 36). The precise physiological function of citrullination is incompletely understood. From the fact that the deimination changes the charge of critical residues of a protein, it is H

O

H

N

Peptidyl arginine deiminase (PAD)

known that citrullination often makes proteins more prone to degradation by proteolytic enzymes (33) (Figure 1). An interesting recent ﬁnding is that proteins undergoing processing in antigenpresenting cells (APCs) can be citrullinated before presentation to T cells (37). The functional consequence of this intriguing observation is not yet understood but warrants further consideration. In human disease, increased citrullination was ﬁrst demonstrated to take place in lining and sublining cells in the joints of patients with RA (32). Subsequently, citrullinated proteins were detected by immunohistochemistry in a number of inﬂamed tissues, including arthritic joints in several different forms of arthritis (38), lungs (26, 39), extraarticular inﬂammatory sites in RA (39), human brain (40), and inﬂamed muscle as well as inﬂamed lymphoid organs (41). No selectivity of citrullination for certain argininecontaining proteins has been demonstrated to date, and citrullination has been observed in many different synovial proteins, including ﬁbrinogen (42), vimentin (43, 44), and CII (M. Hermansson, unpublished observation); furthermore, α-enolase (45) has been demonstrated to colocalize with citrullination in conjunction with joint inﬂammation. O

N

Ca2+ NH H2N+

NH O

NH2

L-arginine residue (+ charged)

PAD: peptidyl arginine deiminase

Change in charge

Different folding More sensitive to degradation

NH2

L-citrulline residue (neutral)

Deimination

Deiminated protein

Figure 1 Deimination of peptidylarginine to peptidylcitrulline is a posttranslational process, also known as citrullination, driven by the calcium-dependent enzyme peptidyl arginine deiminase (PAD). The enzymatic conversion results in the loss of one positive charge for every arginine residue converted to a neutral citrulline. This causes changes in intra- and intermolecular interactions, which could lead to altered protein folding, enhanced degradation by proteases, and exposure of cryptic epitopes. www.annualreviews.org • Immunity to Citrullinated Proteins in Rheumatoid Arthritis

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IMMUNITY TO CITRULLINATED PROTEIN ANTIGENS IN RA AND RELATED CONDITIONS

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The ﬁrst demonstration that antibodies in RA patients display a preferential reactivity with proteins modiﬁed by citrullination came from work by two groups, in France and in the Netherlands. Guy Serre’s group in Toulouse investigated the molecular basis of antibodies that reacted with keratin in the skin (45) and with perinuclear cellular structures (46), which were both used in RA diagnostics, and showed that ﬁlaggrin was the common target of these antibodies (47, 48). Walther van Venrooij’s group in Nijmegen subsequently demonstrated that the ﬁlaggrin-speciﬁc reactivity of RA sera is dependent on deimination (“citrullination”) of arginine residues in ﬁlaggrin-derived peptides (49). This failure to see reactivity against recombinant ﬁlaggrin produced for evaluation in potential immunoassays for RA led to the recognition that the reactivity was dependent on posttranslational citrullination (49). Initially, these ﬁndings were mainly used to develop better diagnostic markers for RA, and several assays were developed that increased the speciﬁcity and sensitivity for RA. The assays most widely used were those with cyclic citrullinated peptides as substrates for the detection of antibodies (15, 18, 50). The initial studies on ACPA reactivity were performed on in vivo citrullinated ﬁlaggrin molecules, and then on intact ﬁbrinogen where arginines had been deiminated in vitro using PAD (51). Investigations on larger groups of RA patients showed comparable reactivity patterns when cyclic citrullinated peptides (CCP), citrullinated ﬁlaggrin (16), or ﬁbrinogen (52) were used as substrates for assays focusing on IgG antibody reactivities. Further studies have demonstrated RA-speciﬁc reactivities against a wide range of citrullinated peptides and proteins. Reactivity to vimentin was originally observed as the so-called antiSa reactivity (53–55), and subsequent assays

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using a mutated variant of vimentin showed reactivities similar to, or with even better sensitivity than, those toward CCP (56) and ﬁbrinogen (44). Reactivities to citrullinated α-enolase represent one recent observation (57), of interest because α-enolase is widely expressed in RA joints and, in immunohistochemical analysis, it colocalizes with citrulline staining. The antibody reactivity to citrullinated CII represents a speciﬁc challenge because CII is the major structural protein in hyaline cartilage and because native CII is the classic autoantigen used to provoke polyarthritis in rodents (58, 59). The relatively low frequency of antibody reactivity to native CII in sera of patients with RA has argued against a pathogenetic role of anticollagen immunity in human disease (60), although there are some reports of higher frequencies of immunity against native CII in joints of patients with RA (61, 62). The current demonstration of a high frequency of antibodies against the citrullinated variants of CII in sera of RA patients, especially certain citrullinated peptides (63), provides a new angle on collagen immunity in RA. The extent to which reactivities of single antibodies are directed toward private epitopes on different citrullinated peptides or proteins and the extent to which they react with public epitopes common to many citrullinated peptides/proteins are incompletely understood, as is the isotype distribution of the ACPA (see Reference 64 for an initial description). The most remarkable features of the data emerging from investigations of different ACPA are the high speciﬁcity for RA and the fact that they deﬁne a distinct subset of RA. Most of these studies have been performed using CCP-based assays. Typically, 50%–70% of early RA patients are anti-CCP positive (18, 19), and the phenotype is thereafter very stable, i.e., very few patients shift from being anti-CCP positive to being anti-CCP negative or vice versa, even after treatment with disease-modifying antirheumatic drugs

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(24, 25). This qualitative phenotypic stability is also seen after treatment with TNFblocking agents, although ACPA concentrations remain stable in some (65, 66), but not in all (67–69), studies. Comparatively few individuals (typically approximately 2%) in a population of healthy controls are positive for anti-CCP antibodies. In contrast to RF, antiCCP antibodies are rather speciﬁc for RA. Thus, only relatively few patients with systemic inﬂammatory diseases, such as systemic lupus erythematosus (SLE), mixed connective ¨ tissue disease (MCTD), Sjogren’s syndrome, or myositis, have anti-CCP antibodies; most investigators have reported fewer than 10% of such patients to be anti-CCP positive. When patients with these diagnoses are subgrouped according to anti-CCP phenotype, those with detectable anti-CCP antibodies often present with RA-like features (including polyarthritic disease, erosions, RF, and HLA association) or can be classiﬁed as RA in addition to other diagnoses. This ambiguity has also been described for patients with psoriatic arthritis (70–73), juvenile idiopathic arthritis (74–77), SLE (78), and MCTD (79). These data suggest a potential new classiﬁcation of arthritis, as some patients with polyarthritis and concomitant features of other systemic rheumatic conditions might well share etiologic features with ACPA-positive classical RA. Further studies of common genetic and environmental determinants for ACPA-positive arthritides may suggest a new ACPA-related classiﬁcation that has a wider inclusion than only those patients who fulﬁll today’s classiﬁcation criteria for RA (1).

EVIDENCE THAT ACPA-POSITIVE AND ACPA-NEGATIVE RA CONSTITUTE TWO CLINICALLY AND GENETICALLY DISTINCT SUBSETS OF RA One of the more exciting features of current work on ACPA in RA is the evidence that

ACPA-positive and ACPA-negative RA constitute two distinct subsets of this criterionbased disease. The ﬁrst conﬁrmation is purely clinical in the sense that ACPA-positive RA patients have a disease course considerably more severe than that of ACPA-negative patients, irrespective of treatment (24, 25, 80, 81). In particular, erosiveness is highly linked to an ACPA-positive status (81–83). A second veriﬁcation comes from treatment, where a recently published study demonstrated how methotrexate induced remission in ACPApositive early arthritis patients but had no effect in ACPA-negative subjects (84). The most compelling data from a pathophysiological perspective, however, demonstrate large differences concerning susceptibility genes for ACPA-positive and ACPA-negative disease. By the late 1970s, the MHC class II genes (5, 6, 85) were already identiﬁed as the major genetic risk factor for RA. Numerous studies on the association between HLA class II genes, in particular the HLA-DRB1∗ shared epitope (SE) alleles and RA, have provided a strong rationale for MHC class II–dependent T cell activation and thus for adaptive immunity having a pathogenic role in RA. This association also differentiated RA from other MHC class II–associated arthritic diseases such as ankylosing spondylitis, psoriatic arthritis, and reactive arthritis (86). The genetic association of RA to HLADRB1 SE has recently been shown to be entirely conﬁned to the ACPA-positive subset (87, 88). This ﬁnding indicates that the implication of MHC class II–dependent T cell activation in the pathogenesis should be limited to ACPA-positive RA and to anticitrulline immunity (see below). In contrast, two studies suggested that ACPA-negative RA may be associated with HLA-DRB1∗ 03, an allele not previously associated with disease susceptibility in the unstratiﬁed RA population (64, 89). Following this initial demonstration of a genetic distinction between ACPA-positive and ACPA-negative RA for HLA-DR genes, a second major genetic risk factor for RA, the

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polymorphism in PTPN22 gene, was also shown to be associated only with the ACPApositive disease (90–92). Other genetic risk factors, in particular variations in the interferon regulating factor 5 (IRF-5) (93) but also polymorphisms in a newly identiﬁed risk gene in the C-type lectin complex (94), were associated exclusively with ACPA-negative disease (93). Taken together, the descriptive studies of disease course and genetic linkages strongly indicate that ACPA reactivity splits RA into two major and clinically relevant subsets of disease. Thus ACPA-positive and ACPAnegative RA should be treated as separate entities when studying the molecular pathophysiology of RA.

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EVIDENCE THAT ANTICITRULLINE IMMUNITY CAN CONTRIBUTE TO DEVELOPMENT OF ARTHRITIS The key issue related to anticitrulline immunity is whether the observed autoantibodies are causally related to the disease, consequences of the disease, or just epiphenomena. Two groups of experiments suggest that anticitrulline immunity may contribute to arthritis. That ACPA may precede clinical RA by years was ﬁrst demonstrated from studies of blood repositories in northern Sweden, where individuals who subsequently developed arthritis had donated blood samples several years before onset of disease. Anti-CCP antibodies were demonstrated up to nine years before clinical onset of RA, and increased frequencies of higher concentrations of antibodies were seen as the individuals approached onset of disease (22). These studies were well in line with a series of earlier investigations from Kimmo Aho and associates in Finland; these authors had shown that rheumatoid factors (95) as well as antikeratin (20) and antiﬁlaggrin antibodies (21) preceded RA development. An independent study from the Netherlands further corroborated the ﬁnding that most individuals who would de656

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velop ACPA-positive RA had already developed their autoantibodies before disease onset (23). Little is yet known about the evolution of ACPA epitope speciﬁcities during arthritis development, information that might shed light on the pathogenic role of different ACPAs. However, the time sequence of ACPA development pre-RA and, in particular, the ﬁnding that very few patients develop ACPA after disease onset, provide indirect evidence for the contribution of anticitrulline immunity, or some other concomitant immunity, in the pathogenesis of ACPA-positive RA. Studies of immunoglobulin isotypes and IgG subclasses in early and long-standing RA also indicate that the isotype repertoire is fully developed by the time of arthritis development, but shows a sustained presence of IgM antiCCP, interpreted as a continuous activation of ACPA-reactive B cells (64). The second line of evidence in support of a direct pathogenic role for anticitrulline immunity comes from experimental animal models. In the mouse, Kristine Kuhn and collaborators found that transfer of monoclonal antibodies to citrullinated ﬁbrinogen enhanced a mild arthritis that was initiated with anti-CII antibody transfer (28). However, antibodies to citrullinated ﬁbrinogen alone were not able to cause arthritis in naive animals with no joint lesions. Furthermore, mice immunized with native CII developed anticitrulline immunity, i.e., reactivity to native as well as citrullinated CII, and administration of citrullinated peptides in a tolerogenic protocol ameliorated the collagen-induced arthritis (CIA). These latter data thus suggested that citrullination of collagen and/or additional proteins might be involved in CIA. In two different reports in the rat, citrullination was shown to enable otherwise nonimmunogenic self molecules to trigger autoantibody production against albumin, CII (29), and ﬁbrinogen (96). Neither the immunity to citrullinated albumin (29) nor to ﬁbrinogen (96) was able to cause arthritis, whereas the immunity to citrullinated CII enhanced

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arthritis in Lewis 1AV1 rats (29). Taken together, these papers document how citrullination may change the immunogenicity of self antigens and suggest that immunity to some citrullinated proteins may contribute to arthritis development. More detailed studies are needed to determine which cells, speciﬁcities, and mechanisms are involved in this process (see below).

WHEN AND HOW IS ANTICITRULLINE IMMUNITY TRIGGERED? The data summarized above indicate that antibodies to autoantigens that have been modiﬁed by citrullination may contribute to arthritis. As citrullination is a common process in many physiological events, including apoptosis and inﬂammation, the rate-limiting steps appear to be whether and when immunity is triggered against these modiﬁed proteins and which epitopes of which proteins are being recognized. Most information on how genes and environmental determinants interact in increasing risk for ACPA-negative as well as ACPApositive RA comes from case control studies in which population-based RA cohorts were compared with healthy individuals from the same population. The most striking ﬁnding from these studies overall is the dichotomy between ACPA-positive and ACPA-negative RA concerning both genetic and environmental determinants. Thus, as noted above, both the major genes, i.e., HLA-DRB1 SE and PTPN22 alleles that predispose for RA, were shown to be risk factors for ACPA-positive disease, but not for ACPA-negative disease. For HLA-DRB1, the susceptibility alleles DRB1∗ 0401 and ∗ 0404 were also closely linked to frequencies as well as levels of ACPA, measured with the CCP assay (87, 88), indicating not only that these genes were susceptibility factors for the disease, but also that they directly inﬂuenced ACPA production. The PTPN22 codes for a tyrosine phosphatase

that is present in many cells, and the allelic difference inﬂuences susceptibility (97) by altering the threshold for activation of PTPN22expressing cells. This is true not only for T cells but also for a number of other cell types (98). The PTPN22 1858 T polymorphism (620W) is speciﬁcally associated with antiCCP-positive RA in many (91, 99, 100), but not in all (101), studies. Recently, a striking gene-gene interaction was demonstrated between HLA-DRB1 SE alleles and the susceptibility allele of the PTPN22 gene, where the combination of two SE alleles and the 620W allele of PTPN22 increased the risk for ACPA-positive RA 20-fold compared to individuals with none of these genetic risk factors (92). These data indicated that both molecular pathways involving MHC class II–dependent T cell activation and tyrosine phosphatase–mediated cell activation are of pathogenic importance in ACPA-positive RA. More recently, additional genetic determinants, notably one gene in the TRAF1-C5 region, have also been shown to associate with ACPA-positive RA (102), but not with ACPAnegative disease (103). Although the susceptibility gene(s) has not yet been deﬁnitely identiﬁed, its possible relation to TRAF1- or C5-mediated functions makes it an additional determinant inﬂuencing a pathway related to adaptive immunity and antibody-mediated effector functions. Smoking, long known to be the major environmental factor in increased risk for RA (104–107), was recently demonstrated to be a signiﬁcant inﬂuence on RF+ disease (104, 106, 108–110). Smoking used to be considered as an unspeciﬁc risk factor, of interest mainly from a public health perspective, rather than a reasonably well-deﬁned trigger of RA, and studies of its actions could help elucidate the molecular pathology of RA. Our investigations showed a striking gene-environment interaction between smoking and the presence of the HLA-DRB1 SE risk alleles as risk factors ﬁrst for RF+ disease (110), and then even more pronounced for ACPA-positive RA (26) (Figure 2). The relative risk of developing

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Figure 2 (a) A major gene-environment interaction between HLA-DR SE and smoking is present in the ACPA-positive RA subpopulation (left) but not in the ACPA-negative RA subpopulation (right). Smoking only confers an increased risk of developing ACPA-positive RA, as does possessing a single copy of the HLA-SE allele or, even more so, two copies of the HLA-SE alleles (RR, relative risk). (Original data published in Reference 26.) (b) The two panels demonstrate the combined effects of the three risk factors (HLA-DRB1 SE, PTPN22 620W allele, and smoking) in ACPA-positive RA (left) and the complete absence of effect of any of these three factors in ACPA-negative RA (right). (Original data published in Reference 92.)

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ACPA-positive RA was over 20 times higher for smokers carrying two copies of the HLADRB1 SE alleles than for nonsmokers with no SE alleles. On the other hand, no increased risk was discerned for development of ACPAnegative RA (26). This gene-environment interaction has subsequently been replicated both in a Dutch study of ACPA-positive and ACPA-negative cases only (27) and in a Danish case control (111) study, which found even higher risk ratios for the combination of smoking and HLA-DRB1 SE than in our original study. Notably, in our continued studies in the Swedish cohorts, we extended the data for gene-environment interactions between smoking and HLA-DRB1 by showing the dose-dependency of smoking, with a combined relative risk of ACPA-positive RA of close to 50 times higher in heavy smokers carrying two copies of HLA-DRB1 SE alleles than in nonsmokers lacking these variations (112). However, no interaction was found between the PTN22 risk alleles and smoking (92). In a recently published study from North America, a more mixed picture emerged on the interaction between smoking and HLADRB1 SE, indicating that other genes or environmental factors may also inﬂuence the risk of RA development (113). The studies described above provide one of the most striking examples of geneenvironment interaction in the risk of developing a speciﬁc diagnosis, where this risk is strictly conﬁned to an immunologically deﬁned subgroup of patients. These observations provide an obvious basis for further molecular studies that would explain the observed effects of genes and environment as well as provide a more general insight into the pathogenesis of ACPA-positive RA. As a ﬁrst step, we initiated studies to determine if and how smoking may inﬂuence citrullination of proteins in lungs. We showed initially that smoking is associated with an increased presence of citrullinated proteins in bronchoalveolar lavage (BAL) cells from healthy smokers and smokers with pulmonary inﬂammation (26). Subsequently, we extended this study to

demonstrate that this increased citrullination may be due to increased expression of PADs, in particular PAD2 in BAL cells from smokers ¨ L. Klareskog, (D. Makrygiannikos, M. Skold, and A. Catrina, submitted for publication). We then proposed the following model for how immunity to citrullinated proteins might be triggered by smoking and how the geneenvironment interactions might be explained (26, 114): Smoking may cause PAD activation and subsequent citrullination in lungs, at the same time as components in smoke act as unspeciﬁc adjuvants activating APCs in the pulmonary compartment. In individuals carrying immune response genes that predispose to a strong immune reaction against certain citrullinated peptides, and where other genetic variants are also present (such as the PTPN22coded tyrosine phosphatase), an immune response with antibody production to citrullinated proteins might be triggered. The validity of this model was further strengthened by a report in a mouse model that immunity to a citrullinated vimentin peptide may be restricted by HLA-DRB1 SE alleles (115).

A POSSIBLE ETIOLOGIC MODEL FOR ARTHRITIS THAT INVOLVES CITRULLINATION AND IMMUNITY TO CITRULLINATED PROTEINS The studies described above provide a framework for molecular studies of adaptive immunity in RA. In the remainder of this review, we use this framework to discuss a potential model for an etiology of ACPA-positive RA that could perhaps be extended to other cases of ACPA-positive arthritis. Notably, most of the issues related to speciﬁcity and regulation of anticitrulline immunity have still been incompletely investigated. The epidemiologic and longitudinal studies referred to above have also produced data to suggest the existence of different stages in a multistep process that leads to ACPA-positive RA. An outline of this model that we discuss below is provided in Figure 3.

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1 Immune response

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Figure 3 A three-stage etiologic model for the development of ACPA-positive RA. Stage 1, the immune response: Environmental risk factors, such as smoking, may induce citrullination of proteins in the lungs. An altered antigen uptake, processing, and presentation of citrullinated antigens could, in genetically susceptible individuals (i.e., HLA-DR SE positive), lead to the production of ACPA. Stage 2, the pathologic inﬂammatory response: An unspeciﬁc arthritis, accompanied by citrullination of proteins in the joints, develops at a later stage. Recruitment of ACPA from the circulation results in the formation of immune complexes. Stage 3, chronic RA: The generation of citrullinated proteins, the inﬂux of immune cells, and the production of cytokines and autoantibodies, as a result of the immune complex formation, perpetuate the joint inﬂammation into chronic RA.

Stage 1: Citrullination and Triggering of Immunity to Citrullinated Proteins Key to understanding anticitrulline autoimmunity in arthritis is the determination of conditions that trigger the occurrence of (a) citrullination and (b) immunity to citrullinated proteins. Activation of PADs and citrullination occurs in inﬂammatory conditions as well as in apoptosis, and many different cells including macrophages and neutrophils can express PADs (30) (Figures 4 and 5). Active immunization with citrullinated self proteins can trigger antibodies directed to citrullinated as well as to the corresponding noncitrullinated proteins. The conditions required to trigger immunity to citrullinated proteins in a more physiological context are less well known. From the relatively rare occurrence of anticitrulline antibodies in healthy individuals (human as 660

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well as mouse) and the frequent occurrence of citrullination already in utero (116) and in inﬂammation (41), the brake of tolerance to citrullinated proteins should be a relatively rare event. In many respects, the occurrence of immunity to citrullinated proteins can be compared with immunity to molecules exposed to the immune system during apoptosis. Here, immunity toward DNA and other constituents of apoptotic blebs may be triggered under certain relatively rare conditions, where both genetic and environmental factors are important (117). We know from studies described above in humans that at least some anticitrulline immunity may be preferentially triggered in the context of certain MHC class II genetic variants, but also other genetic factors should be studied, preferably in a context where anticitrulline immunity can be investigated per se and independently of its possible later involvement in RA pathogenesis.

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Stage 1: Immune response a Influx of cells into the lung

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Figure 4 Stage 1 in the etiological model for the development of ACPA-positive RA: the immune response. (a) Heavy cigarette smoking stimulates an inﬂux of cells into the lungs. (b) Toxic components in the smoke activate the cells and (c, d ) render them more prone to apoptosis. (e) PAD becomes activated during the apoptotic process and ( f ) deiminates proteins present in the lungs. (g) In genetically predisposed individuals, such as those carrying the HLA-DR SE alleles, presentation of citrullinated peptides or other neo-epitopes from citrullinated proteins could (h) activate autoreactive T cells, which in turn could induce B cell help and (i) stimulate the production of ACPA.

Functional polymorphisms in genes coding for the PADs are obvious candidates, and a polymorphism in the PADI4 gene has been reported to increase risk for RA in Japanese (118) and Korean (119) populations, but probably not in Western European populations (120–123). It is, however, not yet formally proven that this polymorphism affects anticitrulline immunity; other possible molecules in pathways related to the citrullination may have variations that predispose to the disease. Of particular interest is whether anti-PAD can be triggered in conjunction with citrullination. A few small studies suggest the exis-

tence of anti-PAD2 and anti-PAD4 antibodies in RA (124). This line of research warrants further attention, particularly given its attractive parallels to the situation in celiac disease, in which the complex of transglutaminase and gluten modiﬁed by this enzyme constitutes the immunogenic protein complex (125). In the ACPA parallel, the entire complex of PAD and a citrullinated antigen may constitute the nonself antigen complex. The possible association between citrullination in lungs and smoking—and the possible triggering of anticitrulline immunity in this context—is a challenging concept. But

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Stage 2: Pathologic inflammatory response a Unknown/unspecific b Influx of cells into the joint

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g IC formation Figure 5 Stage 2 in the etiological model for the development of ACPA-positive RA: the pathologic inﬂammatory response. (a) A second, joint-speciﬁc inﬂammatory event is initiated by an unknown and unspeciﬁc stimulus, for example, infection or trauma. (b) Inﬂammatory cells are recruited to the joint, (c) activated by the unknown “trigger,” and in this inﬂammatory milieu (d ) PAD becomes activated and (e) deiminates proteins present in the joint. ( f ) Circulating ACPA enter the joint, bind to the citrullinated proteins, and (g) form immune complexes (IC).

the model should not be limited to smoking. We might instead consider inﬂammation in lungs driven by various exposures as a potential trigger of citrullination within the airways and development of anticitrulline immunity. Silica dust and mineral oil exposure have both been linked to an increased risk for ACPA-positive RA (L. Klareskog, J. ¨ Ronnelid, K. Lundberg, L. Padyukov, and L. Alfredsson, unpublished results). Several other factors, including air pollutants and maybe also charcoal as in Caplan’s syndrome (126), are also possible agents. The fact that IgA anticitrulline immunity is seen early dur662

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ing development of an anticitrulline immune response (64) indicates that immunity triggered from mucosal surfaces such as the lungs may be involved in triggering of anticitrulline immunity (Figure 4). The nonequal importance of genetic variations in the PADI4 gene for risk of RA in Asian and in Caucasian populations (118–123) is an indication of possible multiple genetic and environmental factors that lead to the production of ACPA. Another trigger of citrullination and citrulline immunity could be infections. Although many pathogens are suggested to be involved in triggering of RA, no solid evidence exists linking infectious agents to RA. The discovery of ACPA provides new opportunities to reinvestigate this issue. Pratesi and colleagues, for example, have recently shown reactivity to citrullinated Epstein-Barr nuclear antigen-1 in RA patients (127). Another pathogen of interest is Porphyromonas gingivalis, the causal agent of adult periodontitis, a disease with many features in common with RA, of which the most striking is an HLASE association (128, 129). Given that P. gingivalis is the only bacterium known to express a functional PAD enzyme, one could hypothesize that infection by P. gingivalis could induce local citrullination and subsequent citrullineimmunity in susceptible individuals, which could lead to the development of RA in a fashion similar to that described for smoking. The seroconversion of a healthy individual from an ACPA-negative to an ACPA-positive state is quantitatively and “risk-wise” the most important step in the series of events that may lead to RA. Thus, for a healthy individual who is positive for ACPA, measured with an antiCCP test, the risk of developing RA in the future is 100-fold greater than that for the general population. This risk is further increased to over 100-fold if HLA-DRB1 and PTPN22 genes are taken into account (100). Little is known to date about the speciﬁcity, as well as afﬁnity maturation, of antibodies during the pre-RA stage of development and whether there is a dynamic in the

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ﬁne speciﬁcity and levels of antibodies during this time. It is of prime importance that such features be investigated, similar to the way studies were conducted in lupus (130). As described above, even less is known for T cell reactivities.

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Stage 2: Development of Unspecific Arthritis and the Possible Further Progression to RA A major feature in the proposed RA scenario is that antibodies to citrullinated proteins occur before onset of disease, and that the occurrence of these antibodies is associated with substantially increased risk of developing RA, even though most individuals with these antibodies do not develop disease. This observation is also compatible with the mouse data described above, in which transfer of anticitrullinated ﬁbrinogen antibodies alone was not able to cause arthritis in naive mice but could enhance arthritis in mice with a lowgrade arthritis caused by other means (28). Antibodies as well as T cells typically cause inﬂammation at sites where the autoantigen(s) is present. No citrullination has yet been demonstrated in normal, uninﬂamed joints or other uninﬂamed tissues (41) except at sites that are relatively inaccessible for the immune system in the stratum corneum (34) or in the CNS (35). A number of different unspeciﬁc proinﬂammatory stimuli appear to cause citrullination in joints, including trauma and postinfectious events (D. Makryannikos, L. Klareskog, A. Catrina, unpublished observations). This suggests that some autoantibodies that are triggered outside the joints, e.g., in the lungs, may bind to autoantigens in joints after an unspeciﬁc “second strike” has caused joint inﬂammation and citrullination. Such a scenario is compatible with observations in both human and mouse: ACPApositive patients with unspeciﬁc mild arthritis are much more likely to develop chronic and long-lasting arthritis than are patients without these antibodies (17, 131). This clinical experience can be compared to the mice that

were subjected to mild CIA and that developed more severe disease after transfer of antibodies to citrullinated ﬁbrinogen (28). Antibodies reacting with citrullinated proteins in the joint may thus enhance arthritis development in human as in mouse. This scenario would favor a three-stage development of ACPA-positive RA: the formation of antibodies, followed by the actual development and chronicity of arthritis (see Figure 6). The hypothesis that immunity to citrullinated proteins in joints may contribute to development of arthritis raises a fundamental question: Why does immunity to citrullinated but ubiquitously expressed proteins such as ﬁbrinogen cause arthritis but not inﬂammation at other sites? Two tentative explanations can be offered: First, the main target molecule for the autoimmune attack may indeed be tissue speciﬁc, whereas reactivities to other proteins are epiphenomena, partly due to crossreactivities. This possibility is nicely illustrated in the putative case of CII immunity and in reports that citrullinated CII is recognized by antibodies from RA patients. This line of reasoning would reactivate the issue of collagen- and cartilage autoimmunity in RA and provide new relevance to the CIA model and collagen autoimmunity. All of this supports calls for greater emphasis in this line of research. The second assumption is that immunity to citrullinated proteins such as ﬁbrinogen, vimentin, and α-enolase, which appear to be present in many sites of inﬂammation, can cause or enhance arthritis. In this scenario, we have to describe how immunity to such common proteins can cause a tissue-speciﬁc inﬂammation in joints. This problem might be parallel to how antibodies to the ubiquitous protein glucose phosphate isomerase (GPI) were shown to cause tissue-speciﬁc joint inﬂammation (132) and to how an initially T cell–driven pathology subsequently became driven by antibodies. A possible explanation for this tissue speciﬁcity (133) is that certain immune complexes containing antibodies

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Stage 3: Chronic rheumatoid arthritis a IC-mediated activation of APCs

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T B

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Figure 6 Stage 3 in the etiological model for the development of ACPA-positive RA: the chronic RA. (a) Immune complexes of ACPA and citrullinated proteins further stimulate antigen-presenting cells (APCs), by binding to complement and Fc receptors (not shown). Activated APCs present more citrullinated antigens, activate more T and B cells, increase the ACPA production but also (b) RF production. (c) Increased production of proinﬂammatory cytokines, including TNF, IL-1, and IL-6, in turn recruits more immune cells into the joint (d ), perpetuating the inﬂammatory process. Activation of PAD generates more citrullinated proteins, establishing a vicious cycle that ultimately leads to (e) the development of chronic RA.

to ubiquitous self molecules may preferentially accumulate in joint tissue. If also valid in human, such a mechanism would provide an option for antibodies to citrullinated forms of ubiquitous proteins to preferentially accumulate in joints and thereby contribute to arthritis development or perpetuation. Further comparative studies between anti-GPI-induced and ACPA-induced arthritis in rodents may show the extent to which lessons from anti-GPI-induced arthritis could help us understand ACPA-associated joint inﬂammation. 664

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Stage 3: Chronicity of Joint Inflammation and Fulfillment of the Diagnostic Criteria for RA Most cases of joint inﬂammation in human, such as synovitis in conjunction with trauma and postinfectious events, are transient and do not lead to chronic disease or to permanent joint damage. There have been some systematic studies of joint inﬂammation in a broad setting, i.e., including a very broad set of individuals with joint inﬂammation in early arthritis clinics. Strikingly, these studies demonstrate that most unspeciﬁc arthritides

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resume spontaneously (134). Patients with early arthritis also demonstrate huge differences in prognosis depending on initial ACPA status (17, 131, 135), as ACPA-positive subjects were more likely to develop RA, whereas ACPA-negative individuals with a clinically comparable picture often went into spontaneous remission. These data are of major clinical interest, as they allow a new clinically relevant classiﬁcation of patients with early unspeciﬁc arthritis. In a biological context, these data support the notion that anticitrulline immunity may indeed enhance development of unspeciﬁc and otherwise transient arthritis into a chronic disease. Notably, chronicity (more than six weeks of disease) is an inherent part of the classiﬁcation criteria for RA (1). It is therefore feasible that a capacity to enhance and/or prolong an otherwise transient arthritis may provide a causative role of anticitrulline immunity.

