Annual Review of Immunology Volume 27 2009

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Annual Review of Immunology

Annu. Rev. Immunol. 2009.27:621-668. Downloaded from arjournals.annualreviews.org by Karolinska Institutet on 03/29/09. For personal use only.

Contents

Volume 27, 2009

Frontispiece Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Translating Molecular Insights in Autoimmunity into Effective Therapy Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Structural Biology of Shared Cytokine Receptors Xinquan Wang, Patrick Lupardus, Sherry L. LaPorte, and K. Christopher Garcia p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Immunity to Respiratory Viruses Jacob E. Kohlmeier and David L. Woodland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 61 Immune Therapy for Cancer Michael Dougan and Glenn Dranoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 83 Microglial Physiology: Unique Stimuli, Specialized Responses Richard M. Ransohoff and V. Hugh Perry p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p119 The Liver as a Lymphoid Organ Ian Nicholas Crispe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Immune and Inﬂammatory Mechanisms of Atherosclerosis Elena Galkina and Klaus Ley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p165 Primary B Cell Immunodeﬁciencies: Comparisons and Contrasts Mary Ellen Conley, A. Kerry Dobbs, Dana M. Farmer, Sebnem Kilic, Kenneth Paris, Soﬁa Grigoriadou, Elaine Coustan-Smith, Vanessa Howard, and Dario Campana p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 The Inﬂammasomes: Guardians of the Body Fabio Martinon, Annick Mayor, and Jürg Tschopp p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p229 Human Marginal Zone B Cells Jean-Claude Weill, Sandra Weller, and Claude-Agn`es Reynaud p p p p p p p p p p p p p p p p p p p p p p267

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Aire Diane Mathis and Christophe Benoist p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p287 Regulatory Lymphocytes and Intestinal Inﬂammation Ana Izcue, Janine L. Coombes, and Fiona Powrie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p313 The Ins and Outs of Leukocyte Integrin Signaling Clare L. Abram and Clifford A. Lowell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p339

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Recent Advances in the Genetics of Autoimmune Disease Peter K. Gregersen and Lina M. Olsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p363 Cell-Mediated Immune Responses in Tuberculosis Andrea M. Cooper p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p393 Enhancing Immunity Through Autophagy Christian Munz ¨ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p423 Alternative Activation of Macrophages: An Immunologic Functional Perspective Fernando O. Martinez, Laura Helming, and Siamon Gordon p p p p p p p p p p p p p p p p p p p p p p p p451 IL-17 and Th17 Cells Thomas Korn, Estelle Bettelli, Mohamed Oukka, and Vijay K. Kuchroo p p p p p p p p p p p p p p485 Immunological and Inﬂammatory Functions of the Interleukin-1 Family Charles A. Dinarello p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p519 Regulatory T Cells in the Control of Host-Microorganism Interactions Yasmine Belkaid and Kristin Tarbell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p551 T Cell Activation Jennifer E. Smith-Garvin, Gary A. Koretzky, and Martha S. Jordan p p p p p p p p p p p p p p p591 Horror Autoinﬂammaticus: The Molecular Pathophysiology of Autoinﬂammatory Disease Seth L. Masters, Anna Simon, Ivona Aksentijevich, and Daniel L. Kastner p p p p p p p p p621 Blood Monocytes: Development, Heterogeneity, and Relationship with Dendritic Cells Cedric Auffray, Michael H. Sieweke, and Frederic Geissmann p p p p p p p p p p p p p p p p p p p p p p p p669 Regulation and Function of NF-κB Transcription Factors in the Immune System Sivakumar Vallabhapurapu and Michael Karin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p693

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Contents

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Review in Advance first posted online on November 13, 2008. (Minor changes may still occur before final publication online and in print.)