CONCLUDING REMARKS AND RESEARCH DIRECTIONS The principal message we hope to convey in this review is how knowledge gained from genetic epidemiology and longitudinal studies of the syndrome called RA can be used to provide a new basis for molecular studies of this disease. The lessons learned are as follows:

RA should be divided into at least two very different subsets, where multiple genetic and environmental determinants distribute dichotomously between the ACPA-positive and ACPAnegative variants of RA. Fundamentally different molecular pathologies must also be assumed for these two variants of disease. Analysis of gene-gene and geneenvironment interaction can provide precise leads to further molecular studies on etiology as well as therapy of distinct subgroups of arthritis. In the case of ACPA-positive RA, these studies point to the contribution of MHC

class II– and T cell–dependent adaptive immunity involving immune reactions toward proteins modiﬁed by citrullination. Longitudinal analyses, from pre-disease states to late disease, help in elucidating potential distinct breakpoints, where progression to disease may depend on different determinants in each of these stages. Future studies of ACPA-positive arthritis should be separate from ACPAnegative RA subjects, as discussed above. To be complete, however, ACPA-positive subjects with RA-like features, but who have other primary diagnoses (70–79), should also be included.

Notably, these lessons have been derived using information from only a very limited part of the mammalian genome, from a very limited set of antibody reactivities, and from limited data on environmental exposures. With new information on genetic variants over the entire genome now burgeoning (102, 136), as well as that from systems detecting multiple antibody reactivities (137), further insight into the molecular pathways involved in various subgroups of RA can be expected. This new information should be incorporated into our models and experimental systems. The implication is thus that any type of experiment aimed at understanding molecular events in RA, in particular when related to adaptive immunity, should be performed using biological specimens or patients’ clinical data for which we have precise knowledge of the proﬁle of ACPA status, genetic determinants, and, if possible, pre-existing environmental triggers, and for which the investigated patients have been subgrouped accordingly. If these measures are taken, the possibilities will be better than ever before to deﬁne arthritis-speciﬁc immune reactions that take place at distinct time periods in disease development. This ability, in turn, should enable

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us to apply the experience garnered from different animal models to the more precise study of pathogenesis and therapies in different groups of RA patients. We conclude that studies on citrullination and immunity against citrullinated proteins, taken together,

may furnish critical knowledge that will allow us to understand and modulate adaptive immunity in newly deﬁned sets of arthritis, including a major subset of RA that is characterized by the presence of antibodies to citrulline-modiﬁed proteins.

SUMMARY POINTS

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1. Antibodies to citrullinated protein antigens (ACPA) constitute a relatively speciﬁc diagnostic tool for RA, as they are present in approximately 60% of an early RA cohort and in approximately 2% of the general population. 2. ACPA-positive and ACPA-negative RA constitute two very different subpopulations of RA concerning the role of major susceptibility genes (HLA-DRB1 and PTPN22) and major environmental risk factors (smoking) as well as clinical course. These two subpopulations, which most likely also have different molecular pathogenesis, should be treated as separate entities in further immunological studies of RA. 3. Antibodies to citrulline-modiﬁed proteins may be causatively involved in the development of arthritis. Support for this proposal comes from data showing the emergence of ACPA before onset of disease and from animal model studies showing that immunity against citrulline-modiﬁed self molecules, such as ﬁbrinogen or collagen type II, can enhance development of arthritis. 4. A new model has been proposed to explain the contributions from genetics and environment (smoking) in causing ACPA and the onset of arthritis. This model includes citrullination induced by environmental agents such as smoking, induction of ACPA in individuals with RA susceptibility genes (including HLA-DRB1 SE), and citrullination of molecules in target tissues. 5. The new model provides a framework for studies of adaptive immunity and of interactions between innate and adaptive immunity in RA. The data and the model emphasize the need to use data on genetic features, on environmental exposures, and on clinical course to subdivide RA in subpopulations before embarking on molecular studies on pathogenesis and treatment.

FUTURE ISSUES 1. The evidence from genetic epidemiology demonstrating the presence of at least two very distinct subsets of RA should be used to deﬁne relevant subsets of arthritis. Hypotheses on etiology in the different subsets of RA, should be tested in molecular immunology using information from genetic epidemiology to properly subdivide patients. 2. Different animal models for arthritis should be used to study different subsets of RA, where these subsets are deﬁned by their genetic, environmental, and clinical characteristics.

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3. Prospective studies on the evolution of citrullination, anticitrulline immunity, and arthritis are needed to determine the different stages and breakpoints, initiation of ACPA, initiation of arthritis, and progression into chronic RA. 4. Further therapeutic and preventive studies can be directed toward molecular pathways deﬁned from increased knowledge on citrullination and anti-citrulline immunity.

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DISCLOSURE STATEMENT The authors do not declare any conﬂicts of interest. The studies from the authors’ laboratories and clinics were supported from multiple and noncommercial sources, including the Swedish Research Council, the Swedish Council for Working Life and Social Research, King Gustaf ¨ V’s 80-Year Foundation, the Swedish Rheumatism Association, the Soderberg Foundation, FAMRI (Flight Attendants Medical Research Academy), the insurance company AFA, and the EU-supported project AutoCure. These sponsors had no inﬂuence on the writing of this manuscript.

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¨ 92. K¨allberg H, Padyukov L, Plenge RM, Ronnelid J, Gregersen PK, et al. 2007. Gene-gene and gene-environment interactions involving HLA-DRB1, PTPN22, and smoking in two subsets of rheumatoid arthritis. Am. J. Hum. Genet. 80:867–75 93. Sigurdsson S, Padyukov L, Kurreeman FA, Liljedahl U, Wiman AC, et al. 2007. Association of a haplotype in the promoter region of the interferon regulatory factor 5 gene with rheumatoid arthritis. Arthritis Rheum. 56:2202–10 ¨ C, B¨ackdahl L, Ribbhammar U, et al. 2007. Association 94. Lorentzen JC, Flomes L, Eklow with arthritis of a gene complex encoding C-type lectin-like receptors. Arthritis Rheum. 56:2620–32 95. Aho K, Heliovaara M, Maatela J, Tuomi T, Palosuo T. 1991. Rheumatoid factors antedating clinical rheumatoid arthritis. J. Rheumatol. 18:1282–84 96. Duplan V, Foulquier C, Clavel C, Al Badine R, Serre G, et al. 2006. In the rat, citrullinated autologous ﬁbrinogen is immunogenic but the induced autoimmune response is not arthritogenic. Clin. Exp. Immunol. 145:502–12 97. Begovich AB, Carlton VE, Honigberg LA, Schrodi SJ, Chokkalingam AP, et al. 2004. A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am. J. Hum. Genet. 75:330– 37 98. Gregersen PK. 2005. Pathways to gene identiﬁcation in rheumatoid arthritis: PTPN22 and beyond. Immunol. Rev. 204:74–86 99. Johansson M, Arlestig L, Hallmans G, Rantapaa-Dahlqvist S. 2006. PTPN22 polymorphism and anticyclic citrullinated peptide antibodies in combination strongly predicts future onset of rheumatoid arthritis and has a speciﬁcity of 100% for the disease. Arthritis Res. Ther. 8:R19 100. Kokkonen H, Johansson M, Innala L, Eriksson C, Jidell E, Rantapaa Dahlqvist S. 2007. The PTPN22 1858C/T polymorphism is associated with anticyclic citrullinated peptide antibody positive early rheumatoid arthritis in northern Sweden. Arthritis Res. Ther. 9: R56 101. Pierer M, Kaltenhauser S, Arnold S, Wahle M, Baerwald C, et al. 2006. Association of PTPN22 1858 single-nucleotide polymorphism with rheumatoid arthritis in a German cohort: higher frequency of the risk allele in male compared to female patients. Arthritis Res. Ther. 8:R75 102. Plenge RM, Seielstad M, Padyukov L, Lee AT, Remmers EF, et al. 2007. Genomewide search identiﬁes TRAF1-C5 as rheumatoid arthritis risk locus. N. Engl. J. Med. 357:1199–209 103. Seielstad M, Padyukov L, Ding B, Plenge RM, Alfredsson L, Klareskog L. 2007. A genome-wide SNP association study identiﬁes novel risk loci for rheumatoid arthritis in Swedish EIRA study. Ann. Rheum. Dis. 66(Suppl. II):680 104. Heliovaara M, Aho K, Aromaa A, Knekt P, Reunanen A. 1993. Smoking and risk of rheumatoid arthritis. J. Rheumatol. 20:1830–35 105. Silman AJ, Newman J, MacGregor AJ. 1996. Cigarette smoking increases the risk of rheumatoid arthritis. Results from a nationwide study of disease-discordant twins. Arthritis Rheum. 39:732–35 106. Uhlig T, Hagen KB, Kvien TK. 1999. Current tobacco smoking, formal education, and the risk of rheumatoid arthritis. J. Rheumatol. 26:47–54 107. Stolt P, Bengtsson C, Nordmark B, Lindblad S, Lundberg I, et al. 2003. Quantiﬁcation of the inﬂuence of cigarette smoking on rheumatoid arthritis: results from a population based case-control study, using incident cases. Ann. Rheum. Dis. 62:835–41 www.annualreviews.org • Immunity to Citrullinated Proteins in Rheumatoid Arthritis

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108. Masdottir B, Jonsson T, Manfredsdottir V, Vikingsson A, Brekkan A, Valdimarsson H. 2000. Smoking, rheumatoid factor isotypes and severity of rheumatoid arthritis. Rheumatology 39:1202–5 109. Wolfe F. 2000. The effect of smoking on clinical, laboratory, and radiographic status in rheumatoid arthritis. J. Rheumatol. 27:630–37 110. Padyukov L, Silva C, Stolt P, Alfredsson L, Klareskog L. 2004. A gene-environment interaction between smoking and shared epitope genes in HLA-DR provides a high risk of seropositive rheumatoid arthritis. Arthritis Rheum. 50:3085–92 111. Pedersen M, Jacobsen S, Garred P, Madsen HO, Klarlund M, et al. 2007. Strong combined gene-environment effects in anticyclic citrullinated peptide-positive rheumatoid arthritis: a nationwide case-control study in Denmark. Arthritis Rheum. 56:1446–53 ¨ 112. K¨allberg H, Padyukov L, Bengtsson C, Ronnelid J, Klareskog L, Alfredsson L. 2007. Smoking is associated with anti-CCP positive RA in a dose dependent manner, results from the Swedish EIRA study. Ann. Rheum. Dis. 66(Suppl. II):291 113. Lee HS, Irigoyen P, Kern M, Lee A, Batliwalla F, et al. 2007. Interaction between smoking, the shared epitope, and anticyclic citrullinated peptide: a mixed picture in three large North American rheumatoid arthritis cohorts. Arthritis Rheum. 56:1745–53 114. Sverdrup B, Kallberg H, Bengtsson C, Lundberg I, Padyukov L, et al. 2005. Association between occupational exposure to mineral oil and rheumatoid arthritis: results from the Swedish EIRA case-control study. Arthritis Res. Ther. 7:R1296–303 115. Hill JA, Southwood S, Sette A, Jevnikar AM, Bell DA, Cairns E. 2003. The conversion of arginine to citrulline allows for a high-afﬁnity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1∗ 0401 MHC class II molecule. J. Immunol. 171:538– 41 116. Tsuji Y, Akiyama M, Arita K, Senshu T, Shimizu H. 2003. Changing pattern of deiminated proteins in developing human epidermis. J. Invest. Dermatol. 120:817–22 117. Casciola-Rosen LA, Anhalt G, Rosen A. 1994. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J. Exp. Med. 179:1317–30 118. Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, et al. 2003. Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat. Genet. 34:395–402 119. Kang CP, Lee HS, Ju H, Cho H, Kang C, Bae SC. 2006. A functional haplotype of the PADI4 gene associated with increased rheumatoid arthritis susceptibility in Koreans. Arthritis Rheum. 54:90–96 120. Barton A, Bowes J, Eyre S, Spreckley K, Hinks A, et al. 2004. A functional haplotype of the PADI4 gene associated with rheumatoid arthritis in a Japanese population is not associated in a United Kingdom population. Arthritis Rheum. 50:1117–21 121. Caponi L, Petit-Teixeira E, Sebbag M, Bongiorni F, Moscato S, et al. 2005. A family based study shows no association between rheumatoid arthritis and the PADI4 gene in a white French population. Ann. Rheum. Dis. 64:587–93 122. Harney SM, Meisel C, Sims AM, Woon PY, Wordsworth BP, Brown MA. 2005. Genetic and genomic studies of PADI4 in rheumatoid arthritis. Rheumatology 44:869–72 123. Martinez A, Valdivia A, Pascual-Salcedo D, Lamas JR, Fernandez-Arquero M, et al. 2005. PADI4 polymorphisms are not associated with rheumatoid arthritis in the Spanish population. Rheumatology 44:1263–66 124. Nissinen R, Paimela L, Julkunen H, Tienari PJ, Leirisalo-Repo M, et al. 2003. Peptidylarginine deiminase, the arginine to citrulline converting enzyme, is frequently recognized

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by sera of patients with rheumatoid arthritis, systemic lupus erythematosus and primary Sjogren syndrome. Scand. J. Rheumatol. 32:337–42 Roth EB, Stenberg P, Book C, Sjoberg K. 2006. Antibodies against transglutaminases, peptidylarginine deiminase and citrulline in rheumatoid arthritis—new pathways to epitope spreading. Clin. Exp. Rheumatol. 24:12–18 Caplan A. 1963. Contribution to discussion on rheumatoid pneumoconiosis. Grundfr. Silikoseforsch. 6:345–49 Pratesi F, Tommasi C, Anzilotti C, Chimenti D, Migliorini P. 2006. Deiminated EpsteinBarr virus nuclear antigen 1 is a target of anticitrullinated protein antibodies in rheumatoid arthritis. Arthritis Rheum. 54:733–41 Katz J, Goultschin J, Benoliel R, Brautbar C. 1987. Human leukocyte antigen (HLA) DR4. Positive association with rapidly progressing periodontitis. J. Periodontol. 58:607– 10 Marotte H, Farge P, Gaudin P, Alexandre C, Mougin B, Miossec P. 2006. The association between periodontal disease and joint destruction in rheumatoid arthritis extends the link between the HLA-DR shared epitope and severity of bone destruction. Ann. Rheum. Dis. 65:905–9 Heinlen LD, McClain MT, Merrill J, Akbarali YW, Edgerton CC, et al. 2007. Clinical criteria for systemic lupus erythematosus precede diagnosis, and associated autoantibodies are present before clinical symptoms. Arthritis Rheum. 56:2344–51 Jansen AL, van der Horst-Bruinsma I, van Schaardenburg D, van de Stadt RJ, de Koning MH, Dijkmans BA. 2002. Rheumatoid factor and antibodies to cyclic citrullinated peptide differentiate rheumatoid arthritis from undifferentiated polyarthritis in patients with early arthritis. J. Rheumatol. 29:2074–76 Matsumoto I, Staub A, Benoist C, Mathis D. 1999. Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme. Science 286:1732–35 Binstadt BA, Patel PR, Alencar H, Nigrovic PA, Lee DM, et al. 2006. Particularities of the vasculature can promote the organ speciﬁcity of autoimmune attack. Nat. Immunol. 7:284–92 Hulsemann JL, Zeidler H. 1995. Undifferentiated arthritis in an early synovitis outpatient clinic. Clin. Exp. Rheumatol. 13:37–43 Nielen MM, van der Horst AR, van Schaardenburg D, van der Horst-Bruinsma IE, van de Stadt RJ, et al. 2005. Antibodies to citrullinated human ﬁbrinogen (ACF) have diagnostic and prognostic value in early arthritis. Ann. Rheum. Dis. 64:1199–204 Wellcome Trust Case Control Consort. 2007. Genome-wide association study of 14,000 cases of seven common diseases and 3000 shared controls. Nature 447:661–78 Hueber W, Kidd BA, Tomooka BH, Lee BJ, Bruce B, et al. 2005. Antigen microarray proﬁling of autoantibodies in rheumatoid arthritis. Arthritis Rheum. 52:2645–55

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:651-675. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir,1 Manish J. Butte,1 Gordon J. Freeman,2 and Arlene H. Sharpe1 1

Department of Pathology, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts 02115-5727; email: arlene [email protected]

2

Department of Medical Oncology, Dana Farber Cancer Institute, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115-6013

Annu. Rev. Immunol. 2008. 26:677–704

Key Words

First published online as a Review in Advance on January 2, 2008

costimulation, T cell, autoimmunity, infectious disease, tumor

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090331 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0677$20.00

Abstract Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, deliver inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. Immune responses to foreign and self-antigens require speciﬁc and balanced responses to clear pathogens and tumors and yet maintain tolerance. Induction and maintenance of T cell tolerance requires PD-1, and its ligand PD-L1 on nonhematopoietic cells can limit effector T cell responses and protect tissues from immune-mediated tissue damage. The PD-1:PD-L pathway also has been usurped by microorganisms and tumors to attenuate antimicrobial or tumor immunity and facilitate chronic infection and tumor survival. The identiﬁcation of B7-1 as an additional binding partner for PD-L1, together with the discovery of an inhibitory bidirectional interaction between PD-L1 and B7-1, reveals new ways the B7:CD28 family regulates T cell activation and tolerance. In this review, we discuss current understanding of the immunoregulatory functions of PD-1 and its ligands and their therapeutic potential.

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INTRODUCTION The concept of T cell costimulation has evolved over time. The two-signal model for T cell activation was proposed by Kevin Lafferty as a model for the activation of naive T cells (1). According to this model, T cells require two signals to become fully activated. The ﬁrst signal, which gives speciﬁcity to the immune response, is provided by the interaction of antigenic peptide−MHC complex with the T cell receptor (TCR). The second, antigen-independent costimulatory signal, is delivered to T cells by antigen-presenting cells (APCs) to promote T cell clonal expansion, cytokine secretion, and effector function. In the absence of the second signal, antigen-speciﬁc lymphocytes fail to respond effectively and are functionally inactivated, or anergic, and resistant to subsequent activation by the antigen. The critical inhibitory function of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4, also known as CD152) was revealed by the fatal lymphoproliferative phenotype of Ctla4−/− mice (2, 3). This function demonstrated that T cell pathways could provide negative as well as positive second signals and provided the ﬁrst indication that negative second signals could regulate T cell tolerance. The discovery of more members of the B7:CD28 family has revealed additional costimulatory pathways that can provide positive and negative second signals to antigenexperienced effector T cells. The functions of these newer pathways have broadened the concept of costimulation. This review focuses on recent advances in our understanding of one of the newer pathways in the B7:CD28 family, the pathway consisting of the programmed death 1 (PD-1; also known as CD279) receptor and its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273). The PD-1 receptor was discovered in 1992 as a gene upregulated in a T cell hybridoma undergoing cell death (4). The important negative regulatory function of PD-1 was revealed by the autoimmuneprone phenotype of Pdcd1−/− mice in 1999

ITIM: immunoreceptor tyrosine-based inhibitory motif

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(5, 6). Since the ligands for PD-1 were identiﬁed in 2000 (7, 8) and 2001 (9, 10), there has been steady progress in understanding the functions of PD-1 and its ligands. Here, we ﬁrst describe the structure and expression of PD-1, PD-L1, and PD-L2. Next, we review recent advances in understanding PD-1 and PD-L signaling. We then summarize recent studies that identify B7-1 (CD80) as a binding partner for PD-L1 and indicate that PDL1 interactions with B7-1 can lead to bidirectional inhibitory responses in T cells (11). Finally, we discuss our current understanding of the roles played by PD-1 and its ligands in regulating T cell activation and tolerance and consider the therapeutic potential of manipulation of PD-1 and its ligands.

STRUCTURE OF GENES PD-1 is a 288 amino acid (aa) type I transmembrane protein composed of one immunoglobulin (Ig) superfamily domain, a ∼20 aa stalk, a transmembrane domain, and an intracellular domain of approximately 95 residues containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) as well as an immunoreceptor tyrosine-based switch motif (ITSM). PD-1 is encoded by the Pdcd1 gene on chromosome 1 in mice and chromosome 2 in humans. In both species, Pdcd1 consists of 5 exons. Exon 1 encodes a short signal sequence, whereas exon 2 encodes an Ig domain. The stalk and transmembrane domains make up exon 3, and exon 4 codes for a short 12 aa sequence that marks the beginning of the cytoplasmic domain. Exon 5 contains the C-terminal intracellular residues and a long 3 UTR. Splice variants of PD-1 have been cloned from activated human T cells (12). These transcripts lack exon 2, exon 3, exons 2 and 3, or exons 2 through 4. All these variants, except for the splice variant lacking exon 3 only (PD-1ex3), are expressed at similar levels as full-length PD-1 in resting peripheral blood mononuclear cells (PBMCs). All variants are

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signiﬁcantly induced upon activation of human T cells with anti-CD3 and anti-CD28. The PD-1ex3 variant encodes an mRNA that lacks the transmembrane domain and resembles soluble CTLA-4, which plays an important role in autoimmunity (13). This variant is enriched in the synovial ﬂuid and sera of patients with rheumatoid arthritis (14). PD-L1 is a 290 aa type I transmembrane protein encoded by the Cd274 gene on mouse chromosome 19 and human chromosome 9. Cd274 comprises seven exons, the ﬁrst of which is noncoding and contains the 5 UTR. The next three exons contain the signal sequence, IgV-like domain, and IgC-like domains, respectively. The transmembrane domain and the intracellular domains are contained in the next two exons (exons 5 and 6). The last exon contains intracellular domain residues plus the 3 UTR. The intracellular domain of PD-L1 is short, only about 30 aa, and highly conserved in all reported species. There is no known function for the intracellular tail of PD-L1. There is one reported splice variant of PDL1 in humans (15) consisting of a sequence lacking the IgV-like domain encoded in exon 2. This mutant should not be able to bind PD1, although the function of this splice variant has not yet been reported. No splice variants have been identiﬁed for mouse PD-L1. PD-L2 is a type I transmembrane protein encoded by the Pdcd1lg2 gene adjacent to Cd274 and separated by only 23 kb of intervening genomic DNA in mouse and 42 kb in human. The gene comprises six exons in mouse and seven in human. Exon 1 is noncoding, whereas the second exon contains the signal sequence. The IgV-like domain is composed of exon 3, the IgC-like domain is exon 4, and exon 5 contains a short stalk, transmembrane region, and the beginning of the cytoplasmic domain. In mouse exon 5, there is a stop codon that results in a cytoplasmic domain of only 4 aa. In human, exon 6 and 7 contain an additional coding region resulting in a cytoplasmic domain of 30 aa. The longer form of the cytoplasmic domain is found in

human, macaque, chimp, dog, cow, pig, and horse but is lost in mouse and rat. The long form of the cytoplasmic domain has no appreciable signaling motifs but is conserved across diverse species, suggesting that the cytoplasmic tail of PD-L2 may have a functional role. There are three PD-L2 splice variants identiﬁed from activated human PBMCs (9, 16). Analogous to the splice variant described in PD-L1, one form drops out the IgV-like exon and presumably loses the capacity to bind PD-1 (9). A second form (called type II) drops out the IgC-like domain. Another form (called type III) loses the IgC-like domain and the transmembrane residues but preserves the intracellular residues. The type II form would be expected to bind PD-1, as most binding activity resides in the IgV-like domain (17), and the type III form might represent a soluble ligand for PD-1.

STRUCTURE OF PROTEINS The unbound three-dimensional structure of PD-1 has been obtained by X-ray crystallography and shows that the β-strands of the Ig superfamily fold are well conserved between CTLA-4 and PD-1 (root mean squared deviation of 1.5 A˚ comparing common α carbons) (18). The CDR3 loop in PD-1 is loosely ordered and does not have conserved amino acids, unlike the binding interface of CTLA4, which is centered on the MYPPPY motif of the CDR3 loop and is highly ordered owing to the consecutive prolines in this motif. None of the PD-1 CDR3 amino acids was found to be important for binding PD-L by scanning mutagenesis. Biophysical studies have addressed the question of self-association of PD-1. Fluorescence resonance energy transfer analyses of full-length PD-1 expressed in CHO cells and analytical ultracentrifugation on a soluble extracellular PD-1 IgVlike domain showed PD-1 to be monomeric. Whether PD-1 requires dimerization to transduce signals is unclear. Molecular models of PD-L1 and PD-L2 have been generated on the basis of the crystal

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structures of B7-1 and B7-2 to guide sitedirected alanine scanning mutagenesis (17). The binding interfaces of both PD-L1 and PD-L2 to PD-1 are on their IgV-like domains. Interestingly, certain mutants of PDL1 and PD-L2 that were unable to bind PD-1 could stimulate T cell proliferation in conHuman

Mouse

PD-1 PD-L1 PD-L2 B7-1

PD-1 PD-L1 PD-L2 B7-1

Naive T cells

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Activated T cells Exhausted CD8+ T cells Naive B cells Activated B cells Peritoneal macrophages Activated macrophages Plasmacytoid DCs Myeloid DCs Monocytes Mast cells Cornea Lung Vascular Endothelium Liver nonparenchymal cells Mesenchymal stem cells Pancreatic islets Placental synctiotrophoblasts Keratinocytes

High expression

No expression

Low expression

No data

Figure 1 Comparison of expression of PD-1, PD-L1, PD-L2, and B7-1 on human and mouse cells. PD-1, PD-L1, PD-L2, and B7-1 are expressed on a wide range of human (7–9, 20, 23, 37, 55, 89, 143, 146–149) and mouse cells (4, 9, 24, 31, 85, 87, 113, 143, 147, 150–153). 680

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junction with anti-CD3. These non-PD-1 binding mutants were also able to stimulate Pdcd1−/− T cells, providing evidence for another receptor for PD-L1 and PD-L2.

EXPRESSION OF PD-1 AND ITS LIGANDS PD-1 can be expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DCs) (Figure 1). PD-1 is not expressed on resting T cells but is inducibly expressed after activation (19). Although PD1 cell surface protein expression can be detected within 24 h of stimulation, functional effects of PD-1 ligation are observed within a few hours following T cell activation (20). Ligation of TCR or BCR can upregulate PD1 on lymphocytes, and the level of mRNA transcription does not strictly correlate with protein production (21). In normal human reactive lymphoid tissue, PD-1 is expressed on germinal center–associated T cells (133). PD-1 compartmentalization in intracellular stores has been described in a regulatory T cell population (22, 22a). PD-1 is inducibly expressed on APCs on myeloid CD11c+ DCs and monocytes in humans (23), but its function on these cells is not clear. There are no data to support a function for PD-1 in the absence of antigen receptor signaling. The two PD-1 ligands differ in their expression patterns. PD-L1 is constitutively expressed on mouse T and B cells, DCs, macrophages, mesenchymal stem cells, and bone marrow–derived mast cells (24). PD-L1 expression is also found on a wide range of nonhematopoietic cells (see Figure 1) and is upregulated on a number of cell types after activation. Both type I and type II interferons (IFNs) upregulate PD-L1 (25, 26). Analyses of the human PD-L1 promoter demonstrate that both constitutive and inducible PD-L1 expression are dependent on two IFN regulatory factor-1 (IRF-1) binding sites that are between 200 and 320 bp upstream of the transcriptional start site (27). These IRF-1 binding sites are also found in mouse,

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although their importance has not been directly tested. Several studies have examined which signaling pathways are required for PD-L1 expression by using pharmacological inhibitors. PD-L1 expression in cell lines is decreased when MyD88, TRAF6, and MEK are inhibited (28). JAK2 has also been implicated in PD-L1 induction (27, 28). Loss or inhibition of phosphatase and tensin homolog (PTEN), a cellular phosphatase that modiﬁes phosphatidylinositol 3-kinase (PI3K) and Akt signaling, increases post-transcriptional PD-L1 expression in cancers (29). PD-L2 expression is much more restricted than PD-L1 expression. PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow–derived mast cells. PD-L2 is also expressed on 50% to 70% of resting peritoneal B1 cells, but not on conventional B2 B cells (30). PD-L2 expression on B1 cells tracks with a restricted VH usage that is skewed toward VH 11/VH 12. PD-L2+ B1 cells bind phosphatidylcholine and may be important for innate immune responses against bacterial antigens. Less is known about transcriptional regulation of PD-L2. Its induction by IFNγ is partially dependent on NF-κB (31). PD-L2 can also be induced on monocytes and macrophages by GM-CSF, IL-4, and IFN-γ (24, 32).