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Translating Molecular Insights in Autoimmunity into Effective Therapy Marc Feldmann Kennedy Institute of Rheumatology Division, Imperial College London, London W6 8LH, UK; email: [email protected]

Annu. Rev. Immunol. 2009. 27:1–27

Key Words

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

cytokines, rheumatoid arthritis, TNF, anti-TNF

This article’s doi: 10.1146/annurev-immunol-082708-100732

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0001$20.00

Autoimmunity and the pathogenesis of autoimmune diseases was a major focus of the Walter and Eliza Hall Institute, where I started my research career. After my initial studies on immune cell culture and immune regulation, I returned to an analysis of the pathogenesis of human autoimmunity in London. Linking upregulated antigen presentation to autoimmunity led to an investigation of the role of cytokines in rheumatoid arthritis (RA), in collaboration with Ravinder Maini. These experiments led to the concept of a TNF-dependent cytokine cascade driving the manifestations of RA, which led to successful clinical trials of anti-TNF monoclonal antibody in RA patients, heralding a major change in medical practice. This success was made possible by enthusiastic support from many laboratory and clinical colleagues and taught us that cytokines are important rate-limiting steps and hence good therapeutic targets. My current scientiﬁc challenge is exploring the hypothesis of whether all major medical needs can be approached via cytokine blockade.

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GROWING UP IN AUSTRALIA

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Postwar France was poor, and despite my Polish father’s French accountancy degree, life was difﬁcult. My Jewish parents thus sought greater opportunities elsewhere. My father had two cousins, both doctors, one in New Haven, Connecticut, in the United States, and the other in Melbourne, Australia, and they both ﬁled immigration papers for my family. The Australian papers came a month before the U.S. papers. Who knows what might have happened had U.S. bureaucracy been speedier. As an eight-year-old, a month’s journey by ship to Australia was an adventure going into the unknown, to a land of kangaroos and much promise, visiting the pyramids en route. Learning English in a land welcoming immigrants was not a major hurdle. Immigrants have a very strong motivation to work hard and succeed. As a child, the image of my father coming home tired from his work as an accountant in a factory, to study anew for his accountancy degree, as his French qualiﬁcation was not recognized, had a profound impact on me. Once qualiﬁed, he built up a prospering sole-proprietorship serving other immigrants. Having helped balance books of accounts on weekends and holidays, I found the thought of following in his footsteps far too boring! My father’s cousin, more ﬂamboyant, was a doctor, and that seemed to my older brother and me a more challenging and possibly more satisfying profession, so we both became medical students at the University of Melbourne. Medical studies opened up new vistas. Some courses were painstaking and meticulous—ﬁve terms of learning and regurgitating anatomy is a chore that is mercifully no longer imposed— but others were exciting and challenging. Biochemistry teachers encouraged successful students to read more widely. My ﬁrst exposure to the Annual Reviews of Biochemistry was an eye-opener, illustrating the questioning and uncertainties of emerging ideas and knowledge, rather than the deﬁnite facts and platitudes that we students usually received in lectures.

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Microbiology provided my ﬁrst glimpse of immunology, with eight lectures on serology taught by a microbiologist on the use of antisera to diagnose infections. A few years later, I learned that this course bore no resemblance to the major discoveries about cellular immunity being made contemporaneously on the other side of the Sydney Road from the university campus, at the Walter and Eliza Hall Institute of Medical Research, by luminaries Jacques Miller, Gus Nossal, and others, who eventually became my mentors. Clinical studies were stressful for a young, immature student like me, who at 17 started medicine straight from high school. We had already experienced death early in our studies, by working with cadavers in anatomy. The University of Melbourne clinical studies were performed in two hospitals: at the Royal Melbourne Hospital, over the road from the university, which took a large group of students, and at St. Vincent’s Hospital in Fitzroy, which took one-ﬁfth the number of students that Royal Melbourne did. I suspect that I am impatient. I got married while still a medical student, and my son was born while I was working in hospital and my daughter while I was completing my PhD. I made what in retrospect was a pivotal decision: to go to St. Vincent’s, the hospital with fewer students, because I was impatient to learn clinical medicine more rapidly by seeing the most interesting patients, which I hoped being part of the smaller group would allow. It was indeed easier to learn clinical medicine, but I paid the price later when, after qualiﬁcation, I and the other young doctors had to see all comers and not just the interesting cases. St. Vincent’s was in a poor, hard-drinking suburb of Melbourne, so almost 50% of patients arriving in casualty (emergency room) were chronic alcoholics, who have neither the most pleasant demeanors nor the most intellectually challenging problems. I therefore began exploring research opportunities earlier than I might have if I had chosen the more academic hospital.