Signaling Through PD-1 Signaling through costimulatory receptors primarily functions to modify antigen receptor signaling. PD-1 typically has greater effects on cytokine production than on cellular proliferation, with signiﬁcant effects on IFN-γ, TNF-α, and IL-2 production. PD1-mediated inhibitory signals depend on the strength of the TCR signal, with greater inhibition delivered at low levels of TCR stimulation. This reduction can be overcome by costimulation through CD28 (8) or IL-2 (33). PD-1 may exert its effects on cell differentiation and survival directly by inhibiting early activation events that are positively regulated by CD28 or indirectly through IL-2

(33). Both CD28 and IL-2 promote cell expansion and survival through effects on antiapoptotic, cell cycle, and cytokine genes. IL2 withdrawal can lead to cell death, another process in which PD-1 has been implicated. There is strong evidence that PD-1 ligation inhibits the induction of the cell survival factor Bcl-xL (20). PD-1 inhibits the expression of transcription factors associated with effector cell function, including GATA-3, Tbet, and Eomes (34). Further studies are required to determine whether PD-1-mediated inhibition is related to its ability to counteract cell survival signals and effector differentiation mediated through CD28, IL-2, Bcl-xL, or a combination of these factors. PD-1 is phosphorylated on its two intracellular tyrosines upon ligand engagement, and then binds phosphatases that downregulate antigen receptor signaling through direct dephosphorylation of signaling intermediates. Two phosphatases, SH2-domain containing tyrosine phosphatase 1 (SHP-1) and SHP-2, can bind to the ITIM and ITSM motifs of PD-1 (35, 36). PD-1 inhibitory function is lost when the ITSM alone is mutated, demonstrating that this tyrosine plays the primary functional role of PD-1 inhibition (20, 36). The association between SHP-1 and PD1 appears to be weaker than the interaction of PD-1 with SHP-2. Together, these studies suggest that PD-1 functions by recruiting SHP-2, and possibly SHP-1, to the antigen receptor signaling complex (35). While the binding of SHP-2 to PD-1 is signiﬁcantly enhanced by PD-1 ligation (20), proximity of PD-1 to the antigen receptor appears to be important for inhibition by PD-1. PD-1 ligation inhibits antigen receptor signaling only in cis and not in trans, indicating that PD-1 ligation must occur close to the site of antigen receptor engagement (37). CTLA-4 moves from an intracellular store to the immunological synapse between an APC and lymphocyte after antigen recognition, depending on the strength of signal (38). In contrast, PD-1 redistributes from uniform cell surface expression to the synapse during

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tor protein SH2 domain–containing molecule 1A (SH2D1A, also known as SAP) (41). Unlike the CD150 family of cell surface receptors, the ITIM/ITSM motifs of PD-1 do not bind SH2D1A (20). Therefore, PD-1 inhibition likely functions solely through direct interaction with SHP-2, or possibly SHP-1, to directly inhibit early events in the antigen receptor signaling cascades (Figure 2). PD-1 interaction with SHP-1 and SHP-2 is further supported by a peptide immunoprecipitation study that investigated binding partners for the PD-1 ITIM and ITSM motifs using mass spectroscopy. The PD-1 ITIM/ITSM motifs also associate with Lck and Csk (35). These ﬁndings suggest that Csk and/or Lck may

T cell–APC interactions (22a). PD-1 could exert its inhibitory effects by bringing SHP-2 into the synapse during antigen receptor signaling, and cross-linking of PD-1 and CD3 increases the amount of SHP-2, but not SHP1, associated with PD-1 (39). PD-1 ligation inhibits PI3K activity and downstream activation of Akt. In contrast, CTLA-4 inhibits Akt activation but does not alter PI3K activity, indicating that these coinhibitory receptors function through distinct mechanisms. PD-1 ligation inhibits phosphorylation of CD3ζ, ZAP70, and PKCθ (40). Other costimulatory receptors, such as CD150, bind SHP-2 through interactions of their ITIM/ITSM domains with the adap-

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Antigen-presenting cell MHC

PD-L

B7 CD28

MY PP PY

Y Y Y Y

PY PP MY

P

Y Y Y Y

PD-1

TCR /CD3 Y

T cell HP-2

ARI

Y

S

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Pl3K

CD28

Akt Bcl-xL, IL-2, IFN-γ

Inhibition of antigen receptor signaling PD-1

Figure 2 Ligation of PD-1 dampens TCR signaling but can be overcome by CD28 costimulation. PD-1 engagement on the cell surface leads to phosphorylation of PD-1 cytoplasmic tyrosines and increases SHP-2 association with the ITSM of PD-1. Recruitment of SHP-2 dephosphorylates signaling through the PI3K pathway and downstream signals through Akt. PD-1 ultimately decreases induction of cytokines, such as IFN-γ, and cell survival proteins, such as Bcl-xL. When signaling through CD28 is delivered at the same time as PD-1 and TCR ligation, inhibitory effects can be overcome, and cytokine production and cell survival is enhanced. 682

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mediate the phosphorylation of PD-1 in T cells, similar to Lyn phosphorylation of PD-1 in a B cell line (36). PD-1 ligation also reduces Erk activation, but this effect can be overcome through cytokine receptor signaling, particularly cytokines that activate STAT5, such as IL-2, IL-7, and IL-15 (37). SHP-2 positively regulates Erk phosphorylation by interacting with Gab2 after IL-2R ligation (42). Both activation of Erk, which is speciﬁcally inhibited by PD-1 ligation, and activation of STAT5, which can overcome PD-1 inhibition, are demonstrated to be involved in the antiapoptotic and proliferative function of IL-2 (43). This suggests a model whereby the association of PD-1 with SHP-2 serves not only to dephosphorylate signaling intermediates, but also perhaps to sequester SHP-2 from its positive signaling role in Erk activation.

Reverse Signaling Through PD-L1 and PD-L2 PD-L1 and PD-L2 not only may inﬂuence responses by engaging PD-1 and modifying TCR or BCR signaling, but also may deliver signals into PD-L1- or PD-L2-expressing cells. The ﬁrst indication that PD-L2 may transmit signals into DCs came from studies using a novel, naturally occurring human IgM antibody (sHIgM12), isolated from a patient with Waldenstrom’s macroglobulinemia, that binds both mouse and human PD-L2 (44). Although DCs treated with sHIgM12 do not upregulate MHC II or B7 costimulatory molecules, they produce greater amounts of proinﬂammatory cytokines, particularly TNF-α and IL-6, and stimulate naive T cell proliferation. Treatment of mice with the sHIgM12 enhances resistance to transplanted B16 melanoma and rapidly induces potent tumor-speciﬁc CTL (45–47). sHIgM12 also completely blocked the development of airway inﬂammatory disease in a mouse model of allergic asthma (48, 49). A recombinantly expressed IgM antibody was generated from sHIgM12 that recapitulates observations with

the patient-derived antibody, arguing for a speciﬁc effect through PD-L2 ligation (50). Further studies are needed to determine how PD-L2 signals into the DC and how these signals inﬂuence immunity or tolerance. Additional evidence for reverse signaling through PD-L1 or PD-L2 on DCs comes from studies of bone marrow–derived DCs cultured with soluble PD-1-Ig fusion protein containing the extracellular domain of mouse PD-1 fused to the constant region of human IgG (51). This soluble PD-1 (sPD-1) inhibited DC activation and increased IL-10 production independent of 2,3-dioxygenase (IDO). These effects could be prevented by preneutralization of sPD-1 with anti-PD-1, suggesting that the acquisition of this suppressive DC phenotype is PD-1-speciﬁc and occurs via PD-L1 or PD-L2. These ﬁndings are consistent with previous studies in which sPD-1 induced IL-10 production by CD4 T cells (52). Bidirectional signaling of PD-1 and PDL may help clarify some of the contradictory results of studies analyzing the PD-1:PD-L pathway. As is discussed in the next section, the discovery of B7-1 as an additional ligand for PD-L1 also may help explain differences observed in functional studies of PD-1 and PD-L1. The identiﬁcation of bidirectional interactions between B7-1 and PD-L1 that inhibit T cell responses further underscores the importance of reverse signaling through PDL1 and demonstrates that PD-L1 can signal into T cells.

sHIgM12: naturally occurring human anti-PD-L2 IgM antibody derived from a patient with Waldenstrom’s macroglobulinemia

B7-1:PD-L1 Interactions Inhibit T Cell Responses A number of lines of evidence have suggested a receptor for PD-L1 or PD-L2, aside from PD-1. B7-1 has recently been identiﬁed as a binding partner for PD-L1 (11). Surface plasmon resonance studies demonstrate speciﬁc and unique interaction between PD-L1 and B7-1, with an afﬁnity (∼1.7 μM) intermediate between the afﬁnities of B7-1 for CD28 (4 μM) and CTLA-4 (0.2 μM), and PD-L1 for

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Table 1 Commonly used mAbs that display neutralizing activity against PD-L1:B7-1, PD-L1:PD-1, or B7-1:CTLA-4 Blocking capacity of mAbs PD-1:PD-L1

B7-1:PD-L1

++

+

−

+

αPD-L1 10F.9G2 10F.2H11

CD28, CTLA-4:B7-1

B7-1:PD-L1

αB7-1 1G10

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16–10A1

+

+

++

−

PD-1 (0.5 μM). B7-2 does not bind to PD-L1 or PD-L2, and PD-L2 does not bind to B7-1. Chemical cross-linking studies indicate that PD-L1 and B7-1 can interact through their IgV-like domains. These domains are known to be important for interactions of B7-1 and PD-L1 with their previously known receptors. On B7-1, there is a partial overlap between the B7-1:PD-L1 interface and that of B7-1:CTLA-4. Likewise, on PD-L1, the PDL1:B7-1 interface overlaps at least partially with the putative PD-L1:PD-1 interface (17). The substantial overlap of B7-1:PD-L1 regions of interaction with those of their previously identiﬁed binding partners suggests that blocking antibodies might interrupt B7-1:PD-L1 binding as well as B7-1:CTLA-4 or PD-L1:PD-1 binding interactions. Adhesion assays tested the capacity of anti-B7-1 and anti-PD-L1 monoclonal antibodies (mAbs) to interfere with PD-L1 and B7-1 binding to B7-1 or PD-L1 300.19 transfectants, respectively. Two classes of B7-1 antibodies could be identiﬁed: one that blocks both B7-1:PDL1 and B7-1:CTLA-4 binding and another that only blocks the B7-1:CTLA-4 interaction (Table 1). Four categories of PD-L1 antibodies have been identiﬁed: those that block only PD-1, those that block only B7-1, those that block both, and those that block neither PD-1 nor B7-1 interactions. B7-1:PD-L1 interactions can induce an inhibitory signal into T cells. Ligation of PD-L1 on CD4 T cells by B7-1, or ligation of B7-1 on 684

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CD4 T cells by PD-L1, delivers a functionally signiﬁcant, inhibitory signal. T cells lacking the previously known receptors for B71 (i.e., T cells lacking CD28 and CTLA-4) showed decreased proliferation and cytokine production when stimulated by anti-CD3 plus B7-1-coated beads. When T cells lacking all the receptors for B7-1 (i.e., T cells lacking CD28, CTLA-4, and PD-L1) were employed, T cell proliferation and cytokine production were no longer inhibited by anti-CD3 plus B7-1-coated beads, indicating that B7-1 acts speciﬁcally through PD-L1 on the T cell in the absence of CD28 and CTLA-4. Similarly, T cells lacking the previously known receptor for PD-L1 (PD-1) showed decreased proliferation and cytokine production when stimulated in the presence of anti-CD3 plus PD-L1-coated beads, demonstrating the inhibitory effect of PD-L1 ligation of B7-1 on T cells. When T cells lacking all known receptors for PD-L1 (i.e., T cells lacking PD-1 and B7-1) were used, T cell proliferation was no longer impaired by anti-CD3 plus PD-L1coated beads. Thus, PD-L1 can exert an inhibitory effect on T cells either through B7-1 or PD-1. Taken together, these results demonstrate a speciﬁc and signiﬁcant bidirectional interaction between B7-1 and PD-L1 that inhibits T cell responses (Figure 3). The identiﬁcation of the PD-L1:B7-1 interaction necessitates a reassessment of the roles of B7-1 and PD-L1 in regulating the activation and inhibition of immune responses, as well as their therapeutic manipulation. Differences in antiPD-L1-blocking antibody speciﬁcities may help explain distinct functional outcomes of blockade of PD-1, PD-L1, and PD-L2 in disease models in vivo. For example, many in vivo studies (discussed below) have found a greater effect of anti-PD-L1 blockade compared with anti-PD-1 or anti-PD-L2 blockade, and this has been attributed to differences in the expression of PD-L1 and PD-L2 or to the half-life or afﬁnities of the antibodies. The 10F.9G2 anti-PD-L1 mAb that has been used in many in vivo studies blocks PD-L1:B7-1

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B7-2 (CD86)

YYYY

MYPPPY

B7-1 (CD80)

MYPPPY

YY CTLA-4 (CD152)

PD-L1

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Antigenpresenting cell

MHC

CD28

YY

PD-L2 (B7-DC)

B7-1

Inhibition

Inhibition

T cell

TCR

PD-L1 (B7-H1)

Activation

PD-1

Inhibition

Inhibition

Figure 3 B7-1:PD-L1 interaction expands pathways in the B7:CD28 family. Recent data demonstrate that PD-L1 and B7-1 productively interact on T cells and can deliver bidirectional inhibitory signals. The identiﬁcation of these costimulatory molecules as binding partners increases our understanding of the interactions that can occur on T cells and APCs and raises the possibility that PD-L1:B7-1 binding may not only deliver signals when ligated, but may also serve to segregate binding away from previously identiﬁed receptors (PD-1, CD28, CTLA-4). IgV-like regions are depicted in blue and IgC-like regions in green, while tyrosine-containing signaling motifs are depicted by Ys.

as well as PD-L1:B7-1 interactions (Table 1). Anti-PD-L1 antibodies that block solely PDL1:PD-1 or PD-L1:B7-1 interactions should have reduced effects on T cell activation as compared with the dual-speciﬁc anti-PD-L1 mAb. Further studies are needed to compare the functional effects of anti-PD-L1 mAb that block solely PD-L1:PD-1 or PD-L1:B7-1 interactions with mAbs that block both interactions. The direct interaction between B7-1 and PD-L1 also compels a revised view of the interactions among molecules within the B7:CD28 family in regulating T cell activation and tolerance and gives increased significance to B7-1 and PD-L1 on T cells. There are limited studies of PD-L1 function on T

cells, but studies of PD-L1−/− T cells indicate that PD-L1 on T cells can downregulate T cell cytokine production (53). Because both PD-L1 and B7-1 are expressed on T cells, B cells, DCs, and macrophages, there is the potential for bidirectional interactions between B7-1 and PD-L1 on these cell types. In addition, PD-L1 on nonhematopoietic cells may interact with B7-1 as well as PD-1 on T cells to regulate cells. In the sections below, we summarize our current understanding of the roles of PD-1 and its ligands in disease models and discuss the outcomes of PD-L1 manipulation in light of the new PD-L1:B7-1 interaction. We also consider the therapeutic potential of manipulation of PD-1 and its ligands.

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AUTOIMMUNITY AND PERIPHERAL TOLERANCE

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The ﬁrst indication that the PD-1 pathway plays a critical role in autoimmunity came from the phenotype of Pdcd1−/− mice. Aged Pdcd1−/− C57BL/6 mice develop a mild glomerulonephritis with low frequency (5). Pdcd1−/− Balb/c mice develop a dilated cardiomyopathy owing to the production of an autoantibody against cardiac troponin (6, 54). Autoimmunity is accelerated by PD-1 deﬁciency on autoimmune-prone backgrounds. These ﬁndings broadly support a role for PD1 in the induction and/or maintenance of tolerance. Subsequent work has examined the mechanisms by which PD-1 and its ligands can control self-reactive T cell responses. PD-1 and PD-L1 provide inhibitory signals that regulate both central and peripheral tolerance in multiple ways. PD-1 is expressed on maturing thymocytes in the course of central tolerance induction. PD-L1 is expressed broadly on the thymic cortex and on thymocytes themselves, whereas PD-L2 expression is limited to the thymic medulla (31, 55). CD4− CD8− (DN) thymocytes start to express PD-1 as they undergo TCRβ rearrangement and begin to display functional preTCRs on the cell surface (19). PD-1:PD-L1 interactions inhibit positive selection during the DN to CD4+ CD8+ (DP) maturational stage (56). PD-1 signaling modiﬁes positive selection signaling thresholds, and loss of either PD-1 or PD-L1 increases DP thymocyte cell numbers (57). PD-1 also can contribute to negative selection (58) and has been identiﬁed as a candidate gene in a microarray analysis of aberrant central tolerance in nonobese diabetic (NOD) mice (59). Together, these ﬁndings point to a role for PD-1 and PD-L1 in central tolerance induction. Self-reactive T cells that escape negative selection are controlled in the periphery by mechanisms of peripheral tolerance. Initial interactions between T cells and APCs, such as DCs, can modify potentially self-reactive responses by the display of self-antigen on

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resting DCs. PD-1 has an important role in controlling the outcome of initial encounters between naive self-reactive T cells and DCs by inhibiting responses of self-reactive T cells. Emerging evidence suggests that immature DCs tolerize T cells, and loss of PD-1 on antigen-speciﬁc T cells increases CD8 T cell responses to antigen-bearing resting DCs (60). Studies in mouse models of autoimmunity and tolerance have revealed that PD-1:PDL interactions not only are important in the initial phase of activation and expansion of self-reactive T cells, but also inﬂuence selfreactive T cell effector function upon antigen reencounter. In the NOD mouse model of autoimmune T cell–mediated diabetes, PDL1 is upregulated in the pancreas on islet cells (31), and loss or blockade of PD-1 or PD-L1 leads to rapid and exacerbated diabetes with accelerated insulitis and proinﬂammatory cytokine production by T cells (61– 63). In a model of antigen-speciﬁc therapy in which administration of antigen-coupled ﬁxed splenocytes induces tolerance and reverses diabetes in NOD mice, PD-1:PD-L1 interactions were required for both the induction and maintenance of CD4 T cell tolerance (64). Notably, blockade of PD-1 or PDL1 reversed anergy in islet-antigen-speciﬁc T cells, whereas CTLA-4 blockade did not break tolerance, indicating a unique function for PD-1:PD-L1 interactions in maintaining T cell anergy. Bone marrow chimera experiments have demonstrated that PD-L1 expression on non–bone marrow–derived cells, including islet cells, inhibits T cell effector function in tissues (63, 65, 66). These studies also suggest a key role for the PD-1:PD-L1 pathway in limiting immune-mediated tissue damage caused by pathogenic T cells upon antigen reencounter in the periphery. Collectively, these ﬁndings demonstrate that PD-1: PD-L1 interactions regulate both the initiation and progression of autoimmune diabetes in NOD mice and identify PD-1:PD-L1 interactions as key mediators of T cell tolerance in tissues.

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In the experimental autoimmune encephalomyelitis (EAE) model of human multiple sclerosis (MS), PD-1 and its ligands also control self-reactive T cells. PD-1, PD-L1, and PD-L2 are all expressed on cellular inﬁltrates within the meninges during active EAE disease in C57BL/6 mice (31). PD-L1 is expressed in the CNS on inﬂammatory cells as well as on astrocytes and vascular endothelial cells. PD-L1 is speciﬁcally induced on CD11b+ APCs by IL-12 (67) and on microglial cells by IFN-γ (68). Initial studies described a role for PD-1 and PD-L2 using neutralizing antibody treatment (69). Anti-PD-1 or anti-PD-L2 mAb administration during the induction of EAE accelerated disease onset and severity, increased CNS inﬂammatory inﬁltrates, and led to increased myelin oligodendrocyte glycoprotein (MOG)-reactive T cells and antibodies. Subsequent studies (70) using blocking antibodies in different mouse strains, such as Balb/c, or gene-deﬁcient animals (53, 71) suggest that PD-1 and PD-L1, but not PD-L2, are predominantly responsible for regulating the severity of disease in most mouse strains. The strain-dependent effects of PD-1 and its ligands in inﬂuencing disease severity in EAE cannot be explained by expression of PDL1 or PD-L2 on APCs or inﬂammatory cells (70). Cytokine production is important in the pathogenesis of EAE, and Pdcd1−/− and Cd274−/− T cells secrete increased amounts of inﬂammatory cytokines, including IL-17 and IFN-γ, in recall responses to myelin antigen (71). Adoptive transfer studies emphasize the critical function for PD-L1 in limiting myelin-reactive pathogenic effector T cells and show that PD-L1 both on the transferred T cell and in the recipient restrains encephalitogenic T cell responses (53). Another important mechanism of peripheral tolerance involves regulatory T cells, which can suppress activated T cell proliferation and cytokine production. Both PD-1 and PD-L1 are highly expressed on these populations and may play a role in regulatory T cell function (72). A number of studies suggest

that PD-L1 may be important for inducing regulatory T cell populations, although the mechanism is not yet clear (73). Experiments in colitis models support the argument for a role of PD-1:PD-L1 on a regulatory cell population and identiﬁed a regulatory subpopulation of CD4+ CD25− PD-1+ T cells that can inhibit the development of colitis (74). PD-L1 is important for in vitro inhibition by another suppressive population of CD4+ DX5+ T cells (75). A role for PD-1:PD-L in humans is suggested by polymorphisms in PDCD1 that have been associated with human autoimmune diseases, including systemic lupus erythematosus (SLE), type 1 diabetes, rheumatoid arthritis, Grave’s disease, and MS (76). Most of these polymorphisms are found in conserved regions in intronic sequences. One intronic single nucleotide polymorphism (G7146A) in PDCD1 is located in a binding site for Runx1 (AML-1), a transcription factor with an important regulatory role in hematopoiesis (77). This polymorphism may alter PD-1 mRNA stability or expression level and is associated with reduced PD-1mediated inhibition of IFN-γ production in German patients with MS (78). A recent study suggests that PDCD1 genetic variation may inﬂuence the risk and expression of SLE, and effects of PD-1 polymorphisms vary according to ethnic background, similar to the effects of mouse PD-1 deﬁciency in different genetic backgrounds (79). A polymorphism in PDCD1LG2 has been described that correlates with SLE (80), but no polymorphisms in CD274 have been linked to human autoimmune diseases. In addition, autoantibodies to PD-L1 that correlate with active disease have been found in rheumatoid arthritis patients and may contribute to disease progression by dysregulating T cell responses (52). It remains to be determined if the signals that hold autoimmunity in check are generated through interactions between PD1 and PD-L1 or between PD-L1 and B7-1. In the NOD mouse model, Cd80−/− NOD mice develop autoimmunity more rapidly

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and with higher incidence than NOD mice (81), although less quickly than Pdcd1−/− and Cd274−/− NOD mice. B7-1-blocking antibodies signiﬁcantly accelerate diabetes onset in NOD mice and induce diabetes in normally resistant male NOD mice, but the two antiB7-1 mAbs appear to differ in their functional effects. The 16–10A1 mAb (which blocks B71:CTLA-4 but not B7-1:PD-L1 interactions) caused more rapid diabetes onset than did the 1G10 mAb (which blocks both B7-1:CTLA4 and B7-1:PD-L1 interactions) (82). Further studies are needed to test the role of PDL1:B7-1 interactions and to understand the mechanisms by which PD-L1 promotes peripheral tolerance. In view of its important role in autoimmune disease, the PD-1:PD-L pathway has become a new therapeutic target. Therapies that increase the expression of PD-L and trigger PD-1 may ameloriate autoimmune diseases. These approaches are only beginning to be evaluated in animal models, but the results appear promising. DCs genetically modiﬁed to overexpress PD-L1 and MOG in the context of MHC II dramatically ameliorate clinical EAE and reduce severity of CNS inﬂammation (83). A recombinant adenovirus expressing full-length mouse PD-L1 partially protects against the development of nephritis in lupus-prone mice (84). IFN-β, an immunemodulatory treatment for MS, can upregulate PD-L1 on APC in vitro and in MS patients in vivo, suggesting that IFN-β may exert its antiinﬂammatory effects in part via upregulation of PD-L1 expression (26).

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INFECTIOUS DISEASE AND MICROBIAL PATHOGENESIS PD-1 and its ligands have important roles in regulating immune defenses against microbes that cause acute and chronic infections. The PD-1:PD-L pathway appears to be a key determinant of the outcome of infection, regulating the delicate balance between effective antimicrobial immune defenses and immunemediated tissue damage. For example, 688

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Pdcd1−/− mice clear an adenovirus infection more rapidly but develop more severe hepatocellular injury than WT mice (85). In a mouse model of herpes stromal keratitis, a blocking anti-PD-L1 mAb exacerbated keratitis, increasing HSV-1-speciﬁc effector CD4 T cell expansion and IFN-γ production and survival (86). These studies suggest that the PD-1:PD-L pathway limits the potentially detrimental consequences of vigorous antipathogen effector T cells. A number of microorganisms that cause chronic infection appear to have exploited the PD-1:PD-L pathway to evade the immune responses and establish persistent infection. Studies in the lymphocytic choriomeningitis virus (LCMV) model of chronic viral infection were the ﬁrst to show a role for the PD-1:PD-L pathway during chronic infection (87). Viruses that cause chronic infections can render virus-speciﬁc T cells nonfunctional and thereby silence the antiviral T cell response (88). Functional dysregulation, or exhaustion, of CD8 T cells is an important reason for ineffective viral control during chronic infections and is characteristic of chronic LCMV infection in mice, as well as of HIV, HBV, HCV, and HTLV infection in humans and SIV infection in primates. There appears to be a hierarchical, progressive loss of function within the phenotype of exhausted virus-speciﬁc CD8 T cells, with cytotoxicity and IL-2 production lost ﬁrst, followed by effector cytokine production (88). PD-1 is upregulated upon activation, and a functionally signiﬁcant high level of expression is maintained by exhausted CD8 T cells in mice chronically infected with LCMV (87). In vivo administration of antibodies that blocked PD-1:PD-L1 interactions enhanced T cell responses. Most importantly, PD-1 and PD-L1 blockade led to a substantial reduction in viral burden. In persistently infected mice that lack CD4 T cell help, blockaded of this pathway restored and maintained the ability of “helpless” CD8 T cells to proliferate, secrete cytokines, kill infected cells, and decrease viral load. Taken together, these studies

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demonstrate that the PD-1:PD-L pathway contributes to T cell dysfunction and lack of viral control in chronic LCMV infection, and they suggest a novel strategy for treating chronic viral infections. Because of the critical role of PD-1:PD-L1 interactions in LCMV, there has been interest in extending this work to chronic viral infection in humans. Several groups have shown that PD-1 expression is high on HIV-speciﬁc (23, 89, 90), HBV-speciﬁc (91, 92), and HCVspeciﬁc T cells (93). PD-L1 is also upregulated on peripheral blood CD14+ monocytes and myeloid DCs in patients with chronic HBV infection (94, 95), and on CD14+ cells and T cells in HIV patients (96). Blocking PD1:PD-L interactions in vitro reverses the exhaustion of HIV-speciﬁc, HBV-speciﬁc (92), HCV-speciﬁc, and SIV-speciﬁc (97) CD8 and CD4 T cells and restores proliferation and cytokine production (23, 89, 90, 93). A mechanistic understanding of PD-1 upregulation during chronic viral infection is at an early stage. Recent work shows that the HCV core, a nucleocapsid protein, can upregulate PD1 and PD-L1 expression on healthy donor T cells and that upregulation of PD-1 is mediated by interaction of the HCV core with the complement receptor C1QBP (98). PD-1 also may serve as a useful marker on virus-speciﬁc CD8 T cells to indicate the degree of T cell exhaustion and disease severity. The level of PD-1 protein per cell is important in regulating T cell dysfunction. For example, the level and percentage of PD-1 expression on HIV-speciﬁc CD8 T cells correlates with viral load, declining CD4 counts, and decreased capacity of CD8 T cells to proliferate in response to HIV antigen in vitro. Similarly, there was a direct correlation between PD-1 expression on HIV-speciﬁc CD4 T cells and viral load (99). Long-term nonprogressors have functional HIV-speciﬁc memory CD8 T cells with markedly lower PD-1 expression, in contrast to typical progressors who express signiﬁcantly upregulated PD-1, which correlates with reduced CD4 T cell number, decreased HIV-speciﬁc effector memory CD8

T cell function, and elevated plasma viral load (100). The PD-1:PD-L pathway also may play a key role in the chronicity of bacterial infections. Helicobacter pylori causes chronic gastritis and gastroduodenal ulcers and is a risk factor for development of gastric cancer. During H. pylori infection, T cell responses are insufﬁcient to clear infection, leading to persistent infection. Gastric epithelial cells express MHC class II molecules and are thought to have important APC function during H. pylori infection. Following exposure to H. pylori in vitro or in vivo, PD-L1 also is upregulated on human gastric epithelial cells. Anti-PD-L1 blocking antibodies enhance T cell proliferation and IL-2 production in cultures of gastric epithelial cells exposed to H. pylori and CD4 T cells, suggesting that PD-L1 may play an important role in inhibiting T cell responses during H. pylori infection (101). PD-L1 is upregulated in gastric mucosal biopsies from H. pylori–infected individuals, who show a marked increase in the CD4+ CD25hi FoxP3+ cell population. Naive T cells cultured with H. pylori–exposed gastric epithelial cells can develop into functional CD4+ CD25hi FoxP3+ regulatory T cells (102). Blocking PD-L1 on in gastric epithelial cells with anti-PDL1 mAb or speciﬁc small interfering RNA (siRNA) prevented generation of the regulatory T cells, suggesting that PD-L1 may promote T cell suppression and persistent infection by controlling the dynamic between regulatory and effector T cells during H. pylori infection. Parasitic worms also have exploited the PD-1:PD-L pathway to induce macrophages with strong suppressive function. During Taenia crassiceps infection in mice, PD-L1 and PD-L2 are upregulated on activated macrophages, and a high percentage of CD4 T cells express PD-1. Blockade of PDL1, PD-L2, or PD-1 signiﬁcantly decreased suppression of in vitro T cell proliferation by macrophages from Taenia-infected mice (103). Similarly, during Schistosoma mansoni infection in mice, macrophages express high

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levels of PD-L1 and more modest levels of PD-L2. Anti-PD-L1 completely abrogated the ability of these macrophages to suppress T cell proliferation in vitro, whereas antiPD-L2 had no effect. PD-L1 expression on macrophages from infected mice declines after 12 weeks of infection, correlating with a break in T cell anergy (104). Thus, an emerging theme is that PD-L1 and PDL2 can mediate the suppressive functions of macrophages during parasite infections. PD-L1 and PD-L2 have distinct roles in the immune response to the protozoan parasite Leishmania mexicana. Cd274−/− 129Sv mice showed resistance to L. mexicana, whereas Pdcd1lg2−/− mice developed exacerbated disease with increased parasite burdens. Cd274−/− mice exhibited a diminished Th2 response, which may explain the increased resistance of Cd274−/− mice. Pdcd1lg2−/− mice exhibited a marked increase in L. mexicana–speciﬁc IgM and IgG2a, which may contribute to the exacerbated disease observed in Pdcd1lg2−/− mice. Increased parasite-speciﬁc IgG production may suppress the healing response through FcγR ligation on macrophages. Further studies are needed to determine whether these distinct outcomes of infection reﬂect impaired PD1 signaling into T cells, B cells, and/or macrophages (105). The key roles of the PD-1:PD-L pathway in reducing T cell responses during chronic viral infections propel development of strategies to manipulate the interaction of PD-1 and its ligands to restore antiviral T cell responses during chronic viral infections. This pathway may have evolved to limit immunemediated damage to the host during infection by turning off pathogen-speciﬁc T cells. We must better understand the immunoregulatory roles of this pathway to determine how to modulate it to activate effective pathogenspeciﬁc T cells while minimizing the risk of autoimmunity and immunopathology. Notably, anti-PD-L1 mAb had greater effects than anti-PD-1 mAb in the chronic LCMV model; the anti-PD-L1 mAb used in these

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studies blocks both PD-L1:PD-1 and PDL1:B7-1 interactions. The therapeutic potential of manipulating PD-1 and PD-L1 to enhance immune responses during chronic infection gives impetus to analyzing the relative effects of blocking PD-1:PD-L1 interactions versus B7-1:PD-L1 interactions during chronic infection.