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SCIENCE IS DIFFERENT FROM MEDICINE: GOLDEN YEARS AT THE WALTER AND ELIZA HALL INSTITUTE The Walter and Eliza Hall Institute of Medical Research (WEHI), across the road from the university, was made famous by the Nobel Prize winner Sir Frank MacFarlane Burnet, who had been a pioneer both in virology and subsequently in immunology. The institute had recently appointed a young and dynamic new director, Gustav Nossal. I went to meet him and to see the institute. The contrast with the rest of the university was stark: WEHI was clearly in a different league. Gus accepted me as a PhD student for the following year, to work on new techniques of tissue culture for generating immune responses in vitro. Little did I know that I had applied far too late, but Gus’s intuition was to make an exception and seek another PhD studentship for me. When I arrived in February, after a summer break recovering from the stresses of endless on-call rotations in the hospital, an arduous apprenticeship compared with today’s European Union work regulation–restricted hours, I was greeted by Erwin Diener, the Swiss scientist

Gus had chosen to supervise my ﬁrst tentative steps into science (see Figure 1). The project was wonderful, optimizing in vitro lymphoid cell cultures that were independently being developed in the United States by Bob Mishell and Dick Dutton (1) and at WEHI by John Marbrook (2) and Erwin Diener (3). The project was wonderful because of its potential inﬂuence on virtually all aspects of immunology. In vitro experiments are truly reductionist, and if that is to your taste, all the elements involved can be controlled: cells puriﬁed and quantitated, antigen concentration maintained precisely, other stimuli controlled. But this control comes at the same price as all the reductionist science still popular today (e.g., gene knockouts): Concepts generated in one precise circumstance often do not extrapolate to complex and nonreductionist reality. With Erwin, I started to improve the current culture methods. It was already possible to generate antibody production from mouse spleen cells. The antigen used, salmonella ﬂagellin, was popular at WEHI, having been used by Gus Nossal and Gordon Ada to help validate Burnet’s clonal selection theory (reviewed in 4). This antigen had been used to demonstrate that one cell produced only one antibody,

Mentors

Jacques Miller

Gustav Nossal

Erwin Diener

Figure 1 My mentors at the Walter and Eliza Hall Institute of Medical Research, Gustav Nossal, Jacques Miller, and Erwin Diener. www.annualreviews.org • Effective Therapy for Autoimmunity

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even from lymph nodes of multiply immunized mice. Assaying ﬂagellin immunity was laborious: Cell suspensions were incubated with bacteria, which adhered to the antibody-forming cells. The suspension was plated on agar, and colonies were grown for a few hours to enable discrimination of cells that had bound multiple bacteria from single bacteria (5). The competing single-cell assay was the hemolytic plaque assay, developed by Niels Jerne (6) and his collaborators. It used complement-mediated lysis, either on agar or between glass slides. An improvement I engineered was to convert the cumbersome bacterial assay to a plaque assay, coating the red cells with protein. For multipurpose use, I developed the technique of using anti-sheep red cell Fab fragments (7), which could be derivatized with haptens, e.g., DNP or proteins, such as, for example, myelin basic protein (MBP), work I did in collaboration with fellow PhD student Vanda Lennon (8). Initially, we performed these cultures on a small scale, 10–20 ﬂasks permitting 3–6 groups, producing 3–6 sets of data to compare. But as techniques improved, many more questions arose, and so the glassware proliferated, as did the need for more washing up, more media, more sera incubators, etc. I needed more resources. Thus, I learned early at WEHI the virtue of collaborations, pooling intellectual and material resources to enhance scientiﬁc productivity. Effective collaboration has been a key part of WEHI’s success as an international scientiﬁc powerhouse over its long history. Erwin had two laboratory technicians, and as he was a reﬂective scientist, not prone to an excessive number of experiments, his technicians were encouraged to assist me. As a beginning PhD student, I found this to be a wonderful situation, as was having Erwin’s patient help in developing my scientiﬁc writing skills. Jacques Miller was at his magniﬁcent prime when I started at WEHI. With Graeme Mitchell, he had just published a series of three landmark papers (9–11) documenting that thymus-derived lymphocytes did not in themselves make antibody or develop into antibodyforming cells, but rather interacted with