THE ROLE OF PD-1:PD-L IN TRANSPLANTATION Negative costimulatory pathways play a key role in regulating rejection of transplanted allogeneic tissues (106–108), and several important lines of evidence demonstrate that PD1:PD-L1 interactions control engraftment of solid organs and graft-versus-host disease (GVHD). Redundancy among negative costimulatory pathways is clearly important for controlling alloreactive T cells, and several studies have demonstrated unique roles for the PD-1:PD-L and CTLA-4:B7 pathways. Both PD-1 and PD-L1 are signiﬁcantly upregulated on alloreactive T cells in transplant recipients (109, 110). The inducible expression of PD-L1 in tissues, such as the heart and pancreatic islets, as well as constitutive expression on other tissues such as the cornea, also may control alloreactive immune responses. Studies in heart transplant models have demonstrated that PD-L1 is upregulated on cardiac allografts as early as one day after heart transplant (111). PD-1 and PD-L2 are induced much later in the response, presumably because they are expressed primarily by inﬁltrating cells. PD-1 upregulation occurs after the onset of GVHD, and PD-L1 is extensively expressed on most cells in GVHD target organs. In heart (112), corneal (113), and skin transplant models (109), administration of PD-L1, but not PD-L2, blocking antibodies accelerated transplant rejection. The absence of a signiﬁcant effect of PD-L2 blocking antibody in transplant models suggests several possibilities. First, PD-L1 and PD-L2 may play different roles in tolerance induction by

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tolerogenic DCs. In support of this view, there is evidence of differential expression of these molecules on tolerogenic DCs in a transplant setting, but there has been no direct test of their respective roles on DCs during rejection (114, 115). In addition, the importance of PD-L1 as a ligand, either on tissues or on lymphocytes themselves, may be central to graft tolerance (63). Recent studies have investigated the role of the PD-1:PD-L pathway in acquired transplantation tolerance using a fully MHC mismatch cardiac transplant model, in which tolerance can be induced by CTLA-4-Ig (116). To examine the role of PD-L1 on the donor versus the recipient in the acquired tolerance model of CTLA-4-Ig, Cd274−/− mice were used as graft donors or recipients of cardiac allografts and given CTLA-4-Ig treatment. Cd274−/− recipients of WT cardiac allografts had accelerated graft rejection compared with WT recipients given CTLA4-Ig therapy. Cd274−/− cardiac allografts were accepted by WT recipients treated with CTLA-4-Ig, yet histologic examination showed evidence of severe chronic rejection and vasculopathy. These data indicate that PD-L1 in the graft protects from local pathology and chronic rejection, whereas PD-L1 expression in the recipient immune system is required for induction and/or maintenance of transplantation tolerance after CTLA-4-Ig therapy. These ﬁndings suggest that PD-1 agonists may be useful for promoting transplant tolerance and preventing local inﬂammation that leads to graft arterial disease (GAD) and chronic rejection. In fully MHC class I and II mismatched allografts, acute rejection occurs within days, and there is a strong induction of PD-1 expression on T cells. Administration of PDL1 blocking antibodies signiﬁcantly hastens rejection in the full mismatch cardiac transplant model (112). The role of PD-1 in regulating alloresponses also appears to depend on the strength of TCR signaling. In heart transplant models in which only MHC class I or II are mismatched, chronic rejection in the

form of GAD is induced. GAD is accompanied by mild PD-1 and PD-L1 upregulation (117), and blockade in the setting of MHC class I or II mismatch does not hasten rejection (110). PD-1 can exert its negative regulatory effect in the presence or absence of positive CD28 costimulation. When fully MHC class I and II mismatched hearts are transplanted into Cd28−/− recipients, positive signals transduced through CD28 are eliminated, which doubles the time to rejection. When PD-1 or PD-L1 blocking antibodies are used in Cd28−/− recipients, rejection is accelerated, indicating that PD-1:PD-L1 interactions are important in acute rejection in the absence of CD28 stimulation. Additionally, PD-1 and CTLA-4 effects are not redundant, as evidenced by ﬁndings in GVHD, where blocking antibodies to PD-1 augment disease induction and coadministration with anti-CTLA-4 substantially accelerates GVHD (118). One mechanism by which PD-L1 blockade may accelerate graft rejection is through prevention of T cell apoptosis. PD-L1, but not PD-1 or PD-L2, blocking antibodies induce accelerated rejection in a skin transplant model using allospeciﬁc TCR transgenic T cells (109). PD-L1 blocking antibody inhibited in vivo apoptosis of alloreactive TCR transgenic T cells, whereas a PD-1 blocking antibody did not. Similar results were obtained in a corneal transplant mode, where only PD-L1, but not PD-1 or PD-L2, blocking antibodies induced rapid rejection (113). This suggests a PD-1-independent role for PD-L1 in promoting tolerance through inducing apoptosis of alloreactive T cells. The mechanism of apoptosis induction, whether it is TCR-dependent or -independent, and whether B7-1 is involved have yet to be determined. Induction of allograft tolerance is dependent on the balance of regulatory and effector T cells, and PD-1 and PD-L1 have been implicated in controlling both regulatory T cell induction as well as increased effector T cell generation in transplant models. PD-L1

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blockade can inhibit allospeciﬁc effector responses in vitro (119), as well as inhibit the induction of allogeneic regulatory T cells by vascular endothelium (73). The acceleration of graft rejection observed after PD-L1 neutralizing antibody administration may depend on regulatory T cells. Administration of anti-PD-L1 on the day of transplantation or on day 60 after transplantation led to cardiac graft rejection in CTLA-4-Ig-treated recipients, with an increase in lymphocyte production of inﬂammatory cytokines and signiﬁcant decreases in regulatory T cell numbers within the allograft. Similar studies in both a skin allograft and a GVHD model have also suggested that PD-1:PD-L1 interactions on regulatory and effector T cells may be important (109, 119). No acceleration of rejection was observed when anti-PD-L1 treatment was given in the absence of CD25+ T cells. The loss of PD-1 and PD-L1 through genetic deletion or antibody blockade also has been demonstrated to increase effector cell generation and cytokine production (110, 118, 119). These studies suggest that PDL1 may regulate transplantation tolerance by controlling the balance between regulatory and pathogenic effector cells, limiting the expansion of alloreactive effector cells in the periphery.

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ALLERGY AND ASTHMA Asthma and allergic responses constitute inappropriately vigorous immune responses to innocuous stimuli. B7 family members are implicated in the regulation of allergy and asthma and are involved in the priming events, helper T cell differentiation, and effector function that underlie the pathology of these diseases. Although a picture of the deﬁnitive role of PD-1 and its ligands in asthma and allergy has yet to emerge, most data point to PD-L2 as a critical molecule in these diseases. PD-L2 is abundantly expressed in DCs in the lung in a mouse model of asthma. In a mouse ovalbumin-induced allergic asthma model, PD-L2 treatment during challenge 692

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but not sensitization resulted in an increase in airway hyperresponsiveness and in Th2 cytokine production (120). There was no effect of either PD-1 or PD-L1 mAbs in either the priming or effector phase. These results suggest that the effector responses of T cells in asthma may be inﬂuenced by PD-L2, but not PD-1. Use of sHIgM12 mAb, which was previously described as inducing reverse signaling through PD-L2, in a mouse model of allergic asthma completely blocked the development of airway inﬂammatory disease, and sHIgM12-treated DCs could mediate these same protective effects when transferred into hyperimmune mice (48, 49). Finally, treatment with PD-L2-Fc prior to sensitization and challenge exacerbated T cell proliferation and cytokine production and increased eosinophilia in vivo but had the opposite effect in vitro, where it inhibited T cell proliferation and cytokine production (121). PDL2 blockade, but not PD-1 or PD-L1 blockade, diminished eosinophil migration to the conjunctiva in a mouse model of allergic conjunctivitis (122). Among the studies that have tested the function of PD-1 and its ligands in allergy and asthma, most have utilized different reagents. Further work is required to understand the role of PD-1 and its ligands in allergic diseases, yet a consistent ﬁnding has been the implication of PD-L2 in these processes.

TUMOR IMMUNITY Tumors express antigens that can be recognized by host T cells, but immunologic clearance of tumors is rare. Part of this failure is due to immune suppression by the tumor microenvironment. PD-L1 expression on many tumors is a component of this suppressive milieu and may act in concert with other immunosuppressive signals. PDL1 expression has been shown in situ on a wide variety of solid tumors including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck (55, 123–131).

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Immunohistochemical staining of normal and malignant breast tissue is contrasted in Figure 4. In addition, PD-1 expression is upregulated on tumor inﬁltrating lymphocytes, and this may also contribute to tumor immunosuppression (58). In ovarian cancer, PD-L1 expression is inversely correlated with intraepithelial, but not stromal, inﬁltrating CD8 T cells, suggesting that PD-L1 inhibits the intratumor migration of CD8 T cells (124). Expression on tumor cell lines is generally lower in vitro, and expression is increased upon adoptive transfer into animals. Translation of PD-L1 mRNA is enhanced by loss of PTEN and the ensuing activation of Akt, a common event in tumorigenesis (29). Most importantly, studies relating PD-L1 expression on tumors to disease outcome show that PDL1 expression strongly correlates with unfavorable prognosis in kidney, ovarian, bladder, breast, gastric, and pancreatic cancer but not small cell lung cancer (124, 126–131). In addition, these studies suggest that higher levels of PD-L1 expression on tumors may facilitate advancement of tumor stage and invasion into deeper tissue structures. The PD-1 pathway may also play a role in hematologic malignancies. PD-1 or PDL1 are rarely expressed on B cell malignancies (55), but PD-L2 has been identiﬁed by microarray analysis as being highly expressed in mantle cell lymphomas (132). PDL1 is expressed on multiple myeloma cells but not on normal plasma cells. T cell expansion in response to myeloma cells is enhanced in vitro by PD-L1 blockade (28). PD-L1 is expressed on some primary T cell lymphomas, particularly anaplastic large cell T lymphomas (55). PD-1 is highly expressed on the T cells of angioimmunoblastic lymphomas, and PD-L1 is expressed on the associated follicular dendritic cell network (133). In nodular lymphocyte-predominant Hodgkin lymphoma, the T cells associated with lymphocytic and/or histiocytic (L&H) cells express PD-1. Microarray analysis using a readout of genes induced by PD-1 ligation suggests that tumor-associated T cells are re-

Figure 4 PD-L1 is upregulated on breast tumor, but not normal, cells Immunohistochemical staining (brown) of a breast ductal carcinoma showing high expression of PD-L1 on neoplastic tissue (thick arrow) and low expression on adjacent normal tissue (thin arrow) (ﬁgure kindly provided by David Dorfman, Brigham and Women’s Hospital). Note that lymphocytes are more abundant in normal tissue areas than in cancer cell nests.

sponding to PD-1 signals in situ in Hodgkin lymphoma (134). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T cell leukemia and lymphoma (135). These tumor cells are hyporesponsive to TCR signals, and PD-1 blockade increased their expression of TNF-α, but not IFN-γ. Studies in animal models demonstrate that PD-L1 on tumors inhibits T cell activation and lysis of tumor cells and in some cases leads to increased tumor-speciﬁc T cell death (123, 136). Treatment with anti-PD-L1 or injection of tumor cells into Pdcd1−/− mice augments antitumor responses, as measured by cytotoxicity and cytokine production (136– 138). Treatment with anti-PD-L1 in vivo delays, but does not halt, tumorigenesis of PDL1-expressing mouse myeloma cell lines (137) PD-L1 blockade can improve the outcome of immunotherapy. PD-L1 expression on the immunogenic tumor P815 causes resistance to immunotherapy, but blockade with antiPD-L1 restores the response to anti-CD137 therapeutic mAb (136). Treatment with antiPD-L1 mAb enhances T cell immunotherapy, and administration of anti-PD-L1 with activated T cells augments rejection of a

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PD-L1-expressing squamous cell carcinoma (125). Tumor-associated APCs can also utilize the PD-1:PD-L pathway to control antitumor T cell responses. PD-L1 expression on a population of tumor-associated myeloid DCs is upregulated by tumor environmental factors, and PD-L1 mAb blockade enhanced DC-mediated T cell activation (139). Plasmacytoid DCs in the tumor-draining lymph node of B16 melanoma express IDO, which strongly activates the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells required cell contact with IDO-expressing DCs and was abrogated by PD-L blockade (140). A fully human PD-1 mAb has been developed and is in Phase 1 clinical trials for cancer. Preclinical studies show that PD-1 is expressed on tumor-speciﬁc human T cells following vaccination with tumor peptide antigen. Blockade of PD-1 with this mAb during in vitro stimulation with melanoma peptide increased the numbers and effector activity of tumor-speciﬁc human T cells (141). Both Th1 and Th2 cytokine production were increased. PD-1 blockade did not change the percentage of apoptotic antigen-speciﬁc human T cells, indicating that the increase in number was due to increased proliferation, not decreased death. In mice, a triple treatment therapy of anti-PD-L1 blockade, depletion of CD4 T cells (primarily regulatory T cells), and irradiated tumor cell vaccination induced complete elimination of large established renal cancer cell (RENCA) tumors with long-lasting tumor-speciﬁc immunity (142), further suggesting that this pathway is a promising target for therapeutic intervention.

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PD-1 AND IMMUNOPATHOLOGY A number of studies point to an important role for PD-L1 in limiting immunopathology. Following infection with LCMV clone 13, WT mice develop a chronic infection, whereas Cd274−/− mice die (87). Blockade or elimination of PD-L1 in mouse models of 694

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autoimmunity leads to exacerbated autoimmunity associated with severe inﬂammation and tissue damage (61, 63–66, 69). Bone marrow chimera studies point to an important role for PD-L1 on non–bone marrow–derived cells in limiting effector T cell responses and immunopathology. The expression of PD-L1 on vascular endothelial cells has led to the hypothesis that PD-L1 on endothelial cells may regulate the activation of T cells that contact the vessel wall, the extravasation of T cells into tissue, and/or limit detrimental consequences of immunopathology. Blockade of PD-L1 on vascular endothelial cells enhances IFN-γ production and cytolytic activity of CD8 T cells in vitro (143). Cd274−/− Pdcd1lg2−/− mice developed severely increased atherosclerotic lesion burden, suggesting that PD-L1 also may play a signiﬁcant role in inﬂammatory diseases in which vascular endothelium and T cells are important for pathogenesis (144). Fibroblastic reticular cells (FRC) express high levels of PD-L1, which can be upregulated during LCMV clone 13 infection (145). Elegant confocal, electron, and intravital microscopy studies demonstrate that the FRC network can regulate T cell access to the paracortex within the lymph node and regulate movement within the LN. In vivo administration of blocking PD-L1 mAb during LCMV clone 13 infection induced signiﬁcant CD8 T cell–mediated damage to the splenic stroma, demonstrating an essential role for this pathway in minimizing virus-induced immunopathology. These studies suggest that PD-L1 on FRC may contribute to viral persistence during chronic infection. Further studies are needed to test whether expression of PD-L1 by FRC may contribute to the inﬂuence of FRC on T cell trafﬁcking.

CONCLUDING REMARKS PD-1:PD-L interactions exert a vital and diverse range of immunoregulatory roles in T cell activation, tolerance, and immune-mediated tissue damage. How do

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PD-1 and its ligands exert their inhibitory effects? One of their most signiﬁcant functions is controlling potentially pathogenic effector T cells, yet PD-1:PD-L can also dampen early activation events when naive T cells encounter antigen in lymph nodes. PD-1 and PD-L1 are also expressed on regulatory T cells and may control their suppression of effector T cells. Recent studies indicate that PD-L1 and PD-L2 can signal bidirectionally by engaging PD-1 on T cells and by delivering signals into PD-L-expressing cells. These bidirectional interactions between PD-1 and its ligands, along with the identiﬁcation of B7-1 as an additional binding partner for PD-L1, may help explain the seemingly contradictory results seen with reagents developed to manipulate this pathway. The discovery of B7-1:PD-L1 interactions also reveals additional ways by which PD-L1 exerts its inhibitory functions.

Because both PD-L1 and B7-1 are expressed on T cells, B cells, DCs, and macrophages, there is the potential for bidirectional interactions between B7-1 and PDL1 on these cell types. Emerging evidence demonstrates a unique and critical role for PD-L1 on nonhematopoetic cells for mediating tissue tolerance as well as protecting tissues from the detrimental consequences of overaggressive effector responses. Microbes and tumors appear to have exploited this pathway to evade eradication by the immune system. These distinctive functions provide therapeutic opportunities for developing PD1/PD-L antagonists to boost antimicrobial and antitumor immunity as well as agonists to control pathogenic T cells in autoimmune diseases and graft rejection. The fundamental and therapeutic importance of the PD-1:PDL pathway gives impetus to further investigation of its functions.

DISCLOSURE STATEMENT A.H.S. and G.J.F. have patents on PD-1 ligands and grants on PD-1 and PD-1 ligands.

ACKNOWLEDGMENTS This work was supported by grants from National Institutes of Health and the Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative (to A.H.S. and G.J.F.) and the National Multiple Sclerosis Society (to A.H.S.). Because of space restrictions, we were able to cite only a fraction of the relevant literature and apologize to colleagues whose contributions may not be appropriately acknowledged in this review.

LITERATURE CITED 1. Lafferty KJ, Cunningham AJ. 1975. A new analysis of allogeneic interactions. Aust. J. Exp. Biol. Med. Sci. 53:27–42 2. Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541–47 3. Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, et al. 1995. Lymphoproliferative disorders with early lethality in mice deﬁcient in Ctla-4. Science 270:985–88 4. Ishida Y, Agata Y, Shibahara K, Honjo T. 1992. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 11:3887–95 www.annualreviews.org • PD-1 and Its Ligands in Tolerance and Immunity

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122. Fukushima A, Yamaguchi T, Azuma M, Yagita H, Ueno H. 2006. Involvement of programmed death-ligand 2 (PD-L2) in the development of experimental allergic conjunctivitis in mice. Br. J. Ophthalmol. 90:1040–45 123. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, et al. 2002. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med. 8:793–800 124. Hamanishi J, Mandai M, Iwasaki M, Okazaki T, Tanaka Y, et al. 2007. Programmed cell death 1 ligand 1 and tumor-inﬁltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer. Proc. Natl. Acad. Sci. USA 104:3360–65 125. Strome SE, Dong H, Tamura H, Voss SG, Flies DB, et al. 2003. B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma. Cancer Res. 63:6501– 5 126. Inman BA, Sebo TJ, Frigola X, Dong H, Bergstralh EJ, et al. 2007. PD-L1 (B7-H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression. Cancer 109:1499–505 127. Konishi J, Yamazaki K, Azuma M, Kinoshita I, Dosaka-Akita H, Nishimura M. 2004. B7-H1 expression on nonsmall cell lung cancer cells and its relationship with tumorinﬁltrating lymphocytes and their PD-1 expression. Clin. Cancer Res. 10:5094–100 128. Nakanishi J, Wada Y, Matsumoto K, Azuma M, Kikuchi K, Ueda S. 2007. Overexpression of B7-H1 (PD-L1) signiﬁcantly associates with tumor grade and postoperative prognosis in human urothelial cancers. Cancer Immunol. Immunother. 56:1173–82 129. Nomi T, Sho M, Akahori T, Hamada K, Kubo A, et al. 2007. Clinical signiﬁcance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer. Clin. Cancer Res. 13:2151–57 130. Thompson RH, Gillett MD, Cheville JC, Lohse CM, Dong H, et al. 2004. Costimulatory B7-H1 in renal cell carcinoma patients: indicator of tumor aggressiveness and potential therapeutic target. Proc. Natl. Acad. Sci. USA 101:17174–79 131. Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. 2006. Immunohistochemical localization of programmed death-1 ligand-1 (PD-L1) in gastric carcinoma and its clinical signiﬁcance. Acta Histochem. 108:19–24 132. Rosenwald A, Wright G, Leroy K, Yu X, Gaulard RD, et al. 2003. Molecular diagnosis of primary mediastinal B cell lymphoma identiﬁes a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J. Exp. Med. 198:851–62 133. Dorfman DM, Brown JA, Shahsafaei A, Freeman GJ. 2006. Programmed death-1 (PD-1) is a marker of germinal center-associated T cells and angioimmunoblastic T-cell lymphoma. Am. J. Surg. Pathol. 30:802–10 134. Chemnitz JM, Eggle D, Driesen J, Classen S, Riley JL, et al. 2007. RNA-ﬁngerprints provide direct evidence for the inhibitory role of TGFβ and PD-1 on CD4+ T cells in Hodgkin’s lymphoma. Blood 110:3226–33 135. Shimauchi T, Kabashima K, Nakashima D, Sugita K, Yamada Y, et al. 2007. Augmented expression of programmed death-1 in both neoplastic and non-neoplastic CD4+ T-cells in adult T-cell leukemia/lymphoma. Int. J. Cancer 121:2585–90 136. Hirano F, Kaneko K, Tamura H, Dong H, Wang S, et al. 2005. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity. Cancer Res. 65:1089–96 137. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. 2002. Involvement of PDL1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc. Natl. Acad. Sci. USA 99:12293–97 www.annualreviews.org • PD-1 and Its Ligands in Tolerance and Immunity

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138. Iwai Y, Terawaki S, Honjo T. 2005. PD-1 blockade inhibits hematogenous spread of poorly immunogenic tumor cells by enhanced recruitment of effector T cells. Int. Immunol. 17:133–44 139. Curiel TJ, Wei S, Dong H, Alvarez X, Cheng P, et al. 2003. Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity. Nat. Med. 9:562–67 140. Sharma MD, Baban B, Chandler P, Hou DY, Singh N, et al. 2007. Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase. J. Clin. Invest. 117:2570–82 141. Wong RM, Scotland RR, Lau RL, Wang C, Korman AJ, et al. 2007. Programmed death1 blockade enhances expansion and functional capacity of human melanoma antigenspeciﬁc CTLs. Int. Immunol. 19:1223–34 142. Webster WS, Thompson RH, Harris KJ, Frigola X, Kuntz S, et al. 2007. Targeting molecular and cellular inhibitory mechanisms for improvement of antitumor memory responses reactivated by tumor cell vaccine. J. Immunol. 179:2860–69 143. Rodig N, Ryan T, Allen JA, Pang H, Grabie N, et al. 2003. Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis. Eur. J. Immunol. 33:3117–26 144. Gotsman I, Grabie N, Dacosta R, Sukhova G, Sharpe A, Lichtman AH. 2007. Proatherogenic immune responses are regulated by the PD-1/PD-L pathway in mice. J. Clin. Invest. 117:2974–82 145. Mueller SN, Matloubian M, Clemens DM, Sharpe AH, Freeman GJ, et al. 2007. Viral targeting of ﬁbroblastic reticular cells contributes to immunosuppression and persistence during chronic infection. Proc. Natl. Acad. Sci. USA 104:15430–35 146. Iwai Y, Okazaki T, Nishimura H, Kawasaki A, Yagita H, Honjo T. 2002. Microanatomical localization of PD-1 in human tonsils. Immunol. Lett. 83:215–20 147. Tsuda M, Matsumoto K, Inoue H, Matsumura M, Nakano T, et al. 2005. Expression of B7-H1 and B7-DC on the airway epithelium is enhanced by double-stranded RNA. Biochem. Biophys. Res. Commun. 330:263–70 148. Mataki N, Kikuchi K, Kawai T, Higashiyama M, Okada Y, et al. 2007. Expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases. Am. J. Gastroenterol. 102:302–12 149. Mazanet MM, Hughes CC. 2002. B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis. J. Immunol. 169:3581–88 150. Nakae S, Suto H, Iikura M, Kakurai M, Sedgwick JD, et al. 2006. Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF. J. Immunol. 176:2238–48 151. Augello A, Tasso R, Negrini SM, Amateis A, Indiveri F, et al. 2005. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway. Eur. J. Immunol. 35:1482–90 152. English K, Barry FP, Field-Corbett CP, Mahon BP. 2007. IFN-γ and TNF-α differentially regulate immunomodulation by murine mesenchymal stem cells. Immunol. Lett. 110:91–100 153. Genomics Institute of the Novartis Research Foundation. Online database. GNF SymAtlas v1.2.4. http://expression.gnf.org

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389

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Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611; email: [email protected], [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2008. 26:705–39

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

mast cells, hypersensitivity responses, adaptive immunity, EAE, MS

This article’s doi: 10.1146/annurev.immunol.26.021607.090320 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0705$20.00

Abstract There are many parallels between allergic and autoimmune responses. Both are considered hypersensitivity responses: pathologies that are elicited by an exuberant reaction to antigens that do not pose any inherent danger to the organism. Although mast cells have long been recognized as central players in allergy, only recently has their role in autoimmunity become apparent. Because of the commonalities of these responses, much of what we have learned about the underlying mast cell–dependent mechanisms of inﬂammatory damage in allergy and asthma can be used to understand autoimmunity. Here we review mast cell biology in the context of autoimmune disease. We discuss the huge diversity in mast cell responses that can exert either proinﬂammatory or antiinﬂammatory activity. We also consider the myriad factors that cause one response to predominate over another in a particular immune setting.

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INTRODUCTION

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Hypersensitivity reactions are a diverse group of pathologic immune responses. The eliciting antigens are not inherently harmful; rather, it is the overzealous immune response that causes the pathology (reviewed in 1). These reactions are classiﬁed into Types I– IV on the basis of the major effector mechanism employed. The term hypersensitivity typically brings to mind the immediate-type allergic reactions (Type I) that include hay fever, atopic dermatitis, venom sensitivity, and systemic anaphylaxis. The mast cell is a central ﬁgure in these responses. When activated by cross-linking of the high-afﬁnity IgE receptor upon allergen encounter, these cells release a plethora of preformed mediators present in granules (hence an immediate-type response) and initiate new production of molecules that can act later to sustain inﬂammation. Other classes of hypersensitivity responses have been considered mast cell independent and result either from the direct interaction of IgG or IgM antibody with a cellular antigen or hapten (Type II) or from immune complex deposition (Type III) or involve CD4+ and CD8+ T cells (Type IV, delayed-type hypersensitivity). However, the recent evidence that mast cells are activated by complement and can modulate both B and T cell responses suggests that Types II, III, and IV hypersensitivities also have mast cell–dependent components. The location of mast cells, the numerous FcεRI-independent activation pathways, and the impressive numbers of cell surface and secreted immunomodulatory molecules expressed by mast cells poise these cells to exert an inﬂuence in most, if not all, immune responses (Figure 1). Indeed, mast cells are implicated in several of the pathologic responses to self-derived antigens associated with autoimmune diseases, many of which fall under the umbrella of hypersensitivity reactions. Our understanding of mast cell biology has undergone a revolution in recent years, and several excellent reviews have covered the newly discovered roles of these cells in innate

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and adaptive responses to infection and allergic disease (2–4). In this review, we focus on what is known about mast cells in hypersensitive autoimmune responses, with an emphasis on how mast cells can affect the adaptive immune system. In the ﬁrst part of this discussion, we review key features of mast cells and describe the range of autoimmune diseases in which mast cells have been implicated. The deﬁned and hypothesized mechanisms of mast cell action are then discussed, including the seemingly paradoxical antiinﬂammatory role of these cells in promoting tolerance. We ﬁnish with a discussion of the genetically determined heterogeneity of mast cells and the implications for disease susceptibility.