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and activated bone marrow–derived antibodyforming cell precursors. He had also recently been elected a fellow of the Royal Society, ahead of the other two scientiﬁc giants at WEHI, Gus Nossal and Don Metcalf. Jacques Miller’s unit studied the function of thymus-derived cells, later renamed T cells by Ivan Roitt et al. (12, 13), whereas Gus Nossal’s unit, where I worked, studied antibody formation from B cells, bursa-equivalent or bone marrow–derived lymphocytes. Growing up in Australia inevitably engendered the love of playing sport. There were no tennis courts in the vicinity of WEHI, but there were squash courts buried in the bowels of the Royal Melbourne Hospital. I played regularly with Tony Basten, a postdoc in Jacques’s unit, and so over sweat and drinks we evolved a collaboration to try to recreate in tissue culture the T-B interactions that Miller and Mitchell had reported in irradiated mice. Tony provided a series of irradiated mice repopulated with thymus cells only, a source of relatively pure T cells (9), and I put them in culture with a variety of other populations, usually adult thymectomized bone marrow– grafted mice (14) that Tony had also provided, where B (but not T) lymphocyte repopulation takes place. To study the process in more detail, I developed a variant of the MarbrookDiener culture system that I had been using to study a variety of immunological processes in vitro, including immunological tolerance. This is illustrated in Figure 2 (15). It permitted separating the two cell populations to assess whether direct cell contact or cell-free mediators were sufﬁcient. The results we obtained (16) were published back-to-back with Anneliese Schimpl/Eberhard Wecker’s (17) results generated in the alternative Mishell-Dutton culture system. Other scientiﬁc interests that I have pursued subsequently were nurtured at WEHI. Ken Shortman was the head of the Biochemistry Unit, but his main focus was cell separation: how to purify cell subsets. Using these techniques enabled me to study lymphocyte-macrophage interactions, the

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process now known as antigen presentation, an acknowledged vital key step in the generation of immune responses. Macrophages are very adherent, so they can be enriched by adherence, and lymphocytes, relatively nonadherent, can also be enriched through this technique. But a more physiological approach was to use recirculating cell populations in vivo enriched in lymphocytes, and access to these cell populations is possible by thoracic duct drainage. John Sprent, a fellow PhD student in Jacques’s unit, was working extremely long hours collecting such cells after the tricky surgery to cannulate the duct and prevent the tiny tube from blocking up. He generously provided these cells, and we showed that thoracic duct cells alone were not able to respond to a particulate antigen, sheep red cells in vitro, unless supplemented by adherent macrophages (18). Vanda Lennon was also my contemporary at WEHI, a PhD student in the Clinical Research Unit headed by Ian Mackay. She studied experimental allergic encephalomyelitis, assisted by Patrick Carnegie. In these animals, we generated autoantibodies to MBP and devised a project to use MBP-coated red sheep cells to detect where the antibody-forming cells to MBP were present in animals. We duly found them in the brain (19). In hindsight, these research interests nurtured at WEHI were recombined in 1983 to help me conceive of a new hypothesis linking upregulated antigen presentation and autoimmunity, triggered by the immunohistological data of Franco Bottazzo (20) and others [e.g., Klareskog & Wigzell (21)] that there was augmented HLA class II expression in autoimmune disease tissues, such as the thyroid in Graves’ disease, or rheumatoid joints. By the time Gus Nossal returned from his sabbatical in Paris, where he had gone to examine reports by Alain Bussard (22) that peritoneal cells could make multifunctional antibody against red cells, I was generating a lot of in vitro culture data. Some months later, when Erwin Diener left to head a new immunology department in Edmonton, Canada, Gus de-

T cells Upper compartment Lower compartment

Nuclepore membrane

B cells Dialysis membrane

M E DI U M

Figure 2 Double-chamber cultures, formed by concentric glass tubes, suspended in a reservoir of medium. T cells were placed in the upper compartment and B cell–containing populations in the lower compartment. Adapted from Reference 15.