WHAT ARE MAST CELLS? Mast cells are members of the innate immune system and are often considered ﬁrst-line responders to immunological insults because of their prevalence in areas highly exposed to the external environment (4). Mature mast cells can be identiﬁed in most tissues as relatively large cells that express high levels of c-kit, the receptor for stem cell factor (SCF), and FcεRI, the high-afﬁnity IgE receptor. Microscopically, these cells are easy to identify with toluidine blue or alcian blue dyes. Despite their relatively immobile nature (mast cells are generally found “ﬁxed” in tissues and do not circulate through the blood), mast cells exert profound and far-reaching effects. They are normally found throughout the skin, mucosa of the genitourinary, respiratory, and gastrointestinal tracts, as well as in most other vascularized tissues of adult mammals, including the lymphoid organs (5). Mast cells are also present in sites directly adjacent to blood vessels and peripheral nerves, as well as in the central nervous system (CNS) where they are most numerous in the leptomeninges, thalamus, and hypothalamus and along nerves in the dura mater of the spinal cord (6). Although some pathologic conditions are associated with increased numbers of mast cells,

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Newly synthesized mediators IL-1, 2, 3, 4, 5, 6, 7, 8, 10 12, 13, 15, 18, 21, 23 Prostaglandins Leukotrienes VEGF, NGF, FGF PAF TGF-β CCL3 CCL2 IFN-α, -β, -γ CRH TSLP CXCL10 TNF

Preformed mediators

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Histamine Proteases Peroxidase Serotonin Heparin IL-4 TNF GM-CSF

T and B cell ligands Receptor-binding agonists IgE + Ag Monomeric IgE IgG + Ag lg light chain Complement hormones Neuropeptides Cytokines Microbial products

Physical activators Heat Cold Pressure

B7 PD-L1 OX40L CD30L CD40L CCL19 4-1BB

Figure 1 Activators and potential responses of mast cells. The versatility of the mast cell lies in its ability to be activated by a variety of stimuli, both receptor-mediated and physical, and produce speciﬁc responses, ranging from the immediate release of preformed mediators to the increased expression of co-stimulatory molecules to the de novo production of cytokines and chemokines.

this expansion might appear minimal when compared to clonally expanding T and B effector cells. Yet, collectively, mast cells comprise a substantial population. It has been estimated that if all human tissue mast cells were amassed together in a single organ, it would equal the size of a normal spleen (D. Metcalfe, personal communication).

tile and act both as effector cells that amplify inﬂammation, as well as regulatory cells that suppress responses. This versatility is reﬂected in the numerous IgE-independent activation pathways that intersect to modulate the quality and magnitude of the mast cell response.

IgE-Dependent Mast Cell Activation MAST CELL ACTIVATION The widely held idea that mast cells evolved primarily to respond vigorously to certain parasites and allergens is a somewhat simplistic notion. Mast cells are extremely versa-

FcεRI-mediated signaling pathways are among the best characterized in mast cells (for review, see 4, 5). Recent studies have also demonstrated the ability of monomeric IgE to activate mast cells (7). Akin to the

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two-signal model of T cell activation, antigen signaling through the FcεRI is often modiﬁed by co-inhibitory or co-stimulatory signals in mast cells (reviewed in 8). For example, concurrent administration of αCD28 enhances TNF (tumor necrosis factor) secretion by bone marrow–derived mast cells (BMMC) activated by IgE + Ag (9). Similarly, simultaneous ligation of the IgE-FcεRI complex and 4-1BB (CD137), a co-stimulatory molecule and member of the TNFR family, induces increased BMMC cytokine production and secretion (10). Some Toll-like receptor (TLR) ligands also synergize with antigen to potentiate cytokine secretion by IgE-FcεRI activation in mast cells (11). Conversely, the IgG receptor FcγRIIB (12), leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4) (formerly gp49B1) (13) and platelet endothelial cell adhesion molecule-1 (PECAM-1) (14) all utilize the Ig-immunoreceptor tyrosine-based inhibitory motif (ITIM) and suppress FcεRIIgE-mediated activation. Mice with targeted disruptions in genes encoding these receptors demonstrate enhanced cytokine release and hyper-responsiveness in IgE-dependent reactions. Given the paradigm of accessory signals in T cell activation and the fact that mast cells express other inhibitory signaling molecules such as B7-H1 (PD-L1) (15) and coactivating molecules such as CD80, CD86, and CD40L (16, 17), we are probably only beginning to understand a more complicated but extremely important picture.

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IgE-Independent Modes of Mast Cell Activation There are limited examples of IgE-dependent mechanisms of mast cell activation that are important in autoimmunity. These include bullous pemphigoid (BP) and Graves’ ophthalmology (18, 19). Of more relevance to this discussion are the IgE-independent modes of activation that include other immunoglobulins, microbial antigens, complement, hormones, peptides, and cytokines. 708

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Both murine and human mast cells express the low-afﬁnity IgG receptors FcγRII and FcγRIII (5) that when cross-linked by IgGantigen complexes are potent inducers of degranulation. Human mast cells also express the high-afﬁnity IgG receptor FcγRI, which is induced after culture with IFN-γ (20). The expression of IgG receptors on mast cells is of particular importance when we consider that autoimmune diseases classiﬁed as Type II and III hypersensitivity reactions are IgGmediated. Ig-free light chains (IgLCs) can also function to elicit antigen-speciﬁc mast cell– dependent hypersensitivity reactions in mice. Cross-linking of mast cell surface proteins with IgLC results in degranulation and mediator release in vitro and in vivo and induces vascular permeability and plasma extravasation into tissue (21). Mast cells express several complement receptors including C3aR, C5aR CR2, CR4, and the recently described collectin/C1qR (2, 22). The importance of some of these receptors was shown in a cecal ligation and puncture model of septic peritonitis in which complement-mediated mast cell activation is necessary for bacterial clearance (23, 24). There is signiﬁcant heterogeneity in the expression of receptors on distinct mast cell subsets as well as differences in the outcome of complement receptor engagement (reviewed in 25). Activated complement contributes to the tissue injury associated with many autoimmune diseases, especially those classiﬁed as Type II and Type III hypersensitivities. It has also been implicated in Type IV autoimmune diseases such as the murine model of multiple sclerosis (26, 27). Pattern recognition receptors (PRRs) are a class of receptors that recognize broad pathogen-speciﬁc motifs, the best studied of which are the TLRs (28). Mast cells from a variety of sources express TLRs 1–4, 6, 7, and 9, the ligands for which include doublestranded viral RNA, peptidoglycan (PGN) from Gram+ bacteria, and lipopolysaccharide (LPS) from Gram− bacteria (2, 29). Mast cell participation in the earliest events in

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immune responses is elicited through these receptors via expression of mediators and costimulatory molecules. Mast cells also express at least one isoform of the nucleotide-binding oligomerization domain proteins, a family of intracellular receptors that recognize bacterial components or their degradation products; a recent study demonstrates that nucleotidebinding oligomerization domain-1 mediates PGN activation of mast cells (30). Other FcεRI-independent entities can inﬂuence mast cell activation outcomes. These include hormones and cytokines, as well as a variety of peptides (5). For example, estrogens, including environmental estrogenic pollutants, can induce mast cell degranulation in vitro (31), whereas progesterone appears to inhibit histamine secretion (32). Neuropeptides, such as substance P (SP), calcitonin gene-related peptide (CGRP), somatostatin, vasoactive intestinal peptide (VIP), and neurotensin, can also induce degranulation of mast cells (33).

Mediator Release from Mast Cells: Degranulation versus New Synthesis Mast cell mediator release is phasic in nature (reviewed in 34). Immediate (early phase) release of preformed mediators during degranulation regulates initial events in an immune response including those exhibited in Type I hypersensitivity reactions. This same signal also generates delayed mediator production. The late-phase response involves the de novo synthesis and release of other mediators including cytokines, lipid mediators, and growth factors (1) (Figure 1). Mast cells can also engage in piecemeal degranulation (or intragranular activation), which involves vesicular transport of the contents of selective secretory granules to the cellular surface (4). For instance, serotonin, rather than histamine, is released in response to IgE-Ag or Compound 48/80 when mast cells are stimulated in the presence of amitriptyline, an inhibitor of serotonin and noradrenaline reuptake (35). Mast cells

MAST CELLS AND B CELLS Little is known about mast cell–B cell interactions in vivo. A major pathway of mast cell activation is through antibody receptors, and mast cells express a number of B cell–modulating molecules. Thus, a functional connection between these cell types is highly likely and important to study in the context of autoimmunity. There are some in vitro data to support this idea. Co-culture with even a small number of unstimulated BMMCs has been shown to activate B cells, an effect that is blocked by treatment with mast cell stabilizers (191, 192). Mast cells also induce synthesis of IgE from B cells through expression of CD40L in the presence of exogenous IL-4 (17). B cells express histamine receptors (H1), and in the presence of histamine demonstrate an increased proliferative response to B cell receptor cross-linking (193), although H1 receptor–deﬁcient B cells appear functional and can produce immunoglobulins IgM, IgE, IgG1, IgG3, and IgG2b (158).

can also selectively release mediators without degranulation (36).

HOW MAST CELLS ARE IMPLICATED IN DISEASE Several indices have been used to implicate mast cells in autoimmune diseases. Obviously, in human disease one must rely largely on correlative evidence. The presence of increased numbers of mast cells, mast cell degranulation, or mast cell expression of implicated molecules at local affected sites in diseased versus normal individuals is supportive, but not deﬁnitive, evidence of mast cell inﬂuence. Among many similar correlative ﬁndings in murine models of autoimmunity, perhaps the most deﬁnitive proof derives from a comparison of disease incidence and severity in mast cell–deﬁcient mice versus their wild-type counterparts. The two most common strains of mast cell–deﬁcient mice currently in use are the (WB X C57BL/6)F1-KitW /KitWv (termed W/Wv ) and KitWsh /KitWsh mice (termed W-sash, C57BL/6 background) (37–39). Both strains carry mutations in the c-kit receptor,

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+/+ 104

IL-3 SCF

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Bone marrow stem cells

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3

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Analyze by flow cytometry

100 0 10

101

102

103

104

FCεRI

Local or IV reconstitution

W/W V

Confirm reconstitution Evaluate disease

Figure 2 Mast cell reconstitution. Mast cell “knock-in” experiments can establish if mast cells contribute to disease. Mast cell populations are typically derived from bone marrow cultured with IL-3+/− SCF. Reconstitution occurs within 4–8 weeks via intravenous or local transfer of these bone marrow–derived mast cells (BMMCs). There are limitations to this approach owing to the inability to efﬁciently reconstitute all tissues in which mast cells normally reside. The CNS and skin are two organs in which mast cells are not typically restored within this time frame (104). To determine whether a speciﬁc mast cell–expressed mediator is essential for a disease phenotype, BMMCs lacking the ability to express the molecule in question can be transferred, and their ability to restore wild-type disease can be measured.

which results in profound deﬁcits in their mast cell populations and can be locally or systemically reconstituted with BMMC (Figure 2). Although the mice, particularly the W/Wv mice, have other mild phenotypic abnormalities, they remain the gold standard for demonstrating mast cell involvement in disease.

MAST CELLS AND THE AUTOIMMUNE HYPERSENSITIVITY RESPONSE Autoimmune diseases result from the coincident loss of tolerance to self in the face of an 710

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unregulated proinﬂammatory response (40). It is our contention that mast cells play a role in both these processes. However, much of the evidence to support this idea is merely correlative and is based, for example, on the presence of activated mast cells in regions of inﬂammation or autoimmune destruction or on the actions of mast cells in other disease processes. To understand fully the extent of mast cell involvement in autoimmunity will require suitable animal models for evaluating the role of mast cells in vivo. Indeed, until such models were available to study multiple sclerosis and rheumatoid arthritis, a role for mast

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cells was considered to be unlikely. In the following sections, we review those autoimmune diseases for which there are considerable and convincing data supporting a role for mast cell involvement. These and other examples of autoimmune diseases in which mast cells are weakly implicated are included in Table 1.

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Mast Cells in Type I Hypersensitivities Much of what we know about mast cells comes from studies of their role in Type I immediate hypersensitivity reactions. As other reviews cover this topic in depth (55), we deal with it only brieﬂy here. The manifestations of Type I reactions are evident within minutes and are dependent on IgE interaction with mast cells. During initial antigen (allergen) sensitization, certain individuals develop an IgE-dominated antibody response for reasons not yet fully understood. Subsequent re-exposure leads to immediate mast cell degranulation and release of mediators that contribute to the early symptoms in allergic and anaphylactic responses including excessive vascular permeability, local swelling, and itch. Late-phase responses can be elicited hours to days later at the site of antigen challenge and are characterized by the inﬂux of inﬂammatory cells to the affected site, tissue remodeling, bronchial hyperreactivity and mucus overproduction in the airways, and local cutaneous swelling.

Mast Cells and Type II Hypersensitivities Type II hypersensitivities are elicited by the interaction of IgG or IgM antibody with a cell surface antigen, followed by the binding of complement and, often, complementmediated cell lysis. Mast cells are well equipped to participate in Type II reactions both directly and indirectly. As described, mast cell activation through IgG or complement receptors causes inﬂammatory mediator release. Mast cells also inﬂuence T cell responses and may be necessary for full T cell

activation that generates T cell help for B cell isotype switching. Among the Type II diseases in which mast cells have a demonstrated role is bullous pemphigoid (BP), a chronic subepidermal blistering skin disease characterized by the presence of IgG autoantibodies to hemidesmosomal antigens BP20 and BP180. Passive transfer of BP20- or BP180-speciﬁc IgG leads to disease in neonatal BALB/c mice and reproduces the key clinical features of human BP, including complement deposition at the junction of the dermis and epidermis, dermal inﬂammation, and subepidermal blistering (56). Mast cell degranulation occurs within 60 min of antibody transfer and elicits neutrophilic inﬁltration and subsequent blistering of the skin in this mouse model (57). Mast cell–deﬁcient or wild-type mice treated with an inhibitor of mast cell degranulation fail to develop disease, but local engraftment of W/Wv mice with BMMCs restores the BP phenotype. Mice deﬁcient for C4 are also resistant to BP but develop disease when pretreated with the mast cell degranulating agent 48/80 (41). In BP, mast cells triggered by complement activation appear to be a crucial source of the potent neutrophil chemoattractant CXCL8 (41, 57). The implication of mast cell–produced CXCL8 is particularly interesting, as BP lesions are often precipitated by exposure to UV light (58), and UV-B irradiation has been shown to selectively and speciﬁcally increase CXCL8 release from mast cells in vitro (59). Human BP is associated with elevated serum levels of IgE autoantibodies and the presence of eosinophils in blisters (which are also recruited in latephase Type I responses) (60, 61). In addition, passive transfer of IgE from BP patients into athymic mice elicits the development of erythematous plaques similar to those observed in BP (18). Taken together, these data support a role for IgE acting through mast cells in human BP. In Graves’ disease (GD), also considered a Type II disease, autoantibodies function in a distinctive manner. In GD, binding of autoantibodies to the thyroid-stimulating hormone

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Mast cells and autoimmune hypersensitivity diseases

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Disease (type of HS)

Proposed self targets

Major animal models

Strongest evidence for mast cell (MC) involvement

References

Graves’ ophthalmology (II or I)

TSH receptor (TSHR)

Transfer of TSHRprimed splenocytes

MC inﬁltrates in orbital tissues in humans and mice

19

Bullous pemphigoid (II or I)

Hemi-desmosomal antigens BP20 and BP180

Transfer of anti-BP20 and -BP180 IgG

MC-deﬁcient mice do not develop disease

41

Pemphigus vulgaris (II)

Desmogleins and desmoplakins

—

Increased MC numbers in human lesions

42

Rheumatoid arthritis (III)

Fc portion of IgG (rheumatoid factor)

Transfer of K/BxN serum

MC blockers used to treat human disease

43

MC-deﬁcient mice not susceptible to disease

44

SLE (III)

Nuclear proteins

Pristane injection

Correlative evidence in human disease

Multiple sclerosis (IV)

CNS myelin proteins

EAE

Correlative evidence in human disease

45

MC-deﬁcient mice have delayed onset and reduced EAE severity Insulin-dependent diabetes mellitus (IV)

Pancreatic β cell-derived Ag (IGRP)

NOD mouse and BB rat

Correlative evidence in human disease and animal models

46

MC-targeted therapy delays disease onset in BB rat Guillain-Barr´e syndrome (IV)

PNS myelin proteins

EAN

Increase in degranulated MC around peripheral nerves in EAN

47

MC-targeted therapy decreases EAN incidence and severity

48

Sjogren’s syndrome (N/A)

RNA-protein complexes, nuclear proteins

NOD and Id3 mice

In human disease, MC found in salivary glands

49

Systemic sclerosis (N/A)

Unclear; nuclear proteins?

“Tight-skin” (TSK) mouse

In human disease, activated MC found in skin

50

MC-targeted therapies improve disease in TSK mice

51–53

Autoimmune vasculitides (II, III, IV)

Unclear; nuclear proteins?

HgCl2 in BN rat

MC-targeted therapies reduce disease in rat model

54

Abbreviations: EAE, encephalomyelitis; EAN, experimental autoimmune neuritis; IGRP, islet-speciﬁc glucose-6-phosphatase catalytic subunit; NON, nonobese diabetic; PNS, peripheral nervous system; SLE, systemic lupus erythematosus.

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(TSH) receptor causes thyrocyte growth and increased thyroid hormone secretion, thus causing clinical symptoms of hyperthyroidism (62). In addition to the lymphocytic inﬁltration of the thyroid, a large number of GD patients have localized connective tissue inﬂammation, which is thought to result from the underlying autoimmune process rather than from thyroid dysfunction. One of the most common forms of this connective tissue inﬂammation is Graves’ ophthalmology, in which inﬂammation in the periorbital space leads to edema, extraocular muscle dysfunction, swelling and redness of the conjunctiva, double vision, and corneal ulceration (63). In Graves’ ophthalmology, mast cells have been found to inﬁltrate the orbital tissue of the eye, and increased circulating levels of IgE (some TSHR-speciﬁc) (19) and SCF are observed in some patients (64). A murine adoptive transfer model of Graves’ ophthalmology also displays mast cell orbital inﬁltration, which precedes the appearance of lymphocytes (65). Mast cells are theorized to function in Graves’ ophthalmology as important sources of chemoattractants (e.g., IL-16 and CCL2), cytokines (e.g., IL-4, IL-5, IL-13), and B cell co-stimulatory signals (e.g., CD154) (19).

Mast Cells and Type III Hypersensitivities Antigen-antibody complexes mediate the major immune destruction in Type III hypersensitivities by depositing in organs and tissues, binding complement, and attracting macrophages and neutrophils. This immune complex formation and deposition can occur during prolonged infection, acute exposure to antigen, or autoimmunity. As in Type II hypersensitivity reactions, there are many reasons to suggest mast cells play a central role in these diseases. These include the ability of both antibodies and complement to activate mast cells resulting in the production of chemoattractant molecules that recruit the pathology-inducing effector cells. In addition, mast cells play an essential role in experi-

mental Arthus reactions. Although not autoimmune in nature, the study of these reactions has provided information on the mechanisms that underlie the common pathology of Type III hypersensitivity diseases. Arthus reactions occur when Ag-Ab complexes deposit in the skin, vasculature, serosa of organs, and the kidney glomeruli (66). They are rare occurrences during vaccination or other instances in which antigen is injected into a sensitized individual. The animal model most often used is the reverse Arthus reaction, in which a dose of antigen is given, followed by passive transfer of antigen-speciﬁc antibody (67). An inﬂammatory response quickly ensues and is characterized by edema, hemorrhage, and neutrophil inﬁltration. Mast cells were ﬁrst implicated in a cutaneous Arthus response using W/Wv mice (68). When compared to wild-type controls, mast cell– deﬁcient mice have decreased edema, neutrophil inﬁltration, and hemorrhage. Mast cell activation through FcγRIII is important for this response (69), as is mast cell activation by complement, which releases neutrophilattracting leukotrienes and TNF (70, 71). Rheumatoid arthritis (RA) is a chronic inﬂammatory disease of the synovial membranes and articular structures of the joints. Rheumatoid factor, an autoantibody against the Fc portion of IgG, is considered an instigating factor in RA and a good marker for disease, although it is neither unique to RA nor present in all patients (72). Both Th1 and Th17 cells exert a major inﬂuence in this disease by helping generate a B cell antibody response and recruiting neutrophils, respectively (73). A commonly used murine model of RA, among several different ones (reviewed in 74), is induced by the injection of immune complex–rich serum from spontaneously arthritic K/BxN mice. This complement- and autoantibody-mediated disease is characterized by synovial inﬂammation and erosion of the joints. Mast cell–deﬁcient mice do not develop disease in this model, although arthritis can be elicited after reconstitution of

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the mast cell compartment (44). Mast cell– derived TNF is implicated in disease pathogenesis, and anti-TNF therapies have been successful in humans (75). However, the ability of some TNF-null mice to develop arthritis after serum transfer suggests that other mediators are required and act in concert with TNF (76). The administration of IL-1 completely restores arthritic disease in W/Wv mice, despite an absence of mast cells (77). Mast cell IL-1 production in this serum transfer model is dependent on activation through the IgG receptor FcγRIII. Mast cells are normally present in the synovial compartments of healthy individuals as well as in RA patients, and in arthritic synovial tissue the mast cells increase in number and appear degranulated (reviewed in 78). High levels of tryptase and histamine have been detected in the synovial ﬂuid of some RA patients. Newer treatments for RA aim to decrease inﬂammation by reducing mast cell numbers and activation. In mice, treatment with the mast cell–inhibiting drugs cromolyn or salbutamol decreases disease severity in susceptible animals (79). Imatinib mesylate, a tyrosine kinase inhibitor developed for the treatment of Bcr/Abl-positive cancers, acts on the c-kit tyrosine kinase receptor and induces apoptosis both in cultured mast cells and in those present in explant cultures of synovial tissue obtained from patients with RA (43). Imatinib prevents disease development and effectively treats established disease in a collagen-induced model of disease (80). Systemic lupus erythematosus (SLE) is a multiorgan autoimmune disease characterized by the production of a variety of antinuclear autoantibodies (reviewed in 81). Symptoms range from rash and arthritis to neuropathies and severe renal disease. Mast cell inﬁltrates are present in affected tissue from patients with SLE-mediated glomerulonephritis, which strongly suggests mast cell inﬂuence (82). Despite this correlative evidence in human disease, results in a pristane-induced model of disease do not support a pathologic role

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for mast cells. Upon intraperitoneal injection of pristane, an isoprenoid alkane, mice develop lupus autoantibodies, including antinRNP/Sm, anti-ribosomal P, anti-Su, antichromatin, anti-single-stranded DNA, and anti-double-stranded DNA (83–85). This is accompanied by the characteristic SLE symptoms including severe glomerulonephritis with immune complex deposition, mesangial proliferation, and proteinuria. In a small-scale study, W/Wv mice develop SLE symptoms that are of equal or greater magnitude to those of their wild-type counterparts (86), suggesting that mast cells have no role in disease or may actually exert a protective inﬂuence. However, pristane induction of disease may bypass the early mast cell–dependent events that initiate SLE in humans. Other models, including those utilizing the lupus-prone NZM or MRL mice, may provide a better system to evaluate disease development (81).

Mast Cells in Type IV Hypersensitivities Type IV hypersensitivity diseases, also called delayed-type hypersensitivities (DTH), are mediated by antigen-speciﬁc CD4+ T cells and generally occur several days following the initial exposure to antigen. Activated T cells migrate to the target tissue site, where they release cytokines and chemokines that recruit other inﬂammatory cells. Contact dermatitis (e.g., a poison ivy response) and the clinical tuberculin test are common examples of DTH, as are some classic T cell–dependent autoimmune disorders. Murine models have shown that mast cells can make important contributions to cutaneous DTH (87, 88), suggesting they may play a role in other T cell–dependent diseases. Multiple sclerosis (MS), a CNS demyelinating disease, is among the most common of the autoimmune diseases (reviewed in 89). MS patients exhibit multicentric CNS lesions that correspond to destruction of the myelin sheaths surrounding nerve axons. These myelin sheaths insulate nerve axons,

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facilitating the saltatory conduction necessary for transmitting nerve impulses over long distances. This loss of nerve impulse transmission accounts for the clinical problems deﬁning MS including loss of visual acuity and other sensory disturbances, weakness, difﬁculty with ﬁne and gross motor movements, as well as bowel and bladder incontinence. MS and the prototypic murine model of the disease, experimental autoimmune (also termed allergic) encephalomyelitis (EAE), are both CD4+ T cell–mediated diseases that demonstrate MHC-linked and gender-linked susceptibility (89). Although the eliciting agent is not known in MS, immunization with a myelin peptide (MBP, myelin basic protein; PLP, proteolipid protein; or MOG, myelin oligodendrocyte glycoprotein) in complete Freund’s adjuvant (CFA) generates EAE in genetically susceptible rodents. Activation of naive myelin-speciﬁc T cells occurs primarily in peripheral lymphoid organs in humans as well as mice, and the accompanying increase in blood-brain barrier (BBB) permeability allows a massive inﬂux of inﬂammatory cells, including both antigen-speciﬁc and naive T cells, macrophages, and dendritic cells (DCs), into the CNS (90). Stimulation of autoreactive T cells by myelin epitopes in the brain and spinal cord initiates a local inﬂammatory response, profoundly damaging CNS tissues. The clinical disease phenotype varies from a chronic progressive to relapsing-remitting course. In mice this is dependent on the genetic strain and the peptide used to induce disease (91). There are several differences between the rodent model of disease and its human counterpart, including unique sites of demyelination and a different composition of the cellular inﬁltrate present in pathological plaques (92). Still, EAE is considered by most immunologists to be a useful model of MS pathology and has allowed identiﬁcation of several therapeutic targets (93). Adoptive transfer of IFN-γ-producing autoreactive CD4+ T cells into naive mice causes disease and has been cited repeatedly as proof of the dogma that Th1 (IFN-γ-

producing) cells are the major effector cells in EAE (and presumably MS). However, that IFN-γ−/− mice paradoxically develop worse disease complicates this paradigm (94). Recently, Th17 cells were shown to orchestrate the inﬂammatory cascade in the CNS in EAE, presumably by recruiting monocytes and other innate immune cells to the site of inﬂammation (95). Th17 cells originate from the same naive precursors as Th1 and Th2 cells, but differentiate under the inﬂuence of TGF-β and IL-6, rather than IL-12 and IL-4, respectively (96–98). The Th17 pathway appears to be exclusive; addition of IFN-γ or IL4 to the culture medium inhibits production of IL-17 (97, 98). Although IFN-γ-producing cells may contribute to established inﬂammation, loss of potential for Th1 differentiation that occurs in IFN-γ−/− mice may “shunt” T cells toward the Th17 pathway and thus worsen disease. The data supporting a role for mast cells in the pathogenesis of MS and EAE are compelling. In both the murine and human disease, mast cells accumulate at sites of inﬂammatory demyelination in the brain and spinal cord and are often found degranulated (99). High levels of tryptase, a mast cell–speciﬁc proteolytic enzyme, and histamine are found in the cerebrospinal ﬂuid of MS patients, suggesting mast cell activation (100, 101). Gene-expression proﬁling demonstrates that transcripts encoding the histamine 1 (H1) receptor, as well as the mast cell–speciﬁc genes tryptase and FcεRI, are highly expressed in CNS plaques of patients with chronic MS (102). The most direct evidence that mast cells are involved in this disease syndrome comes from studies using mast cell–deﬁcient mice. Under deﬁned disease induction conditions, W/Wv mice display a very mild disease and delayed onset when compared to their wildtype littermates (Figure 3) (45). The entry of CD4+ and CD8+ T cells into the CNS also appears compromised in W/Wv mice (103). Selective mast cell reconstitution of W/Wv mice through intravenous transfer of BMMCs

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Mean clinical score

4.0 3.5 3.0

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2.5 2.0 1.5 1.0 0.5 p < 0.001 0 0 5 10

15

20

25

30

35

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Figure 3 Mast cell–deﬁcient mice exhibit reduced EAE disease severity. EAE was induced by immunization with MOG35−55 in CFA (complete Freund’s adjuvant). Wild-type, W/Wv , and W/Wv mice reconstituted with BMMCs were scored daily according to the following clinical scoring system: 0, no clinical disease; 1, tail ﬂaccidity; 2, hind limb weakness; 3, hind limb paralysis; 4, fore limb paralysis or loss of ability to right from supine; 5, death (45). (Figure reproduced from J. Exp. Med. 2000, 191:813–22, with permission.)

restores severe disease susceptibility. This occurs despite the inability of intravenous mast cell reconstitution to appreciably repopulate CNS mast cells, indicating that mast cells can exert effects at other sites outside the target tissue in this experimental model (104). Indeed, as discussed below, mast cells exert profound effects on the initiation of peripheral T cell responses as well as on their effector function. Insulin-dependent diabetes mellitus (IDDM), also called type 1 diabetes, is a chronic metabolic disorder that develops in two discrete phases and is mediated in part by CD8+ T cells (reviewed in 105). Prior to clinical signs of disease, a mixed population of leukocytes invades the pancreatic islets resulting in insulitis. The consequent destruction of the insulin-producing β cells of the pancreas leads to hyperglycemia. There are several rodent models of IDDM (106). In experimental chemical diabetes, toxins such as alloxan or streptozotocin are injected into an animal, selectively killing 716

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insulin-producing pancreatic β cells. Both the nonobese diabetic (NOD) mouse and the BioBreeding (BB) rat spontaneously develop insulitis that ultimately progresses to IDDM at a high frequency. There are several reasons to suspect that mast cells are implicated in IDDM. Mast cells are normal residents within the pancreatic ducts, placing them in close proximity to the pancreatic islets (107). There is a striking increase in the frequency of mast cells in the acinar parenchyma in inﬂammatory diseases of the pancreas such as chronic pancreatitis and pancreatic cancer, and this cellular increase correlates positively with the extent of inﬂammation (107–109). Mast cells produce several mediators that could affect the development of diabetes, including oxygen intermediates and proteases, which may have direct actions on the destruction of the β islet cells. Release of leukotriene B4 (LTB4), a mast cell–derived chemoattractant that may be essential for efﬁcient recruitment and/or retention of autoreactive T cells in the target organ, is increased in type 1 diabetes (110). The ability of mast cells to regulate the development of CD8+ T cell responses in other disease models suggests another possible mode of action in diabetes (103). Of most signiﬁcance is the ﬁnding that more mast cells are present in the pancreatic lymph nodes of lymphopenic diabetic BB rats prior to disease onset (compared to lymphosufﬁcient littermates). This correlates with overexpression of mast cell transcripts in these mice (46). In addition, treatment of spontaneously diabetic BB rats with cromolyn delays disease onset. The inﬂuence of mast cells in IDDM may be more complicated than simply increasing inﬂammation or enhancing CTL function. Mast cells produce TNF and TGF-β, cytokines that can be proinﬂammatory but also regulate T cell tolerance to autoantigens in some models of diabetes (111, 112). Mast cells also express PD-L1 (15), an inhibitory molecule implicated in protection from disease (113).

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Guillain-Barr´e syndrome (GBS), a T celldependent demyelinating autoimmune disease of the peripheral nervous system (PNS), is usually preceded by illness, most commonly infection with Campylobacter jejuni, cytomegalovirus, Epstein-Barr virus, or Mycoplasma pneumoniae (114). All of these pathogens possess lipo-oligosaccharides in their bacterial walls that resemble gangliosides found in peripheral nervous tissue, indicating that molecular mimicry is important in the initiation of this disease (115). As a result, innate immune cells are activated and B and T cells respond to both the microorganism and its conjugate gangliosides in the host tissue, leading to eventual peripheral nerve myelin destruction. Experimental autoimmune neuritis, the rodent model of GBS, is generated through immunization with a PNS myelin peptide in CFA (116). After immunization, inﬂammatory cells inﬁltrate the PNS, leading to demyelination of axons, weakness, and, occasionally, paralysis. Directly preceding onset of symptoms, there is a marked increase in the number of degranulated mast cells around the affected peripheral nerves (47). The activation and proliferation of nerve-associated mast cells are also observed after adoptive transfer of a neuritogenic P2 protein-speciﬁc T cell line into Lewis rats (117), and treatment with a mast cell–stabilizing drug decreases the incidence and severity of disease (48).

MAST CELLS IN OTHER AUTOIMMUNE DISORDERS There is evidence for mast cell involvement in a variety of other diseases with autoimmune components that do not ﬁt neatly into the Type I–IV hypersensitivity classiﬁcation. Systemic sclerosis (SSc), or scleroderma, is characterized by symptoms that include excessive collagen accumulation and vascular lesions in the skin and the internal organs, causing thick, indurated skin and often progressing to pulmonary hypertension or renal crisis (118). Antinuclear and antinucleo-

lar autoantibodies, along with putative new pathogenic autoantibodies, including antiendothelial cell antibodies and antiplateletderived growth factor receptor antibodies, are present in a high percentage of SSc patients (119). Activated tryptase-producing mast cells have been found in the skin of human patients with SSc (50) and have been implicated as a source of ﬁbrogenic cytokines, including TGF-β, and ﬁbroblast-attracting proteases (120). Increased mast cell density and degranulation are observed in the “tight skin” mouse model of SSc (121), and dermal ﬁbrosis is reduced after treatment with mast cell stabilizers (52, 53) or a mast cell chymase inhibitor (51). The latter acts to prevent the cleavage of TGF-β into its active form. Mast cells also appear to play important roles in inﬂammatory bowel disease, namely ulcerative colitis (UC) and Crohn’s disease (CD). Although there is some debate on whether these diseases are “true” autoimmune diseases, as the eliciting antigens are hypothesized to be part of the normal gut ﬂora, these diseases clearly result from exaggerated immune responses to nonpathogenic antigens in genetically susceptible individuals (reviewed in 122). Tissue from UC patients shows mast cell aggregation along the line of demarcation dividing inﬂamed from healthy tissue (123). An increase in mast cell density in involved tissue of CD patients has been well documented (124), and mast cells are thought to modify the inﬂammatory CD process through release of histamine (125), TNF (126), and the T cell chemoattractants XCL-1 (127) and IL-16 (128).

HOW DO MAST CELLS INFLUENCE AUTOIMMUNE DISEASE? Despite substantial evidence that mast cells are critical players in the pathogenesis of autoimmunity, their precise mode of action is, for the most part, still a subject of speculation. We know a great deal about how mast cells inﬂuence innate and adaptive immunity in

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MAST CELLS, STRESS, AND AUTOIMMUNITY Another link between autoimmunity and mast cells lies in the tendency for many of these diseases to ﬂare during times of stress. Corticotropin-releasing hormone (CRH) is produced by the paraventricular nucleus of the hypothalamus in response to stress and initiates a hormonal cascade that results in activation of the sympathetic system and release of epinephrine and norepinephrine. CRH is localized in inﬂamed tissues, and mast cells possess both receptors for CRH (194) and the ability to release CRH de novo (195). CRH causes release of VEGF (vascular endothelial growth factor) from mast cells in vitro (196). The promotion of angiogenesis and subsequent regional inﬂammation is another way by which mast cells can contribute to disease pathology.

infection and allergy. Assuming that there are commonalities in all immune responses, much of this information can be applied to understanding the role of these cells in autoimmunity. Although not required for all responses, mast cells are perfectly situated to directly affect many cell types and play a responseamplifying role. Of importance, mast cells are not just proinﬂammatory but can also dampen an immune response directly through expression of certain cytokines such as IL-10 or TGF-β or indirectly by facilitating T regulatory cell (Treg) activity.

Overview of a Prototypic Adaptive Immune Response During an infection, immature DCs residing in peripheral tissues such as the skin, airways, and gastrointestinal tract capture and process microbial antigens during routine sampling of the environment. Pathogens also activate DCs via cell surface or intracellular PRRs, triggering DC maturation and migration to the secondary lymphoid organs. Here DCs present microbial-derived peptides in the context of the class II MHC to naive T cells initiating T cell activation, clonal expansion, and dif718

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ferentiation (Figure 4) (129). DCs are also crucial for priming cytotoxic CD8+ T cells through the presentation of exogenous Ag by class I MHC. Mature DCs secrete cytokines such as IL-12, IL-23, TGF-β, and IL-6, providing CD4+ T cells with differentiation signals for at least four alternative pathways: Th1 (IL-2- and IFN-γ-producing), Th2 (IL-4-, IL-5-, and IL-13-producing), Th17 (IL-17producing), or Treg (Foxp3-expressing) cells. The differentiation process gives rise to effector T cells that have acquired the ability to leave the lymph nodes and, informed by cellderived mediators in the tissue, home to sites of inﬂammation. Similar events generate the antigenspeciﬁc T and B cells that mediate autoimmune responses. However, beyond the distinct target antigens in such responses, it is not clear what distinguishes pathologic from protective responses. Self-proteins are not normally immunogenic because of multiple mechanisms that act to maintain tolerance. In addition, how the DC co-stimulatory signals required for generating fully activated T cells are elicited is unclear, although there is evidence that infection may be involved and can initiate or exacerbate both allergic and autoimmune responses (130).

Mast Cells Exert Influence at Multiple Disease Checkpoints in EAE The study of mast cell inﬂuence on EAE disease course provides an excellent framework for dissecting mast cell actions on adaptive immune responses. It is postulated that mast cells act at several checkpoints in disease development. These include events at the peripheral sites of antigen exposure that regulate DC function, in secondary lymphoid organs where T cells are primed and differentiated, at the BBB where inﬂammatory cell efﬂux into the CNS is controlled, and within the CNS where myelin and nerve damage occurs (131). Several aspects of the T cell response to myelin peptide in MOG35−55 –induced EAE

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Secondary lymphoid organs Migration to sites of inflammation

Ag capture Migration and maturation

Ag:MHC presentation with CD80/86

Clonal expansion and acquisition of T cell effector function

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TLR activation

DC release of IL-12, IL-23, IL-6, or TGF-β regulates Th cell fate decision

Figure 4 A prototypic adaptive T cell immune response. At the onset of a typical infection, dendritic cells (DCs) in peripheral tissues capture and process antigens before migrating to local secondary lymphoid organs where they present antigen to T cells and elicit T cell differentiation and proliferation. The effector T cells then leave the lymph node and home to areas of inﬂammation. Mast cells have the potential to affect all of these events.

appear to be defective in W/Wv mice and can be corrected by mast cell reconstitution (103). First, there is a marked absence of normal lymph node hypertrophy after immunization, due in part to reduced numbers of T cells that enter and undergo clonal expansion. There is a reproducible though not statistically signiﬁcant defect in the induction of MOG-speciﬁc IFN-γ and IL-17 production by CD4+ T cells postimmunization in the lymph node and spleen of W/Wv mice (103; B.A. Sayed & M.A. Brown, unpublished observation). Ex vivo analysis of whole spleen cultures reveals that both IL-4 and IFN-γ levels are signiﬁcantly diminished in W/Wv mice compared to their wild-type littermates. The normal activation-induced increases in CD11a, CD44, and CD54 are also reduced. Most striking are the differences in antigen-speciﬁc T cell inﬂux into the CNS beginning as early as day 8 post disease induction (B.A. Sayed & M.A. Brown, unpublished observation). W/Wv mice exhibit ﬁve- to seven fold decreases in the numbers of CD4+ and CD8+ T cells that enter the CNS, an observation that corresponds to reduced inﬂammation and

demyelination in the brain and spinal cord (45, 103). It has been deﬁnitively established that these differences in T cell response are not due to an inherent defect in W/Wv T cell function: W/Wv mice display normal T cell development in the thymus and numbers of peripheral CD4+ and CD8+ T cells (103) that are comparable to those of wild-type littermates. Furthermore, in vitro differentiation of W/Wv - or wild-type-derived T cells shows that similar Th1 and Th17 cytokine production and transfer of self-reactive encephalitogenic CD4+ T cells from W/Wv or wild-type mice cause equivalent disease in naive T cell–deﬁcient hosts (103; B.A. Sayed & M.A. Brown, unpublished observation).

DC–Mast Cell Interactions in the Initiation of an Immune Response DCs are likely to be one of the earliest target cells of mast cell inﬂuence. Mast cells are found in close apposition to DCs in all tissues except blood and are thus subject to the same microbial activation signals as DCs during infection. The induction of EAE depends on the

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concurrent administration of myelin peptide and CFA. Heat-killed Mycobacterium tuberculosis, a component of CFA, serves as an initial DC- and mast cell–activating stimulus in the periphery. LPS, PGN, zymosan, and dsRNA are among the microbial-related agonists that may be relevant in other infection-associated autoimmune responses. A large body of evidence documents the effects of mast cells on DCs in infection and contact hypersensitivity settings, where mast cell activation through PRRs affects DC migration, maturation, and function. This is a probable mechanism whereby mast cells exert a major effect on T cell responses in EAE and other autoimmune diseases (Figure 5). Like mast cells, DCs are quite heterogeneous (reviewed in 132). Thus the responses of DCs to mast cell interaction are likely to be variable as well. They are broadly classiﬁed into either myeloid (mDCs) or lymphoid (lDCs) based on the precursor cell from which they are derived. DCs present in the thymus are thought to develop from both myeloid and lymphoid precursors, whereas peripheral DCs arise almost solely from myeloid precursors. mDCs are considered most important for both T cell priming and tolerance; however, there appears to be some plasticity between the phenotype and function of the two subsets. The myeloid subset of DCs can be further classiﬁed into Langerhans cells (LCs), found in skin and other stratiﬁed epithelia, and interstitial DCs (iDCs), found in most other organs. A third DC subset, plasmacytoid DCs (pDCs), may originate from either lymphoid or myeloid precursors and are located with iDCs in blood and peripheral tissues. Upon activation, there are impressive changes in the expression of co-stimulatory markers and mediators in mast cells (see Table 2) that are particularly relevant to DC function. For example, mast cells can release relatively large amounts of both PGE2 and TNF within minutes of activation. Both of these mediators have the ability to alter DC cell migration and lymph node hypertrophy (133, 134). The importance of mast cell–

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derived TNF was shown in a footpad infection model in Escherichia coli, in which mast cell–deﬁcient mice fail to exhibit the characteristic hypertrophy of draining lymph nodes (135). Reconstitution with wild-type but not TNF-deﬁcient mast cells restores the wildtype phenotype. Similarly, following subcutaneous injection of PGN, lymph node hypertrophy and DC mobilization in vivo are mast cell–dependent phenomena, although this response appears to be TNF-independent (136). Contact hypersensitivity (CHS) responses to chemical haptens are highly dependent on the local density and migratory properties of epidermal LCs. In these responses, the mast cell can be activated through FcεRI crosslinking or via monomeric IgE binding to this receptor (158, 159). Following sensitization and challenge with the hapten FITC, both W/Wv and TNF−/− mice exhibit deﬁcits in the CHS response and show signiﬁcant delays in the migration of haptenated DCs into draining lymph nodes (133). Engraftment of mast cell–deﬁcient mice with wild-type but not TNF−/− BMMCs repairs the DC migration defect. Histamine released from mast cells may also contribute to LC migration from the skin to draining lymph nodes. After passive sensitization with IgE, mast cell– deﬁcient mice challenged with antigen show signiﬁcant defects in LC activation and trafﬁcking to draining lymph nodes, which are repaired by mast cell reconstitution (160). Treatment of wild-type mice with cimetidine, an H2 (histamine 2) receptor antagonist, inhibits the observed IgE/Ag-induced LC migration, implicating mast cell–released histamine. DC migration goes hand in hand with maturation, deﬁned as inducible changes in cytokine, chemokine, or cell surface marker expression. Mast cells also affect DC maturation and ultimately inﬂuence the ability of DCs to direct the quality of Th cell differentiation through the expression of inducible mediators. Histamine, perhaps the best-known mast cell mediator (although other cells can make it), has profound effects on DCs and Th

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a

Mast cell

TLR activation

(–)

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Ag:MHC II

(+) Histamine IL-10 TGF-β PGD2, PGE2

TNF IL-4 IL-12 TSLP

CD40L CD40

Maturation

No maturation Dendritic cell (immature)

b

CD80/86 Ag:MHC II

PD-L2 OX40L

Th1-inducing IL-4, IL-12, IL-25, TSLP Th2-inducing Histamine, PGD2, PGE2, IL-18

Mast cell

DC

Naive CD4+ T cell

Regulatory T-inducing TGF-β, IL-10 Th17-inducing ? Figure 5 (a) A model for mast cell inﬂuence on DC responses I. Both mast cells and DCs are activated by microbial antigens through TLRs. The outcome of the response, DC maturation or suppression of the maturation process, is regulated by the predominant mediators and cell surface molecules expressed by locally activated mast cells. (b) Mast cell inﬂuence on DC responses II. Mast cells express mediators that act to “polarize” the DC and therefore ultimately inﬂuence naive CD4+ T cell fate decisions.

cell fate decisions. Immature and mature DCs (from human blood) express all four histamine receptors (H1–H4) (161). Histamine as well as PGD2 , PDE2 , and leukotrienes suppress DC IL-12 production in vitro (134, 140, 144, 145). Histamine also inhibits TLR-mediated

induction of other DC-produced cytokines, including IFN-α, IL-1α, IL-1β, IL-18, IL-6, CXCL10, CCL20, CCL5, and TNF and increases the expression of CXCL8 and IL-10 (140, 162). Antigen uptake and presentation by DCs (163) and DC chemotaxis (137)

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Mast cell-derived factors and their effects on DCs

Mast cell-derived factor

Effect on DCs

Leukotrienes Prostaglandins

IL-4

137

Promotes Th2 skewing of T cellsa

138–140

Enhances migrationa,b,c

141–143

Increases IL-10 and decreases IL-12c

144

PGD2 : decreases IL-12 and promotes Th2 skewingc

145

PGE2 : increases IL-10c

134

migrationa

146

Essential for

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References

Decreases IL-12a

Histamine

Increases production of Th2-attracting chemokinesa

147

Inhibits maturation of DC during infectionb

148

Decreases IL-10 leading to increased IL-12c

149

Decreases prostaglandin E productiona

150

Enhances DC migrationb

TNF

133, 136

Produces tolerogenic DC that induce Tregsc TGF-β

Promotes DC generation of

CD4+ CD25+

Tregsc

151 152

Increases expression of chemokine receptorsa

153

Essential for generation of LCsb

154

TSLP

Promotes inﬂammatory Th2-skewing of T cells by ex vivo human DC

155

Serotonin

Differentiates DC with reduced stimulatory capacity but increased cytokine productiona

156

Heparin

Differentiates CD1a+ DCa

157

a

Human monocyte-derived DC, in vitro. Murine DC, in vivo. c Murine DC, in vitro. b

are enhanced by histamine as well. In vivo, protocols that induce mast cell degranulation (and elicit histamine release) coincident with immunization result in a polarized Th2 response (164). In contrast, mast cell– derived IL-4 is necessary for severe disease and an optimal Th1 response in MOG35−55 –induced EAE (165). The ﬁnding that IL-4 promotes IL-12 expression by inhibiting IL-10 transcription in DCs (149) may explain this seemingly paradoxical observation. Activated mast cells release many cytokines that alter the expression of DC activation markers as well. For example, TNF induces increases in IFN-γR, CD40, ICAM1 (CD54), MHC class II, and CD86 in vivo, a ﬁnding that corresponds to accelerated autoimmune disease in NOD mice (166). Thymic stromal lymphopoietin (TSLP), an 722

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epithelial-derived cytokine, is also expressed by mast cells and has potent effects on DCs. These effects include ability to induce OX40 ligand expression resulting in a DC population that preferentially converts DCs to a Th2-polarizing antigen-presenting cell (167). Although a mast cell source for these cytokines has not been conﬁrmed in vivo, the coculture of activated mast cells and human DCs in vitro results in impressive increases in CD80, CD86, CD83, CCR7, and MHC class I and class II expression on DCs (139).

Direct Mast Cell Effects on Initiating the T Cell Response Mast cells may directly regulate T cell priming in a number of ways (Figure 6). Peripheral mast cells in the skin and mucosa and secondary lymphoid system are the most

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Mast cell

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Ag presentation? Co-stimulation

Naive CD4+ T cell TGF-β IL-6 IL-21 IL-23 IL-12 IL-4

TGF-β IL-10, IL-2 IL-12, IL-4 IL-6, IFN-γ

IL-4 IL-12

IFN-γ IL-12 Histamine IL-4

Th17

Th1

Regulatory T cell

Th2

Figure 6 Direct mast cell–T cell interactions. MHC class II and T cell co-stimulatory molecule expression by mast cells provides potential antigen-presenting cell capability. Mast cells also express most cytokines that control T cell differentiation pathways and directly regulate T cell fate decisions.

likely orchestrators of these early effects. Some mast cell lines can be induced to express MHC class II molecules and have been shown to have antigen-presenting capability in in vitro settings (168, 169). Mast cells could also provide direct costimulation to T cells within the secondary lymphoid organs via their expression of CD80, CD86, CD153, ICOSL, 41BB, CD40L, and OX40L or enhance T cell proliferation and expression of activation markers through elaboration of cytokines such as IL-2, IL-4, IL-7, and IL-15 (15). Mast cells express all of the cytokines (IL-12, IL-6, IL-4, IL-1, IL-21, IL-23, IL25, and TGF-β) implicated in Th polariza-

tion and may augment DC-derived cytokine contributions to Th differentiation decisions. Furthermore, histamine has been shown to have direct effects on T cell differentiation (170). CD8+ T cells have been implicated in both the exacerbation and suppression of EAE and MS in a number of studies (171, 172), and the effects of mast cells are not limited to CD4+ T cell responses in EAE. W/Wv mice also generate a diminished primary CD8+ T cell response. CD8+ T cell cells primed in mast cell– deﬁcient mice express signiﬁcantly decreased amounts of IFN-γ and CD44 post-MOG immunization (103). The requirement of mast

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cells for the initiation of strong CD8+ T cell responses has implications not only for EAE but also for diseases such as type I diabetes, where CD8+ T cells are among the major effector cells and play a role in the direct cytotoxic destruction of insulin-producing pancreatic β cells.

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In addition to inﬂuencing migration to lymph nodes, mast cells can recruit DCs to sites of tissue inﬂammation. Cross-linking of FcεRI on mast cells induces the release of the CCL1 chemokine, which acts to recruit LCs bearing the CCL1 receptor, CCR8, to sites of allergic inﬂammation (173). Mast cells also express CCL19 (174), the ligand for CCR7, a chemokine receptor required for DC migration (175) and highly expressed by myeloid DC present in inﬂamed CNS lesions in MS (176). These ﬁndings may have relevance to relapsing-remitting models of EAE, including Theiler’s murine encephalitis virus– and PLP-induced disease in SJL mice. In these models, the activation of naive myelin-speciﬁc T cells in the CNS is dependent on the directed migration of peripheral myeloid DCs to the CNS (177). Here the DCs present new myelin epitopes distinct from those used to induce disease. This directed migration and subsequent activation of T cells with new antigen speciﬁcities are thought to make a major contribution to epitope spreading. Mast cells also appear to orchestrate the migration of T cells (Figure 7). Entry of autoreactive T cells into the CNS is a checkpoint in disease development. The ability of both antigen-speciﬁc and bystander CD4+ and CD8+ T cell populations to migrate to the CNS is compromised in W/Wv mice (103; B.A. Sayed & M.A. Brown, unpublished observation). This defect correlates with decreased peripheral T cell expression of early activation/homing markers including CD44, CD11a, and CD54, molecules that

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regulate leukocyte adhesion and are implicated in EAE, in W/Wv mice. Mast cells could inﬂuence T cell migration into the CNS in several ways: (a) During the initiation of the response, mast cells may affect DC maturation and indirectly regulate the activation state of T cells. In the absence of mast cells, DCs may not effectively prime T cells, resulting in defects in T cell homing potential and cytokine production. (b) Resident mast cells in the target organ (in the case of EAE, the CNS) may become activated and release chemoattractants such as LTB4, CCL3, CCL2, CCXCL10, CCL19, and CCL21, all of which have been implicated in T cell recruitment in EAE and MS (178). Mast cell– derived LTB4 is essential for the recruitment of both CD4+ and CD8+ effector cells to sites of inﬂammation through interaction with BLT1 expressed on T cells in an in vivo model of airway hyperresponsiveness (179). In vitro T cell migration studies support this ﬁnding (180). (c) Local perivascular mast cells may release vasoactive amines including histamine and serotonin that increase the BBB permeability, allowing the more efﬁcient inﬂux of T (and other) cells to the CNS. (d ) Mast cells express matrix metalloproteinases (MMPs) and, via chymase and tryptase, convert them to active forms that promote collagen breakdown at the underlying basement membrane, allowing transendothelial passage (181). It is postulated that upon disease induction in W/Wv mice, T cells do not acquire adequate expression of relevant homing molecules, and we speculate that the lack of chemotactic factors and factors regulating endothelial permeability normally produced by resident CNS mast cells leads to inefﬁcient breach of the BBB. Thus, the inﬁltration of the CNS by inﬂammatory cells is compromised. Because IL-17 acts as a chemotactic factor for many innate inﬂammatory cells, inefﬁcient migration of Th17 cells into the CNS may limit the extent of inﬂammation, myelin degradation, and disease pathology.

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Vessel lumen

BBB

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Activation of naive T cells

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CNS

Basement membrane degradation (proteases)

Vascular permeabilization (histamine, serotonin)

New myelin epitope release

Reactivation of effector T cells

Chemotaxis (CCL3, CCL2, CXCL8, LTB4) Myelin repair (NGF)

Figure 7 Model for mast cell inﬂuence on inﬂammatory events in the target tissues in autoimmunity. Mast cell mediators produced at the site of inﬂammation regulate immune cell effector function subsequent to initial T cell activation events. Proteases (chymase and tryptase) facilitate the degradation of endothelial cell basement membranes by cleaving MMPs to active forms. Together with histamine and serotonin, which increase vascular permeability, and chemotactic factors, these mediators regulate entry of immune cells into the target tissue (in this case the CNS). Mast cell proteases can act to promote epitope spreading by directly degrading the myelin sheath, resulting in tissue damage and release of new myelin epitopes that are presented by resident or inﬁltrating DCs to naive or effector T cells. Paradoxically, mast cells also release nerve growth factor (NGF), which could play a role in nerve axon remyelination.

Direct and Indirect Influence of Mast Cells on T Cell Tolerance In autoimmunity, there is a breakdown in one or both of the two major systems that operate to prevent large-scale self-reactive T cell– mediated responses. The ﬁrst occurs during T cell development when mechanisms exist to delete such T cells in the thymus, collectively referred to as central tolerance or negative selection (182). This process is imperfect, as self-reactive T cells often escape deletion in healthy individuals. Central tolerance is augmented by peripheral tolerance mechanisms that either induce anergy (nonresponsiveness) in relevant T cells or utilize specialized populations of Tregs that inhibit effector T cell function (reviewed in 183). Virtually nothing is known about mast cells in central tolerance, though they populate the

thymus and could conceivably play a role in negative selection. However, recent work has clearly implicated mast cells in peripheral tolerance through their effects on a subset of Tregs. The Tregs encompasses a variety of cellular entities. Tregs can be divided into two general classes: naturally occurring thymic– derived Tregs and inducible or adaptive Tregs (183). The suppressor phenotype of naturally occurring Tregs and some inducible Tregs is controlled by the expression of the forkhead/winged-helix transcription factor, Foxp3. Mutations in the Foxp3 gene lead to hyperactivation of the immune system and multiorgan autoimmune disease in humans (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: IPEX) and in mice (scurfy) (184, 185).

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The best evidence for mast cell–dependent Treg-mediated suppression comes from studies in a murine skin transplant model in which mast cells are crucial intermediaries in maintaining CD4+ CD25+ Foxp3+ Treg– dependent tolerance (186). In this model, mice receive an intravenous injection of allogeneic donor lymphocytes together with anti-CD154, followed by a skin transplant from the allogeneic donor. Serial analyses of gene expression experiments demonstrated that genes encoding several mast cell products including IL-10, FcεRI, CCL2, and CCL12 are highly expressed in cultures of tolerant allografts, a ﬁnding that coincides with an inﬂux of mast cells to the graft. Mast cell–deﬁcient mice cannot be rendered tolerant and are unable to sustain allografts. Graft tolerance coincides with cross-talk between Tregs and mast cells; Treg secretion of IL-9, a mast cell growth factor, promotes mast cell recruitment to and activation in tolerant grafts. How mast cells then promote Treg expansion and/or survival is not yet clear.

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Other Antiinflammatory Roles of Mast Cells Mast cells can also directly suppress inﬂammation through the expression of IL-10. Mast cell–deﬁcient mice (both W/Wv and W-sash) exhibit stronger CHS responses to urushiol, the allergen-bearing sap derived from poison oak (Toxicodendron diversilobum) and poison ivy (Trichomanes radicans), than their wild-type counterparts (187). At days 5 and 10 post challenge, untreated mast cell–deﬁcient mice and those reconstituted with IL-10−/− BMMCs showed increased epidermal ulceration and necrosis and dermal leukocytic inﬁltration as compared to controls, revealing that mast cell production of IL-10 is required for limiting inﬂammation and decreasing the swelling associated with urushiol. Engraftment of mast cell–deﬁcient mice with FcRγ−/− BMMCs, which lack expression of both FcεRI and FcγRIII, fails to reconstitute the suppressive effect, indicating 726

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that mast cells are activated through Fc receptors in this model. Dermal mast cells and mast cell–produced IL-10 plays a similar suppressive role in reducing inﬂammation in response to chronic UV-B irradiation.

Mast Cells at Sites of Tissue Destruction Among the several ways that mast cells can act directly within target tissue to regulate autoimmune disease severity is the ability to bestow direct damage on local tissue structures. Mast cells are the sole source of several proteolytic enzymes such as tryptase and chymase. Microarray analysis revealed that there is a substantial increase in expression of the gene encoding tryptase in plaques of MS patients when compared to nondiseased areas, suggesting that this enzyme has a critical role in local pathology (102). Mast cell proteases can degrade myelin in vitro, which leads us to speculate that mast cell activation in the CNS during the effector phase of disease (through peptides, cytokines, antibody, or complement, for example) contributes to the release of new myelin epitopes and epitope spreading (188) (Figure 4). Tryptase may also act through PAR2, a protease-activated receptor. PAR2 is expressed on several types of immune cells as well as on dorsal root ganglion neurons and spinal afferent nerve ﬁbers (189). In neurons, tryptase elicits release of neuropeptides such as SP and CGRP. These in turn have the potential to promote activation of many other immune cells, resulting in the initiation or exacerbation of the inﬂammatory cascade. As in the periphery, when naive cells activated within the CNS come under the inﬂuence of speciﬁc cytokines, they undergo differentiation.

Variable Mast Cell Responses: Can This Explain Diversity in Mast Cell Influence? The major themes in this review encompass the idea that mast cells are multifunctional

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cells, are widely distributed in many tissues, and express a large array of mediators and costimulatory molecules. Most important, mast cells are tuneable; that is, depending on the physiologic setting, mast cells will respond variably (4). These differences in responsiveness translate to the distinct effects mast cells exert in speciﬁc disease conditions. For example, most features of mast cells are consistent with their primary role as proinﬂammatory cells and players in the breakdown of peripheral tolerance, yet two models of “tolerogenic” mast cells provide convincing evidence otherwise. There are many examples of this variability, but because most mast cell responses are assessed in vitro or ex vivo, the picture we have now is probably still very simplistic. At least four key factors inﬂuence the heterogeneous responses of mast cells: (a) The anatomic sites of speciﬁc mast cell populations confer a unique tissue-speciﬁc phenotype. A well-known illustration of this is the classiﬁcation of connective tissue versus mucosal mast cells that differ not only in their preferred sites of residence but also in the expression of granule proteases and relative life span. (b) Related to the distinct locations of mast cells are the unique subsets of target cells in different tissues adding to response variability. Distinct populations of DCs could have unique phenotypic responses to the same mast cell products. (c) A vast number of agonists are able to activate mast cells, and these can elicit distinct responses. It is the convergence of multiple signals that dictates the ﬁnal response of a single mast cell. For example, the classical FcεR-mediated response activation can be modiﬁed by co-stimulatory pathways, such as those triggered by 4-1BB agonists (10). In vivo, the tolerogenic response of mast cells in the skin transplant model occurs in the absence of discernible infection, unlike most proinﬂammatory responses. In this setting, Tregs may “educate” and expand pop-

NERVE GROWTH FACTOR: A MECHANISM OF MAST CELL PROTECTION? Nerve growth factor (NGF) is a cytokine produced by several cell types, including mast cells, that has protective effects in EAE and MS (197, 198). Administration of NGF systemically or locally within the CNS reduces EAE disease severity (199). It is highly expressed in cerebral spinal ﬂuid during acute EAE and MS and levels decrease coincident with remitting episodes. NGF is involved in the survival and differentiation of both CNS and peripheral neurons (200) and has been implicated in myelin repair and regulating Th1 and Th2 balance within the CNS in EAE and MS (201). Thus, in contrast to their many proinﬂammatory effects, mast cells may act to negate the immune-mediated damage under certain circumstances through expression of nerve growth factor. ulations of tolerogenic mast cells by secretion of IL-9 and other factors. Conversely, pathogen-derived stimulatory signals, as in EAE, may push mast cells toward generating predominately proinﬂammatory signals. (d ) Genetically determined variability in mast cell numbers and responses may confer strikingly distinct response outcomes. SJL mice are estimated to have 4–5 times more mast cells than C57BL/6 mice have (190). A study of Th2 cytokine expression in C57BL/6, Balb/c, and SJL mice showed striking differences in the ability of BMMCs to express IL-4 and IL-5 (165). Although all these variables can explain the unique responses mast cells exert in different settings, there is still much to learn. It is worth reiterating that animal studies provide imperfect models of human disease. If the heterogeneity in mast cell populations between various murine strains affects disease susceptibility, this effect is likely ampliﬁed in mast cell populations from unique outbred humans. Thus, caution must be exercised when developing paradigms of mast cell function.