cided that I could keep the two technicians in Erwin’s charge, which enabled me to carry on with a wider range of projects and not confront the classic PhD student’s dilemma of too many ideas for the time and limited resources. Gus’s decision had some interesting implications, and it was not popular with other staff. Still a PhD student, I was heading a little group working in immune tissue culture. In the methodological aspects, Alan Harris, who had trained with Renato Dulbecco in culturing tumor cells at the Salk Institute, was very helpful, querying the methods we used with spleen cells, compared with his own work with cancer cells. Gus offered me new students, and I had the opportunity to initiate John Schrader into the intricacies of immune cell culture. It was a challenge, from which both John and I escaped unscathed (23). Hermann Wagner was the ﬁrst of a group of talented young German medical scientists who came to WEHI to train, followed by Martin Rollinghof and Harold von Boehmer. Hermann had worked in complement and was intrigued www.annualreviews.org • Effective Therapy for Autoimmunity

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by immune cell killing; he wanted to develop an in vitro system for generating cytotoxic T cells (CTL). We set about doing this using the Marbrook-Diener culture system; the experiments succeeded and resulted in an in vitro generation of CTL. Hermann developed this research path enthusiastically over the ensuing years (24). Wunderlich and Canty at NIH had previously generated similar results using the Mishell-Dutton system (25). Taking part in the collaborative atmosphere at WEHI was an incredible learning experience. Gus had a saying that I paraphrase: “Not publishing your data is a luxury few can afford.” I took that to heart and suspect few PhD students have published more from their thesis time than I did, owing to the multiple collaborations evaluating immune responses in vitro. Many of these papers were written late at night, fuelled by coffee and the music of the Rolling Stones.

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MOVING TO LONDON Gus was a friend and contemporary of most senior immunologists, so contemplating a postdoctoral position in the laboratory of another famous scientist was a realistic possibility for me. High on my list were Gerald Edelman’s lab in New York and Avrion Mitchison’s lab in London. Because of funding problems in Australia (regular occurrences in all laboratories), Gus decided to delegate to me attendance at a small, intimate immunology conference at Brook Lodge, a retreat in Kalamazoo, Michigan, owned by the Upjohn Company and used for conferences. I was very privileged to be able to take part; so many luminaries whose papers I had read, such as Baruj Benacerraf, James Gowans, Fritz Bach, and Mel Cohn, were present. On the way, I visited Avrion (Av) Mitchison and Gerald Edelman and compared which lab might be more suitable for me. Av was a most charming host, inviting me to stay in his house and give a talk to his colleagues at the National Institute of Medical Research (NIMR) at Mill Hill, where there was a wonderful intellectual and friendly 6

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atmosphere (e.g., 26, 27). So I decided to join Av’s group, but not at NIMR, but rather at the new Imperial Cancer Research Fund (ICRF) Immunology Unit he was starting at University College. Talented young colleagues there from the beginning, Marty Raff, Mel Greaves, and Nancy Hogg, were soon joined by Peter Beverley, Reg Gorczynski, Robert Tigelar, and Geoff Shellam, with Liz Simpson a frequent visitor. Subsequently, Mike Owen, Benny Chain, and Mary Collins joined us. All have gone on to major scientiﬁc careers and contributions. It was an exciting place to be, rich in intellectual resources and modern scientiﬁc equipment, with Av’s friendship with the Herzenbergs securing him one of the very ﬁrst ﬂuorescence-activated cell sorters (FACS) (28). Scientiﬁc visitors abounded, to give seminars in the crowded, small seminar room in the Department of Zoology, with Av, lying back in the front row with his feet up, eyes almost closed, as they gave their seminar, but very much awake, as question time revealed. Memorable was a young Peter Doherty coming to tell the world of his very surprising ﬁndings with Rolf Zinkernagel (29) of the genetic restrictions in CTL activation and of the various interpretations they were exploring. Moving to London had amazing advantages. No longer was there the tyranny of distance and isolation that have so preoccupied legions of Australian scientists to this day, and the United States, where almost half of science was being performed, was now only between 7 and 10 hours away and could be visited for a few days. The exhausting 24-hour trips from Melbourne to the scientiﬁc centers of Europe or the United States were no longer necessary. For example, there was no need to choose which of two major conferences to attend: Travel time and exhaustion level were no longer deciding factors. I was invited to many conferences, and I went to a lot. There was more money for research than in Australia, and more scientists were available for the skilled, laborintensive immunology research. Added to these career beneﬁts were the numerous cultural attractions of London. Av readily organized an