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SUMMARY POINTS 1. Mast cells are present in many tissues and collectively constitute a signiﬁcant cell population. They are activated by a multitude of IgE-independent agonists including antigen-IgG complexes, complement, and microorganisms and are a rich source of many secreted and cell surface–expressed immune-modulating molecules. 2. Mast cells have been extensively studied in the context of Type I (immediate-type) hypersensitivities but also have a signiﬁcant role in Types II–IV hypersensitivity responses that are associated with many autoimmune diseases. Annu. Rev. Immunol. 2008.26:705-739. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

3. Several sites of mast cell inﬂuence likely affect both the initiation and effector phase of autoimmune disease. These range from promoting DC maturation and migration to secondary lymphoid organs to directing T cell differentiation to orchestrating the migration of T cells and other immune cells to the sites of tissue inﬂammation. 4. In addition to proinﬂammatory actions, mast cells have suppressive effects on some immune responses. 5. The variable effects of mast cells are related to the activating stimulus, the tissue site of the mast cell, and the proximal target cells as well as to the genetically determined variability in mast cell mediator production.

FUTURE ISSUES 1. Mast cell–deﬁcient mice need to be developed on diverse genetic backgrounds to expand the range of diseases that can be studied in vivo. 2. Relevant mast cell activators and mediators in autoimmune diseases must be determined. 3. Investigators must determine the molecular basis for proinﬂammatory and tolerogenic actions of mast cells in disease. 4. Investigators must examine the extent of mast cell response heterogeneity in disease.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENT The authors thank Dr. Kavitha Rao for the critical reading of this manuscript.

LITERATURE CITED 1. Galli SJ, Lantz CS. 1999. Allergy. In Fundamental Immunology, ed. WE Paul, pp. 1137–84. Philadelphia: Lippincott-Raven. 4th ed. 2. Marshall JS. 2004. Mast-cell responses to pathogens. Nat. Rev. Immunol. 4:787–99 728

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177. Bailey SL, Schreiner B, McMahon EJ, Miller SD. 2007. CNS myeloid DCs presenting endogenous myelin peptides ‘preferentially’ polarize CD4+ T(H)-17 cells in relapsing EAE. Nat. Immunol. 8:172–80 178. Engelhardt B. 2006. Molecular mechanisms involved in T cell migration across the bloodbrain barrier. J. Neural. Transm. 113:477–85 179. Taube C, Miyahara N, Ott V, Swanson B, Takeda K, et al. 2006. The leukotriene B4 receptor (BLT1) is required for effector CD8+ T cell-mediated, mast cell-dependent airway hyperresponsiveness. J. Immunol. 176:3157–64 180. Ott VL, Cambier JC, Kappler J, Marrack P, Swanson BJ. 2003. Mast cell-dependent migration of effector CD8+ T cells through production of leukotriene B4. Nat. Immunol. 4:974–81 181. Di Girolamo N, Indoh I, Jackson N, Wakeﬁeld D, McNeil HP, et al. 2006. Human mast cell-derived gelatinase B (matrix metalloproteinase-9) is regulated by inﬂammatory cytokines: role in cell migration. J. Immunol. 177:2638–50 182. Kappler JW, Roehm N, Marrack P. 1987. T cell tolerance by clonal elimination in the thymus. Cell 49:273–80 183. Shevach EM. 2006. From vanilla to 28 ﬂavors: multiple varieties of T regulatory cells. Immunity 25:195–201 184. Chatila TA, Blaeser F, Ho N, Lederman HM, Voulgaropoulos C, et al. 2000. JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunity-allergic disregulation syndrome. J. Clin. Invest. 106:R75–81 185. Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, et al. 2001. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27:18–20 186. Lu LF, Lind EF, Gondek DC, Bennett KA, Gleeson MW, et al. 2006. Mast cells are essential intermediaries in regulatory T-cell tolerance. Nature 442:997–1002 187. Grimbaldeston MA, Nakae S, Kalesnikoff J, Tsai M, Galli SJ. 2007. Mast cell-derived interleukin 10 limits skin pathology in contact dermatitis and chronic irradiation with UV B. Nat. Immunol. 8:1095–104 188. Johnson D, Seeldrayers PA, Weiner HL. 1988. The role of mast cells in demyelination. 1. Myelin proteins are degraded by mast cell proteases and myelin basic protein and P2 can stimulate mast cell degranulation. Brain Res. 44:195–98 189. O’Brien PJ, Molino M, Kahn M, Brass LF. 2001. Protease activated receptors: theme and variations. Oncogene 20:1570–81 190. Johnson D, Yasui D, Seeldrayers P. 1991. An analysis of mast cell frequency in the rodent nervous system: Numbers vary between different strains and can be reconstituted in mast cell–deﬁcient mice. J. Neuropathol. Exp. Neurol. 50:227–34 191. Skokos D, Goubran-Botros H, Roa M, Mecheri S. 2002. Immunoregulatory properties of mast cell-derived exosomes. Mol. Immunol. 38:1359–62 192. Tkaczyk C, Frandji P, Botros HG, Poncet P, Lapeyre J, et al. 1996. Mouse bone marrowderived mast cells and mast cell lines constitutively produce B cell growth and differentiation activities. J. Immunol. 157:1720–28 193. Banu Y, Watanabe T. 1999. Augmentation of antigen receptor-mediated responses by histamine H1 receptor signaling. J. Exp. Med. 189:673–82 194. Cao J, Papadopoulou N, Kempuraj D, Boucher WS, Sugimoto K, et al. 2005. Human mast cells express corticotropin-releasing hormone (CRH) receptors and CRH leads to selective secretion of vascular endothelial growth factor. J. Immunol. 174:7665–75

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195. Kempuraj D, Papadopoulou NG, Lytinas M, Huang M, Kandere-Grzybowska K, et al. 2004. Corticotropin-releasing hormone and its structurally related urocortin are synthesized and secreted by human mast cells. Endocrinology 145:43–48 196. Cao J, Cetrulo CL, Theoharides TC. 2006. Corticotropin-releasing hormone induces vascular endothelial growth factor release from human mast cells via the cAMP/protein kinase A/p38 mitogen-activated protein kinase pathway. Mol. Pharmacol. 69:998–1006 197. Micera A, Lambiase A, Rama P, Aloe L. 1999. Altered nerve growth factor level in the optic nerve of patients affected by multiple sclerosis. Mult. Scler. 5:389–94 198. Micera A, Vigneti E, Aloe L. 1998. Changes of NGF presence in nonneuronal cells in response to experimental allergic encephalomyelitis in Lewis rats. Exp. Neurol. 154:41–46 199. Arredondo LR, Deng C, Ratts RB, Lovett-Racke AE, Holtzman DM, Racke MK. 2001. Role of nerve growth factor in experimental autoimmune encephalomyelitis. Eur. J. Immunol. 31:625–33 200. Levi-Montalcini R, Skaper SD, Dal Toso R, Petrelli L, Leon A. 1996. Nerve growth factor: from neurotrophin to neurokine. Trends Neurosci. 19:514–20 201. Zhang J, Li Y, Lu M, Cui Y, Chen J, et al. 2006. Bone marrow stromal cells reduce axonal loss in experimental autoimmune encephalomyelitis mice. J. Neurosci. Res. 84:587–95

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:705-739. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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T Follicular Helper (TFH) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay Immunology and Inﬂammation Research Program, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia; email: [email protected], [email protected], [email protected]

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Key Words

First published online as a Review in Advance on January 2, 2008

T cell help, autoimmunity, IL-21, chemokines, immunodeﬁciency, antibodies

The Annual Review of Immunology is online at immunol.annualreviews.org This article’s doi: 10.1146/annurev.immunol.26.021607.090344 c 2008 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0741$20.00

Abstract T cell help for antibody production is a fundamental aspect of immune responses. Only recently has a better understanding of the cellular and molecular mechanisms for T cell help emerged. A subset of T cells, termed T follicular helper cells (TFH cells), provides a helper function to B cells and represents one of the most numerous and important subsets of effector T cells in lymphoid tissues. TFH cells are distinguishable from Th1 and Th2 cells by several criteria, including chemokine receptor expression (CXCR5), location/migration (B cell follicles), and function (B cell help). Central to the function of CD4+ T cells is IL-21, a “helper” cytokine produced by TFH cells that potently stimulates the differentiation of B cells into Ab-forming cells through IL-21R. Consequently, dysregulation of TFH cell function, and over- or underexpression of TFH cell–associated molecules such as ICOS or IL-21, most likely contributes to the pathogenesis of certain autoimmune diseases or immunodeﬁciencies.

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INTRODUCTION

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One of the fundamental discoveries of modern immunology was the ﬁnding that antibody (Ab) responses require “help” from cells of thymic origin. In these classic experiments (1, 2), cells from different sources were transferred to irradiated recipients. Because irradiation destroyed the recipient’s endogenous immune system, any effect could be attributed to the donor cells. Neither bone marrow cells nor thymocytes alone were sufﬁcient to reconstitute an Ab response to sheep red blood cells. However, irradiated mice that received cells from both bone marrow and thymus responded well. These interacting cells later became known as B and T cells, respectively. Because the bone marrow–derived (B) cells but not the T cells were identiﬁed as the actual Ab-forming cells, the response was referred to as T-dependent, and thymus-derived cells were called T “helper” (Th) cells. The generation of Ab-forming cells occurs during a germinal center (GC) reaction (3, 4). GCs are specialized structures that develop within B cell follicles of secondary lymphoid tissues, such as lymph nodes, spleen, tonsils, and the Peyer’s patches of mucosal-associated lymphoid tissues. It is within the GC that critical processes such as somatic hypermutation, class switch recombination, and selection of high-afﬁnity B cells occur (3, 5, 6). The GC reaction is essentially the engine that drives T cell–dependent Ab responses, and the GC is an important site where T cells provide direct help to antigen-speciﬁc naive B cells. Importantly, CD4+ T cells are essential for the formation of a GC reaction (7), and they provide developmental cues for the differentiation of antigen-selected high-afﬁnity GC B cells into memory B cells or plasma cells, which together sustain long-term humoral immunity (6–8). Despite the fundamental role of CD4+ T cells in B cell responses and humoral immunity, there was, until recently, little appreciation of the cellular and molecular mechanisms for T cell help. Th2 cells, which produce cytokines such as IL-4 and direct B cells 742

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to undergo Ig isotype switching to IgG and IgE, were long considered to be the cells that provided help to B cells. It is now apparent that a subset of nonpolarized CD4+ T cells termed follicular B helper T cells (TFH ) (also referred to simply as T follicular helper cells) are the true helper cells for Ab responses, although other T cells such as Th2 cells, γδ T cells, and NKT cells may also contribute. The important developments that have driven this ﬁeld of study have been the discovery of numerous molecules that participate in providing T cell help to B cells. The interaction between CD40 on B cells and CD40L (CD154), transiently expressed on activated CD4+ T cells, stimulates B cell proliferation and, in the presence of appropriate cytokines, isotype switching as well (9). Inducible costimulator (ICOS) is another essential costimulatory molecule expressed on activated CD4+ T cells that, when engaged by its ligand (ICOS-L) on antigen-presenting cells (APCs) including B cells, induces the production of helper cytokines such as IL-2, IL-4, and especially IL-10 (10). However, it was the identiﬁcation of the chemokine receptor CXCR5 that has provided a particular impetus for the understanding of T cell help for B cells, as CXCR5 serves as a marker for TFH cells and promotes the colocalization of T and B cells in lymphoid follicles. Here, we discuss some of the controversial questions concerning TFH cell differentiation, the relation of TFH cells to other T cell subsets such as Th2 and/or Th17, and the inﬂuence of several molecules that are highly expressed by TFH cells and most likely participate in T cell–dependent B cell differentiation. One molecule we discuss in detail is IL-21, which has all the hallmarks of a “helper” cytokine (a separate article in this volume covers the biology of IL-21; see Reference 11). IL-21, despite its roles in other systems, is a TFH cell–secreted cytokine and is one of the most important stimulators of B cell proliferation, isotype switching, and differentiation (12). Indeed, the ﬁelds of IL-21 and CD4+ T cell help are rapidly converging.

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Finally, we argue that many immunological disorders, including autoimmune diseases, immunodeﬁciencies, and malignancies, relate to dysfunction of TFH cells or their associated effector molecules. Thus, targeting of TFH cell molecules should offer new opportunities for the therapeutic manipulation of immune responses.

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CXCR5 AND THE IDENTIFICATION OF TFH CELLS The identiﬁcation of the true CD4+ helper T cell for B cells followed the discovery of CXCR5. CXCR5 is a chemokine receptor expressed by all mature B cells as well as a subset of antigen-experienced CD4+ T cells in lymphoid tissues. CXCR5 is largely absent from CD8+ T cells and naive CD4+ T cells. CXCR5 is responsible for the positioning of B and T cells in the follicular areas of lymphoid tissues (13) through recognition of its ligand, the chemokine CXCL13, which is produced by follicular stromal cells, including follicular dendritic cells (FDCs) (14, 15). Remarkably, ∼50% of CD4+ T cells in activated lymphoid tissues, such as human tonsils, are CXCR5+ , and a subset of these localizes to the GC (16–18). Indeed, following antigen stimulation, the quantity of CXCR5+ CD4+ T cells in lymphoid tissues far exceeds that of any other type of effector T cell. CXCR5+ CD4+ T cells isolated from human tonsils are efﬁcient at providing help to B cells, thereby facilitating their differentiation into plasma cells (17– 21). Because of their ability to provide B cell help, their clear phenotypic distinction from Th1 and Th2 cells, and their predominant localization to B cell follicles, CD4+ CXCR5+ T cells were termed T follicular helper (TFH ) cells (17–19).

ORIGIN OF TFH CELLS AND THEIR RELATION TO Th1, Th2, Th17, AND Treg CELLS Perhaps the most pertinent yet unresolved issue pertaining to TFH cells is their relation-

ship to other T cell subsets, such as Th1, Th2, Th17, and T regulatory (Treg) cells (see Figure 1). Various transcription factors determine effector T cell differentiation, and the ﬁve main effector T cell subsets express distinct transcription factors (Figure 1). The ﬁve effector T cell subsets depicted in Figure 1 secrete different cytokines, express different chemokine receptors, and localize to diverse sites. These functions relate to the cells’ specialized roles in immune defense. For instance, Th2 cells produce cytokines associated with defense against large extracellular parasites (Figure 1). T cell help for B cells was long attributed to Th2 cells because Th2 clones support Ab production in vitro better than Th1 clones (22) and because IL-4, a Th2 cytokine, stimulates B cell proliferation and class switching and induces upregulation of costimulatory molecules such as CD40 and MHC class II. However, B cell help still occurs in the absence of IL-4, inasmuch as IL-4deﬁcient mice can generate T-dependent Ab responses (23, 24). A revised model for T cell help shows that TFH cells are the predominant helper cells for B cells, and that Th1 or Th2 cytokines serve to skew responses in particular directions. For instance, IL-4 promotes Ig isotype switching to IgE, which is important for antiparasite responses, whereas IFN-γ favors isotypes important for antiviral immunity. Figure 1 depicts a relatively simple model for the differentiation of the various T effector subsets; however, the point at which functional TFH cells emerge is uncertain. The development of a follicular homing capability by activated T cells is the ﬁrst event in the process. Naive T cells are CXCR5− and use the chemokine receptor CCR7 to enter secondary lymphoid organs and migrate to T cell zones (25). CXCR5 is transiently upregulated on CD4+ T cells following their activation, thereby endowing them with a follicular homing capability. This upregulation occurs prior to proliferation and differentiation and is dependent on costimulatory signals delivered through CD28, OX40, and ICOS www.annualreviews.org • T Follicular Helper Cells

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Function

Th1 T-bet

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IFN-γ TNF

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IL-4 IL-13

Immunity to extracellular parasites

TGF-β

Regulation/ tolerance

IL-17

Inflammation, fungal immunity

IL-21

T cell help for B cells

CXCR3 CCR5

GATA-3 STAT-6 CrTh2

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TGF-β

Foxp3

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Figure 1 Effector T cell differentiation and the expression of transcription factors, effector cytokines, and chemokine receptors. Transcription factors for each subset have been placed in the nucleus (note that the role of Bcl-6 in TFH cell development still requires conﬁrmation). The list of chemokine receptors, or cytokines, for each of the subsets is not exhaustive. Adapted with additions from Reference 32.

(26–30) (see also Figure 3 below). However, CXCR5 expression on activated T cells is only transient, and fully polarized Th1 and Th2 cells do not express this chemokine receptor (31). Several different fates are possible for activated T cells. Many lose expression of CXCR5 and either follow the Th1/Th2 (or Th17/Treg) differentiation pathways or leave the secondary lymphoid tissues to become circulating memory T cells. A fraction of cells, however, retains CXCR5 expression. These antigen-primed cells develop into effector T cells that can home to B cell follicles 744

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and, ultimately, provide help to B cells. The capacity to provide help must relate to acquisition of the relevant molecules— such as ICOS, CD40L, and IL-21—that have functional roles in humoral immunity (discussed below). However, these molecules can also be expressed by other T cell subsets, and so the differentiation steps leading to the generation of TFH effector cells are still largely unclear. A possible scenario for the generation of TFH cells has all T helper subsets (i.e., Th1, Th2, Th17, and TFH cells) arising independently from naive T cells, as suggested in Figure 1. In this

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scenario, activated CXCR5+ T cells serve as an intermediate cell type that gives rise to all the effector subsets depending on additional stimuli. Nevertheless, it must be stressed that a precise understanding of how Th cell fate is determined is still unclear. Activation of transcription factors, which determine cellular fate, must occur at certain points, and we propose that Bcl-6 is the transcription factor most likely to be required for the development of TFH cells. It is likely that after the initial activation by antigen-loaded APCs in the T cell zone, T cells destined to become TFH cells must receive additional signal(s) provided by cells located in or close to the B cell follicles. Two distinct cell types are excellent candidates for this role: B cells and CD4+ CD3− accessory cells. B cells may inﬂuence the differentiation of T cells into TFH cells. Abundant evidence also conﬁrms that B cells direct T cell development, polarization, and memory (33–37). B cells may also inﬂuence further differentiation of TFH cells into Th1 or Th2 effector cells, as B cells are capable of producing IL-12 and IL-4 (38, 39), which may induce T cell polarization. CD4+ CD3− accessory cells could also provide the secondary signals for the differentiation of activated CXCR5+ T cells into TFH effector cells (40). These cells are of a nondendritic lineage and interact with antigen-speciﬁc CD4+ T cells that have been previously primed by dendritic cells (DCs). This interaction may depend on signals through OX40 and CD30. Furthermore, they are localized at the sites of T-B collaboration: B cell follicles and the T-B interface (40). A particularly controversial topic is the relationship of TFH cells to Th17 cells. Th17 cells rely on the transcription factor RORγt and cytokines such as TGF-β together with either IL-6 or IL-21 for their differentiation and function, which are clearly distinct from those used by Th1 or Th2 cells (Figure 1). Even though Th17 cells were not deﬁned when TFH cells were discovered, TFH cells do show some striking similarities

to Th17 cells, particularly autocrine stimulation by IL-21 and provision of B cell help (41) (discussed below). Nevertheless, TFH cells are clearly distinguishable from Th17 cells in that they do not express Th17 cytokines such as IL-17 and IL-22, and Th17 cells do not express CXCR5 (120). However, many of the molecules that characterize TFH cells are also involved in functions associated with other effector subsets, including Th17 cells. Our analysis of TFH cell gene transcription using Affymetrix microarrays has failed to identify many of the gene transcripts associated with Th17 cells, such as RORγt, IL-17, IL-22, IL23R, or CCR6 (C.R. Mackay, unpublished data).

MOLECULES ASSOCIATED WITH TFH CELL FUNCTION The features of CXCR5+ TFH cells that distinguish them from Th1, Th2, Th17, or other T cells (42) are summarized in Figure 2. Like other effector T cells, TFH cells in lymphoid tissues express markers indicative of activation, such as CD69, CD95, and ICOS, and also express low levels of CCR7 and CD62L (similar to Th1 and Th2 cells). They also have effector function, namely provision of help for Ab production (16–18, 20, 21). Furthermore, although CXCR5+ T cells have a very limited repertoire of cytokine secretion, they do express IL-21, which, despite its association with NKT cells (43) and Th17 cells (44, 45), is highly associated with TFH cells (Figure 2) (discussed below). The primary function of TFH cells—provision of help for the differentiation of B cells into effector cells during a GC reaction—is substantiated by reports that TFH cells, compared with nonTFH cells, express increased levels of CD40L, ICOS, and IL-10 (17, 18, 20, 46). These molecules positively regulate B cell differentiation, and humoral immune responses are compromised in mice or humans with mutations in the CD40L and ICOS genes (47–50). www.annualreviews.org • T Follicular Helper Cells

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TFH cell Other important TFH-expressed molecules PD-1 Bcl-6 CD200 CD30L

CD57 Fyn CDK5r1 GPR18

IL-6R IL-21R BTLA Sialyltransferase 8A

Figure 2 Important molecules for TFH cell function. (Left) Human tonsil stained for IL-21 expression using an anti-IL-21 mAb. Regions of the tonsil are indicated, including T cell areas (T), the B cell follicle (B), and the germinal center (GC). Note the positioning of IL-21-expressing cells at the rim of the GC. IL-21 expression is not restricted to the GC, and numerous cells in the T cell zone express IL-21. Whether these represent TFH -like cells or Th17 cells remains to be determined. (Right) Some of the best-characterized interacting molecules for TFH and B cells. Listed below are numerous other molecules overexpressed in TFH cells that presumably play an important role in their function.

Interestingly, circulating TFH cells are deﬁcient in humans with mutations in CD40L or ICOS genes (29). Recent microarray analyses showed that TFH cells express an extensive array of genes that distinguishes them from Th1- and Th2type CD4+ T cells (51–54). For instance, compared with conventional CD4+ T cells, human TFH cells express higher levels of IL-21, the intracellular adaptor protein SAP (SLAM-associating protein), SAP-associating transmembrane receptors CD84 and CD229 (Ly9), the protein tyrosine kinase FynT, as well as the GC-restricted transcription factor Bcl-6 (51, 52, 54) (see also Figure 2). Using the Roquin mouse, which has expanded TFH cells as a result of a defect in degradation of ICOS transcripts (discussed below), Vinuesa et al. (53) showed that mouse TFH cells have a molecular proﬁle remarkably similar to that of human TFH cells, as they also prefer746

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entially express transcripts for IL-21, CD84, CXCL13, Bcl-6, PD-1, and many others. The relevance of some of the more important TFH cell-expressed molecules is discussed below.

Bcl-6 Transcription factors determine T cell fate, and the important elements that drive Th1, Th2, Th17, and Treg cell differentiation have now been identiﬁed (see Figure 1). For instance, T-bet determines Th1 cell lineage commitment and cytokine production, and GATA-3 and c-Maf drive Th2 cytokine production (55). Th17 differentiation is initiated by TGF-β and either IL-6 or IL-21, which activate signal transduction and activator of transcription (STAT) 3 and induce expression of the transcription factor RORγt, ultimately resulting in lineage speciﬁcation (56, 57). Currently there is no deﬁnitive

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evidence for a particular transcription factor directing the commitment of naive CD4+ T cells to the TFH cell lineage, although circumstantial evidence favors Bcl-6. The expression of Bcl-6 is tightly regulated and is largely conﬁned to GC B cells (58–60). Also, Bcl-6 is preferentially expressed by TFH cells but not by Th1 or Th2 cells (51). In B cells, Bcl-6 is a master regulator of GC lineage commitment and is a suppressor of plasma cell differentiation (61). Bcl-6-deﬁcient mice display defective T-dependent Ab responses and have limited Ab afﬁnity maturation owing to the absence of GCs (62, 63). Bcl-6 expression also represses GATA-3 and downregulates secretion of IL-4 by T cells, thus providing further support of a dynamic role for Bcl-6 in T cell differentiation and fate (64). However, expression of Bcl-6 by T cells in follicles is by no means uniform: Only 10%–15% of CD4+ T cells in GCs appear to express Bcl-6 protein (65). It is conceivable that Bcl-6 determines TFH commitment at stages before or after Th1, Th2, or Th17 differentiation.

ICOS ICOS is a CD28-like costimulatory molecule with an important role in T-dependent Ab responses. Expression of ICOS is regulated during T cell activation and differentiation, such that ICOS upregulation occurs following the receipt of signals during T cell activation (Figure 3). T cells stimulated through the T cell receptor (TCR), CD28, and ICOS proliferate and produce cytokines, such as IL4 and IL-10, that facilitate T:B interactions and antibody production (10, 37, 66). ICOS is expressed at very high levels on TFH cells within the light zone of GCs (10, 17, 67), and a direct correlation exists between the expression of ICOS by CD4+ T cells and the amount of IL-10 produced (37, 68). This correlation suggests an important role for this pair of effector molecules in regulating B cell responses. Indeed, ICOS deﬁciency is associated with impaired IL-10 production in human and murine CD4+ T cells (48, 69, 70).

ICOS is also expressed by other T cells, notably Th2 cells, whereas its ligand, ICOSL, is expressed on APCs, including B cells (71, 72). In contrast to CD28, ICOS is not constitutively expressed, but is instead induced after T cell activation (10) (Figure 3). ICOS signaling enhances T cell proliferation, the secretion of cytokines, especially IL-10, as well as the upregulation of cell-cell interaction molecules. The genes upregulated following engagement of ICOS are similar to those upregulated by CD28 in both human- and murine-stimulated CD4+ T cells, although the magnitude of gene upregulation induced by ICOS is less than that induced by CD28 (73, 74). Probably the most important difference between signals delivered through CD28 and ICOS is that their ligands are expressed at high levels on different cell types (DCs and B cells, respectively). This difference relates to the speciﬁc roles of these two molecules, ﬁrst for the regulation of T cell priming, and subsequently for T-B interactions (see Figure 3). Homozygous loss of ICOS is associated with late-onset common variable immunodeﬁciency (CVID) in humans (49). Patients with CVID due to ICOS deﬁciency lack GC in their lymph nodes, consequently show a dramatic reduction in the number of IgM-expressing memory B cells, and are essentially devoid of isotype-switched memory cells. Interestingly, these patients also have a deﬁciency in the number of CXCR5+ CD4+ T cells in their peripheral blood. This ﬁnding may explain the reduced production of IL-10 and IL-17 by their CD4+ T cells (29, 49). Consistent with these results, mice deﬁcient in ICOS or ICOS-L also have impaired GC formation and isotype switching, diminished TFH cell numbers in their spleens, and reduced production of B cell helper cytokines, such as IL-4 and IL-10 (29, 30, 48, 78–81). These ﬁndings suggest that ICOS signaling is important for the maintenance and/or generation of TFH cells (29, 30) and reinforce the association between ICOS expression and IL-10 production. In support of this www.annualreviews.org • T Follicular Helper Cells

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TFH cell

CCR7

T cell

IL-21R

IL-21R

CD40

B7 pMHCII

FDC

B cell APC

BCR

d Plasma cell

Figure 3 Multiple signals and steps for the generation of TFH cells and T-dependent antibody responses. CD28 and ICOS, together with IL-21, costimulate T cells activated through the T cell receptor (TCR). The ordered expression of the various costimulatory molecules, cytokines, and receptors facilitates the various steps in the process of TFH cell differentiation and function. (a) In the T cell zone of lymphoid tissues, mature DCs expressing B7.1 and B7.2 present peptide-MHC class II (pMHCII) ligand to the TCR of naive CD4+ T cells, which constitutively express CD28 (CD28c). CD28 provides the early costimulatory signal that, with TCR stimulation, induces expression of other costimulatory receptors belonging to the Ig and TNFR/TNF superfamilies (75). Ligation of CD28 ampliﬁes the TCR signal rather than delivering a qualitatively different signal (75, 76). (b) Activated CD4+ T cells produce IL-21 and induce expression of CD28 (CD28i) and ICOS. (c) Sustained signaling of activated CD4+ T cells through the TCR, CD28, and IL-21R in the T zone and at the T-B cell interface leads to modulation of the expression of molecules important for migration, such as CXCR5, CCR7, and costimulatory receptors including ICOS, CD40L, and OX40 (28, 75, 77). (d ) Migration of functional TFH cells to follicles and delivery of T cell help support the selection of activated Ig-secreting B cells in germinal centers.

association, one study showed that the B helper activity of tonsillar TFH cells corresponded with a high level of expression of ICOS, such that only the CXCR5hi ICOShi CD4+ T cells were potent inducers of IgG production by cocultured B cells (82). Thus, the reduced GC response associated with ICOS deﬁciency might simply result from reduced numbers of TFH cells. The high expression of both ICOS and IL-21 are deﬁning features of TFH cells, and ICOS expression deﬁnes the subsets of Th cells that are proliﬁc producers of IL-21. However, where these two axes meet at the 748

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level of TFH cell generation remains unclear. On the one hand, IL-21 may drive ICOS expression. We think this is unlikely because IL21-deﬁcient mice have a defect in TFH cell generation but express an abundance of ICOS on all CXCR5− CD4+ T cells (A. Vogelzang & C. King, submitted manuscript). On the other hand, the assumption that ICOS expression is necessary for IL-21 production is also unlikely because blockade of ICOS:ICOSL interactions signiﬁcantly reduces but does not eliminate IL-21 production, and T cells from ICOS-deﬁcient mice can produce limited amounts of IL-21 (H. McGuire

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& C. King, submitted manuscript). Our view is that both IL-21 and ICOS are necessary for TFH cell generation and that T helper cells utilize ICOS:ICOS-L interactions that quantitatively contribute to IL-21 production.

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CD57 Immunohistochemical studies performed over the past two decades identiﬁed a small population of CD4+ T cells that were distinguishable by their coexpression of the NK cell marker CD57. These cells comprise ∼10% of all tonsil CD4+ T cells and localize to GCs (20, 21, 83, 84). Interestingly, all tonsillar CD4+ CD57+ T cells also express CXCR5 and thus represent ∼20% of the CXCR5+ subset (20, 21). Furthermore, CD4+ CXCR5+ CD57+ T cells in human tonsils express the activation molecules CD69, ICOS, HLA-DR, PD-1, OX40, and CD45RO and downregulate CCR7 and CD62L (20, 85; S. G. Tangye, unpublished observations), suggestive of an activated phenotype and/or effector function. Accordingly, CD57 has been described as a deﬁning marker for T helper cells for B cells (20, 83). Despite these observations, the function of the CD57 molecule remains uncertain, and inconsistent ﬁndings have prompted researchers to question whether CD57 is actually a valid marker for TFH cells. For instance, CD57+ CD4+ T cells are incapable of producing B cell helper cytokines such as IL-4 and IL-10, and they are inefﬁcient at inducing differentiation of cocultured autologous B cells into Ig-secreting cells in vitro (85–91). These ﬁndings are consistent with the proposal that CD57+ GC T cells are anergic (88) and exhibit regulatory activity by suppressing the function of CD4+ CD57− T cells (92). In contrast, other recent studies have reported that CXCR5+ CD57+ CD4+ T cells isolated from human tonsils exhibit increased cytokine production and induce greater B cell differentiation than do CXCR5+ CD57− CD4+ T cells (20, 46, 83, 93, 94). The

use of CD57+ CD4+ T cells from blood in some studies and from tonsils in others may have produced some of these differences. The fact that CXCR5+ and CXCR5− CD4+ T cells in blood and lymphoid tissues differ substantially with respect to their function and phenotype may have also contributed to the varying results. Our own studies have revealed that both tonsillar CXCR5+ CD57− and CXCR5+ CD57+ CD4+ T cells can induce differentiation of autologous B cells in vitro, whereas CXCR5− CD57− CD4+ T cells cannot. However, more Ig secretion was detected in cultures containing CXCR5+ CD57− T cells than in those with CXCR5+ CD57+ T cells (21). The most likely cause of these differences is the fact that CXCR5+ CD57+ CD4+ T cells undergo apoptosis more rapidly than CXCR5+ CD57− CD4+ T cells (46, 54, 95). Furthermore, expression of CD95 and PD-1 is higher on CXCR5+ CD57+ T cells than on CXCR5+ CD57− CD4+ T cells (S.G. Tangye, unpublished data), suggesting that in addition to the heightened intrinsic susceptibility of CXCR5+ CD57+ T cells to death, these cells may undergo greater apoptosis following engagement of death receptors by ligands expressed on B and T cells. High expression of ICOS, rather than CD57, probably better deﬁnes TFH cells capable of inducing Ig secretion from B cells (46, 51). Because ICOS is expressed at the highest level on CXCR5+ CD57+ T cells (S.G. Tangye, unpublished data), CXCR5+ CD57+ and CXCR5+ ICOShi + CD4 T cells probably share many phenotypic and functional characteristics.

IL-21: A TFH CELL–EXPRESSED HELPER CYTOKINE IL-21 was recently identiﬁed as a cytokine that costimulates lymphocyte proliferation and drives the differentiation of NK cells in vitro (96). IL-21 is a member of the common γ chain (γc)-signaling family of cytokines (96; see also Reference 11, in this volume). The www.annualreviews.org • T Follicular Helper Cells

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receptor for IL-21 (IL-21R) can be expressed by many cell types (97), but it is found predominantly on B cells as well as on some NK cells and T cells (51, 96, 98, 99). The IL-21R consists of a unique cytokine-binding protein (IL-21Rα) and the γc (98, 100), which is also a component of receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15 (101). In vivo and in vitro studies have demonstrated a central role for IL-21 in lymphocyte activation, survival, and differentiation. Initial studies of the in vitro effects of IL-21 focused on its ability to enhance effector functions, such as IFN-γ production and cytotoxic function of CD8+ T cells and NK cells (102). More recently, IL-21 was shown to play a role in NKT cell and CD4+ T cell effector function (43). Notably, IL-21 greatly enhanced proliferation of human CD4+ CD25− T cells, rendering them resistant to Treg-mediated suppression (103). The ability of IL-21 to enhance the proliferation of T cells is in marked contrast to other γc-signaling cytokines such as IL-2 and IL-15 because IL-21 does not induce proliferation in the absence of stimulation through the TCR (96). Thus, IL-21 is a genuine T cell costimulator, and its potential for amplifying signals through the TCR is supported by evidence demonstrating that IL-21:IL-21R interactions generate signals through the Jak-STAT, MAPK, and PI3K pathways (104). The study of the role of IL-21 in Th cell differentiation is a rapidly evolving ﬁeld, and the ﬁndings from a number of studies resonate with the important role of IL-21 in humoral responses. Recent studies have revealed that IL-21 is important for the generation and migration of Th2 cells (105–107), and TFH cells, in turn, express signiﬁcantly greater amounts of IL-21 than do Th1 or Th2 subsets (51). Our own work with IL-21and IL-21R-deﬁcient mice indicates that IL21 is an autocrine growth factor for TFH cells (Figure 3) (A. Vogelzang & C. King, submitted manuscript). IL-21 is a crucial component of TFH cell generation through its abil-

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ity to modulate expression of the chemokine receptors CXCR5 and CCR7 on activated T cells (A. Vogelzang & C. King, submitted manuscript). A defect in TFH cell generation in IL-21-deﬁcient mice suggests a role for IL21 that precedes the upregulation of CXCR5 and movement of TFH cells into the B cell follicle. Collectively, these studies indicate that IL-21 has a fundamental role in T helper cell differentiation. One major target of IL-21 is B cells. Studies of ex vivo isolated human and murine B cells have demonstrated that IL-21 greatly enhances proliferation induced by ligation of CD40 (96, 108–110). In contrast, IL-21 inhibits proliferation of murine B cells induced by TLR ligands, namely LPS and CpG, by inducing apoptosis (108, 111, 112). Interestingly, some of the pro- and antiproliferative effects of IL-21 on murine B cells appear to be strain speciﬁc (108). IL-21 also modulates isotype switching and Ig production by human and murine B cells, as it can act as a switch factor for secretion of IgG1 and IgG3 by human B cells stimulated with anti-CD40 mAb (113), and induces differentiation of murine and human naive, GC and memory B cells into Ig-secreting cells (21, 110, 114). Although IL-10 had been considered the most potent inducer of plasma cell differentiation on human B cells, IL-21 exceeds its effects on CD40-stimulated human B cells by up to 100-fold (21). Human B cells stimulated with CD40 mAb and IL-21 are induced to express the enzyme activationinduced cytidine deaminase (AID) and the transcription factor B-lymphocyte maturation protein-1 (Blimp-1) (110), which play critical roles in Ig isotype switching and commitment to the plasma cell lineage, respectively. Importantly, the ability of human tonsillar TFH cells to provide B cell help for their differentiation into plasma cells is IL-21 dependent, as this effect was greatly reduced by neutralizing endogenous IL-21 present in these cultures (21). The action of IL-21 on the differentiation of human naive B cells into Ig-secreting cells could be reduced by

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IL-4 (21, 110)—through the ability of IL-4 to antagonize the IL-21-induced expression of Blimp-1 (21)—whereas the combination of IL-4 and IL-21 resulted in increased production of IgE in cultures of enriched human B cells (113, 115). In contrast to its effects on human B cells, IL-21 inhibited IgE production by murine B cells stimulated with LPS and IL4 (116). Together, these ﬁndings demonstrate a dynamic interplay among several different γc-binding cytokines. Several of the ﬁndings from in vitro cultures have been conﬁrmed in vivo. For instance, mice transgenic for expression of human IL-21 have increased numbers of splenic B cells, including Ig isotype–switched cells and plasma cells (114). However, a deﬁciency of either IL-21 or its receptor results in an impaired ability to produce antigenspeciﬁc IgG following immunization with a T cell–dependent antigen. Furthermore, the levels of total as well as antigen-speciﬁc IgE are elevated in the serum of these mice (117, 118). This ﬁnding conﬁrms the negative regulatory effects of IL-21 on IgE production by murine B cells (116, 117). Notably, Tdependent immune responses are completely abrogated in mice deﬁcient in IL-4 and IL21R (117), suggesting that signaling through both IL-4R and IL-21R is necessary for optimal humoral immune responses. Recently, IL-21 was described as an essential autocrine factor for the generation and differentiation of Th17 cells (44, 45, 119), challenging a previous study that demonstrated unimpeded Th17-driven experimental autoimmune myocarditis in the absence of IL-21R signaling (105). These dramatic new ﬁndings bring into question the precise role of IL-21 and the relationship between TFH cells and Th17 cells. If Th17 cells produce copious amounts of IL-21, as indicated, it would imply that these T cells may also deliver a helper signal to B cells. However, one factor that may limit the interaction of Th17 cells with B cells is their expression of chemokine receptors: Th17 cells express CCR4 and CCR6 but lack CXCR5 (41, 120), which would recruit

Th17 cells to sites of inﬂammation but would exclude them from entry into B cell follicles.

MIGRATION AND LOCALIZATION OF TFH EFFECTOR AND MEMORY CELLS Chemoattraction to the CXCR5 ligand CXCL13 allows TFH cells to localize to B cell follicles, where they can directly provide help to B cells. CXCR5 is also expressed by most B cells and is required for the development of B cell follicles in secondary lymphoid tissues. Mice lacking either CXCR5 or CXCL13 show major aberrations in follicular architecture and reduced numbers of lymph nodes and Peyer’s patches (13, 121). Expression of CXCR5 is transiently upregulated when T cells interact with peptide-MHC class II and with costimulatory molecules on APCs (122, 123), and levels of CXCR5 expression following priming may reﬂect qualitative or quantitative aspects of this stimulation. For instance, antigen-speciﬁc TCRtransgenic CD4+ T cells exhibit a higher and more homogenous expression of CXCR5 following immunization than do their polyclonal counterparts (123). One factor that distinguishes TFH from recently activated CD4+ T cells is their continued expression of CXCR5. Further interaction with peptideMHC class II ligand and costimulatory molecules on B cells at the B cell/T cell interface and within the GC is likely to preserve CXCR5 expression on TFH cells. Interaction between effector T cells and B cells occurs ﬁrst in T cell areas and later in B cell follicles within secondary lymphoid organs (3, 4, 124). After the ﬁrst engagement, B cells either migrate to extrafollicular foci, where they differentiate into plasma cells that rapidly secrete low-afﬁnity antibodies, or they migrate (along with T cells) into follicles and form GCs, where somatic mutation and afﬁnity maturation occur. As emphasized previously, the GC reaction is strongly dependent on help from antigen-speciﬁc T cells, www.annualreviews.org • T Follicular Helper Cells

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which must migrate into the follicle to support this response (123–126). The chemoattractant receptor characteristically expressed by TFH cells is CXCR5, which facilitates TFH cell migration to follicles. However, the conditions under which TFH cells localize to follicles may vary. Naive T cells that recirculate through lymphoid tissues gain entry primarily through their expression of L-selectin and the chemokine receptor CCR7. T cells colocalize with DCs in the T cell zone, allowing unprimed T cells to ﬁrst receive signals from professional APCs rather than from B cells. A remarkable sequence of cellular movements follows successful T cell priming (15): Activated T cells (which have upregulated CXCR5) migrate to the B cell follicles and position themselves at the edge of the follicle, where they meet antigen-primed B cells that have speciﬁcally migrated outward. Physical interactions then facilitate CD40-CD40L-dependent B cell activation. Thereafter, antigen-primed T cells are distributed throughout the entire follicle, including the GC, to provide helper signals to B cells. Whether TFH cells can enter follicles directly from the blood is an interesting but unresolved question. Although TFH cells have all the hallmarks of an activated, effector subset of T cells, unresolved issues remain: The exact proportion of CXCR5+ CD4+ T cells in lymphoid tissues that can provide help to B cells and the precise location of these T cells are unknown. Certainly, CXCR5+ CD4+ T cells in GC can be considered true TFH cells; however, T cells closely related in phenotype and function most likely exist outside of follicles (for instance, note the large numbers of IL-21+ cells in the T cell zone shown in Figure 2). Moreover, at least six different follicular T cell subsets have been identiﬁed on the basis of surface markers and/or anatomical location (reviewed in Reference 42). In human tonsils, follicular T cells are located in the mantle zone, the outer zone, and the light zone of the GC. Although they are all deﬁned by CXCR5 expression, they differentially ex-

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press other markers such as CD57 and ICOS (discussed below). A subset of CXCR5+ CD4+ T cells also exists in blood (∼10%) (16), but these cells are in a resting state and are incapable of providing help to B cells. They coexpress CD62L and CCR7 and probably represent a subset of TFH cell–derived memory T cells. The fate of TFH cells following the GC response has signiﬁcant implications for immunological memory, vaccine development, and autoimmunity. Until recently, TFH cells were considered fully differentiated cells, prone to apoptosis due to high expression of CD95 (54). However, a recent study demonstrated that a local memory CXCR5+ CD69+ TFH cell compartment is retained in B cell follicles after resolution of the GC response (127). A population of high-afﬁnity CXCR5+ T cells remained for an extended period of time in the proximity of CXCL13-expressing FDCs and were reactivated upon secondary immunization (127). Thus, immunological memory associated with TFH cells probably differs markedly from memory associated with other types of T cells. Furthermore, because the site for TFH cell function is lymphoid tissue, it would not be surprising if immunological memory in this T cell population were more sessile than that in, for instance, a CD8+ T cell or Th1 population. Although the possibility that CXCR5 expression on TFH memory cells becomes ﬁxed during differentiation remains, it is more likely that the microenvironment supplies some factor(s) that maintains their CXCR5hi phenotype. TFH memory cells were found in the vicinity of peptide-MHC class II depots in draining lymph nodes after immunization (127), suggesting that local triggering of TCR maintains expression of CXCR5 and CD69 on TFH cells. The maintenance of CXCR5 expression on TFH cells would be consistent with a continued interaction between a small number of remaining antigen-speciﬁc T cells and B cells, or other APCs, near the FDC network after resolution of the GC. However, the level of ongoing stimulation was not

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adequate to maintain expression of TFH effector cell molecules such as ICOS and IL21, which require antigen challenge for their reexpression. Thus, TFH effector cells differ markedly in their localization and recirculation patterns compared with other effector T cell subsets (128). Whereas most effector T cells, such as IFN-γ-producing Th1 cells or cytotoxic CD8+ T cells, localize to peripheral tissues or inﬂammatory lesions, TFH effector or memory cells localize to lymphoid tissues, particularly B cell follicles.

CXCR5+ γδ T CELLS Although T cell help for B cells has historically been viewed in the context of conventional CD4+ αβ TCR+ cells, the fact that αβ T cell–deﬁcient mice can mount Ab responses to T-dependent Ag suggests that other lymphocytes may provide help for B cells (129). Subsequent studies demonstrated that γδ T cells underlie the ability of αβ T cell– deﬁcient mice to produce GCs and relatively normal levels of serum Ig (129). Researchers (130) also recently found that ∼15% and 50% of γδ T cells present in peripheral blood and tonsils, respectively, express CXCR5. These frequencies are similar to those of CXCR5+ CD4+ T cells in these sites. Furthermore, CXCR5+ γδ T cells in tonsils display a phenotype, helper cytokine proﬁle, and effector function that resemble those of tonsillar CXCR5+ CD4+ T cells: CXCR5+ γδ T cells express CD45RO, CD27, HLA-DR, CD40L, and ICOS, but not CCR7 and CD62L (130); they produce vast amounts of IL-4 and IL10, but low levels of IFN-γ and TNF-α; and they have the ability to induce differentiation of cocultured B cells (130). The in vitro ﬁndings are consistent with the detection of γδ T cells within the FDC network of established GC in human lymphoid tissues (131). Determining whether IL-21 plays any role in the helper function of CXCR5+ γδ T cells may provide some interesting insights. Of note, γδ T cells express IL-21R and CXCL13 (a signature molecule of TFH cells)

when stimulated with IL-21 (132). Autocrine production of IL-21 by TFH cells may also regulate their production of CXCL13, and together these molecules guide both γδ and αβ CXCR5+ T cells to follicular areas of lymphoid tissues, where they would be positioned to provide B cell help. Because αβ and γδ T cells recognize different repertoires of Ag, the compartmentalization of a subset of each of these populations capable of inducing Ig secretion would provide a mechanism whereby humoral immune responses could be elicited against a diverse array of Ag irrespective of the type of responding T cell.

TFH CELL DYSREGULATION AND DEVELOPMENT OF AUTOIMMUNITY New B cell receptor speciﬁcities, including those with autoreactivity, arise continuously during the GC reaction. Ab-forming cells that emerge from the GC reaction need to be tightly controlled, owing to their longevity and production of high-afﬁnity antibodies. Consequently, most B cell responses depend on T cell help. The absence of T cell help during B cell priming leads to apoptosis, rather than differentiation of B cells into GC cells or plasma cells. Thus, self-reactive follicular B cells are usually precluded from differentiation within GCs and are normally absent from the memory and plasma cell compartments (133). B cells must compete for T cell help, and the presence of self-reactive T cells leads to the emergence of high-afﬁnity self-reactive B cells. Accordingly, any dysregulation of T cell function or tolerance induction can have a signiﬁcant effect on the selection of Ab speciﬁcities. There is substantial evidence for a T cell basis for autoantibody responses in certain autoimmune diseases. GC formation has been described in many autoimmune strains of mice, and these arise during onset of autoantibody production (134). In patients with autoimmune diseases, for example systemic lupus erythematosus (SLE), www.annualreviews.org • T Follicular Helper Cells

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self-reactive B cells survive within GCs and differentiate to plasma cells and memory cells (133). The involvement of TFH cells in autoantibody production is evident in various strains of mice that either over- or underexpress important TFH cell–associated molecules such as CD40L, ICOS, SAP, and IL-21. Blocking CD40CD40L interactions prevents GC formation, autoantibody production, and the aberrant accumulation of GC-like B cells and plasmablasts in the peripheral blood of patients with SLE (135) as well as in murine models of lupus (134). Moreover, two different murine models of human lupus are characterized by a TFH cell–like transcriptome in their spleens (53, 136). IL-21 is also overexpressed in BXSB mice, which develop murine lupus (114). Interestingly, human SLE patients and lupus-prone mice have an increased frequency of ICOS+ CD4+ T cells in their peripheral blood (137) and spleens (138), respectively, suggesting an expansion of TFH cells in these pathogenic conditions. Likewise, Roquin−/− mice exhibit a clear increase in numbers of TFH cells as a result of excessive signaling through ICOS. It is unclear whether dysregulated signaling though ICOS results in the characteristic increase in il-21 message noted for lupus-prone mice. However, because blocking ICOS/ICOS-L interactions (138) or neutralizing IL-21 (139, 140) ameliorates disease in animal models of human lupus and rheumatoid arthritis, increased expression of ICOS may augment production of IL-21, which subsequently promotes B cell activation and secretion of pathogenic autoantibodies. In summary, immunological tolerance among T cells appears to be particularly relevant for the control of autoimmune antibody speciﬁcities, and it is likely that TFH cells provide inappropriate helper signals to selfreactive B cells in cases of antibody-mediated autoimmune diseases. Beyond their restricted role in T cell help for B cells, TFH cells may also promote chronic inﬂammation. Prototypic TFH cells express CXCR5 and localize to B cell folli-

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cles in lymphoid tissues. These two factors distinguish them from Th17 cells, which localize to nonlymphoid tissues. However, in dysregulated immune responses, TFH cells have been identiﬁed in nonlymphoid tissues, particularly in autoimmune diseases; this may result from CXCL13 production at these sites. TFH and Th17 cells both express high levels of ICOS, and the costimulation provided by IL-21 promotes their generation (see Figure 3). In this context, dysregulated TFH cells producing large amounts of IL-21 at nonlymphoid sites could elicit widespread effects that contribute to chronic T cell–mediated autoimmune inﬂammation, including the bystander generation of Th17 cells. Human autoimmune diseases mostly affect nonlymphoid tissues, which often contain large numbers of inﬁltrating, activated lymphocytes (141), as well as lymphoidlike tissues with an ectopic GC (134, 142– 145). These ectopic GCs may be a site for generating high-afﬁnity antibodies through somatic hypermutation. Understanding the mechanisms that enable the development of these outposts of lymphoid tissue in peripheral tissues, and the mechanisms that favor their maintenance rather than resolution, remains relevant for also understanding the pathogenesis of certain autoimmune diseases. Chemokines produced in ectopic GCs as well as in other lymphoid tissues are likely to be instrumental for the development and maintenance of these structures. TNF-α and lymphotoxin play a well-established role in lymphoid neogenesis, mediated in part through regulated production of the homeostatic chemokines CXCL13, CCL19, and CCL21 (146). Analysis of CXCL13, CCL19, and CCL21 expression in synovium of patients with rheumatoid arthritis suggests that the formation of a GC is dependent on the concentrations of both CXCL13 and lymphotoxin (147). CXCL13 is usually produced by FDCs in secondary lymphoid tissues. However, in rheumatoid arthritis, CXCL13producing cells were identiﬁed as vascular

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endothelium and synoviocytes (147, 148). A possible scenario within inﬂamed tissue is that TNF-α, secreted by inﬁltrating inﬂammatory cells, induces production of CXCL13 by vascular endothelium and synoviocytes, and the CXCL13 then recruits circulating B cells and TFH cells and induces expression of lymphotoxin and development of ectopic GC.

MECHANISM FOR REGULATION OF TFH CELLS Normally, various mechanisms operate to prevent delivery of inappropriate help to selfreactive B cells. For instance, follicular CD4+ CD25+ CD69− Tregs that express CXCR5 localize to the follicles and suppress the helper effects of follicular CXCR5+ CD57+ cells for Ab production by GC B cells (149). Another mechanism hinges on the suppressive effects of Roquin, a ubiquitin ligase that prevents differentiation and activation of self-reactive TFH cells (53) Large numbers of T cells in sanroque mice (which bear a single base pair substitution in roquin) develop a TFH phenotype and accumulate in the follicles. These T cells resemble human TFH cells in that they express high levels of ICOS, CXCR5, CD200 and PD1 and transcripts for IL-21 (53). sanroque mice form spontaneous GCs and develop an autoimmune-like syndrome associated with high levels of autoantibodies. Roquin negatively regulates ICOS expression in T cells by promoting the degradation of Icos mRNA. A conserved segment in the Icos 3 untranslated mRNA is important for regulation by Roquin. This segment comprises a 47–base pair region complementary to T cell–expressed microRNAs. The repressive activity of this segment is disrupted by base pair inversions in sanroque mice, which are predicted to abrogate miR-101 binding (150). These ﬁndings highlight the central role of ICOS mRNA regulation (and, indirectly, TFH cells) in the pathogenesis of T-dependent Ab-mediated autoimmune diseases.

TFH CELLS AND IMMUNODEFICIENCIES Ineffective T cell help to B cells appears to underlie certain humoral immunodeﬁciencies. Examples of such immunodeﬁciencies include X-linked lymphoproliferative disease (XLP), ICOS deﬁciency, and CVID, conditions in which affected individuals experience progressive hypogammaglobulinemia as well as an impaired ability to form GCs and generate long-lived memory B cells and serological immunity (49, 69, 70, 151, 152). Such defects are also observed in mice that have been rendered deﬁcient in genes associated with TFH cells, such as CXCR5, CD40/CD40L, ICOS/ICOS-L, and SAP (79, 153–155). Interestingly, CXCR5+ CD4+ T cells are deﬁcient in mice and humans lacking functional CD40-L (29), ICOS (29, 30), or CD28 (28). Furthermore, CD4+ T cells from patients with XLP and from sap−/− mice are unable to upregulate ICOS expression in vivo and in vitro and are defective in production of B helper cytokines, such as IL-4 and IL-10 (70, 152, 156). These ﬁndings have shed light on some of the molecular requirements for the generation and effector function of TFH cells. Because TFH cells express an array of molecules involved in the provision of B cell help—ICOS, CD40L, OX40, IL-21, SAP— it is likely that compromised development or function of TFH cells contributes to impaired B cell differentiation and humoral immunity in conditions of immunodeﬁciency.

TFH CELLS AND LYMPHOMAS Human TFH cells have a distinct phenotype and genotype (Figure 2) (discussed above). Accordingly, several recent studies that have compared the transcriptome or phenotype of malignant T cells have recognized that the malignant cell phenotype in angioimmunoblastic T cell lymphoma (AITL) shares many similarities with that of TFH cells. Speciﬁcally, malignant AITL cells are CD4+ T cells that express Bcl-6 (65, 157, 158), www.annualreviews.org • T Follicular Helper Cells

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CXCR5, CD40L, OX40, and PD1 (159–161) and produce CXCL13 (162). However, the malignant cells in AITL are unique in their expression of CD10 (163). This marker may therefore serve as a means of discriminating between normal and malignant TFH cells. Hallmarks of AITL are B cell activation, hyperplastic B cell follicles within lymph nodes, hypergammaglobulinemia, and proliferation of FDCs (163). Furthermore, within reactive lymph nodes of patients with AITL, the malignant T cells are in close association with activated B cell follicles and FDCs (163). For these reasons, researchers (159, 162) recently proposed that the dysregulated production of CXCL13 and constitutive expression

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of CD40L by malignant TFH cells may result in the increased recruitment of B cells into follicles, their aberrant activation, and subsequent hypergammaglobulinemia. It is certainly possible that CXCL13 produced by malignant CD4+ TFH cells is responsible for recruiting large numbers of B cells into reactive follicles in lymph nodes of patients with AITL. However, given the potent effects of IL-21 on the activation and differentiation of human B cells into Ig-secreting cells (109, 110, 113), the sustained production of IL-21, rather than CXCL13, by TFH cells may more likely underlie the exaggerated B cell activation and hypergammaglobulinemia characteristic of this disease.

SUMMARY POINTS 1. TFH cells are one of the most numerous and important subsets of effector T cells. TFH cells are distinguishable from Th1 and Th2 cells in several respects: chemokine receptor expression (CXCR5), location and migration (follicles), function (B cell help), cytokine production (IL-21), and expression of transcription factors (Bcl-6). 2. IL-21 serves as a “helper” cytokine produced by TFH cells that stimulates B cells through IL-21R. IL-21 may also serve as an autocrine factor for TFH cells. 3. Many of the important molecules for TFH cell function, such as ICOS and IL-21, contribute to the pathogenesis of autoimmune diseases or immunodeﬁciencies. 4. The identiﬁcation of TFH cells and the molecules they express provides opportunities for new therapeutic approaches to autoimmune diseases. Indeed, a number of companies are targeting TFH -associated molecules such as IL-21 and ICOS.

FUTURE ISSUES 1. Is Bcl-6 a transcription factor for TFH cells and does it determine their differentiation? 2. TFH cells are only now becoming accepted as a bona ﬁde subset distinct from Th2 or Th1 cells; however, their relation to Th17 requires further clariﬁcation. 3. The study of the precise role of IL-21 in TFH cell differentiation and function, and its effects on other cell types, is a rapidly evolving ﬁeld. IL-21 has been implicated in the function of numerous T cell subsets. Is IL-21 a classical “helper” cytokine that facilitates T cell help to many lymphocyte types, other than B cells? 4. How effective will inhibitors of TFH cell effector molecules be for the treatment of human autoimmune diseases?

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DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENT This work was supported by the Australian National Health and Medical Research Council and the Juvenile Diabetes Research Foundation.

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Annual Review of Immunology

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Contents

Volume 26, 2008

Frontispiece K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Doing What I Like K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Protein Tyrosine Phosphatases in Autoimmunity Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert, and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57 Forward Genetic Dissection of Immunity to Infection in the Mouse S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81 Regulation and Functions of Blimp-1 in T and B Lymphocytes Gislâine Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p133 Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin, and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171 T Cell Trafﬁcking in Allergic Asthma: The Ins and Outs Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p205 The Actin Cytoskeleton in T Cell Activation Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p233 Mechanism and Regulation of Class Switch Recombination Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p261 Migration of Dendritic Cell Subsets and their Precursors Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Sánchez p p p p p p p p p p p p p293

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The APOBEC3 Cytidine Deaminases: An Innate Defensive Network Opposing Exogenous Retroviruses and Endogenous Retroelements Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317 Thymus Organogenesis Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p355 Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p389 Annu. Rev. Immunol. 2008.26:741-766. Downloaded from arjournals.annualreviews.org by Shanghai Information Center for Life Sciences on 04/28/08. For personal use only.

Monocyte-Mediated Defense Against Microbial Pathogens Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p421 The Biology of Interleukin-2 Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p453 The Biochemistry of Somatic Hypermutation Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa, Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p481 Anti-Inﬂammatory Actions of Intravenous Immunoglobulin Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p513 The IRF Family Transcription Factors in Immunity and Oncogenesis Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p535 Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p585 Development of Secondary Lymphoid Organs Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p627 Immunity to Citrullinated Proteins in Rheumatoid Arthritis Lars Klareskog, Johan Rönnelid, Karin Lundberg, Leonid Padyukov, and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651 PD-1 and Its Ligands in Tolerance and Immunity Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p677 The Master Switch: The Role of Mast Cells in Autoimmunity and Tolerance Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p705 T Follicular Helper (TFH ) Cells in Normal and Dysregulated Immune Responses Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p741

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