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appointment for me to the research staff of ICRF, and my planned return to Australia after a two-year fellowship was postponed, indeﬁnitely as it turned out. I did make trips back with my family so that my children could visit their grandparents. On one of these trips, I bought my ﬁrst souvenir of tribal art from Australia, an aboriginal bark painting, from a Melbourne dealer. A love of tribal art, encountered ﬁrst in Fiji on the way to London, has remained with me. My ofﬁce is now crowded with African and New Guinea masks and sculptures. I believe (an untested hypothesis) that being surrounded by original creations fosters creativity!

THE EARLY MAGICAL DAYS OF CYTOKINES In the early 1970s, I developed a multichambered culture ﬂask to study whether cell contact was necessary, and this generated a keen interest in intercellular mediators (15). The technology back then for identifying these mediators was far inferior to today’s. And so although important biological activities were present in supernatants and were given names to reﬂect that [e.g., osteoclast-activating factor, macrophageactivating factor, T cell growth factor, B cell– stimulating factor (30–33)], their molecular identity remained a mystery. An approximate molecular weight was as far as the effort got, as the potency of cytokines meant that there was very little protein. The number of potential mediators described was growing fast, and to try to make sense of this, Joe Oppenheim and others initiated the ﬁrst of a series of conferences that grew into the Cytokine Conferences. The ﬁrst was near NIH in 1977, and the second was at Ermatigen, Switzerland, in 1979. The ﬁrst conference focused on clarifying the problems of the ﬁeld; by the second, attendees suggested that some bioactivities might coexist within the same or related molecular species. We agreed upon the nomenclature interleukin, with IL-1 potentially encompassing lymphocyte-activating factor, osteoclastactivating factor, and endogenous pyrogen, all

based chieﬂy on similar molecular weight and origin, and IL-2 being T cell growth factor. A consensus paper was published from this conference chieﬂy reﬂecting input from Kendall Smith and Joe Oppenheim (34–37). But the real turning point in this ﬁeld came with the use of new technology, driven by perceived clinical need. Molecular biology techniques had invaded immunology in the mid- to late 1970s and had instigated real progress, such as cloning of antibody genes (38) and clarifying the generation of antibody diversity. Interferons (anti-viral mediators) were considered to be potential cancer cures (39), and so a lot of work was emerging in the late 1970s to scale up their production. By 1979 (40), Tada Taniguchi had cloned the ﬁrst type I interferon (IFN) cDNA, closely followed by David Goeddel and Sidney Pestka (41) and Shigekazu Nagata and Charles Weissman (42). Cloning of interferon was closely followed by the cloning of IFN-γ and other important interleukins, IL1 (43), IL-2 (44), IL-4 (45), IL-6, etc., in the 1980s. By 1984, the cloning of tumor necrosis factor (TNF) and lymphotoxin were reported, ﬁrst presented at a Cytokine Conference at Schloss Elmau by David Goeddel. By this time, I had started collaborating with Ravinder (known usually as Tiny) Maini (see Figure 3) on the role of cytokines in the pathogenesis of rheumatoid arthritis (RA). The properties of pure TNF described by Goeddel (46) were highly suggestive of those relevant to RA. The molecular biologists were providing new tools for elucidating the properties and function of cytokines, and, with that, many aspects of pathology and medicine were to change dramatically.

CYTOKINES AND UNCOVERING MOLECULAR CLUES TO AUTOIMMUNITY Science progresses by testing new ideas or hypotheses. In the early 1980s, there was increasing realization that in various autoimmune disease sites, there was upregulation of major histocompatibility complex (MHC) www.annualreviews.org • Effective Therapy for Autoimmunity

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Key collaborators

Ravinder (Tiny) Maini

Fionula Brennan

Mike Shepard

Jim Woody

Figure 3 Important collaborators throughout my career: Ravinder Maini, Fionula Brennan, Mike Shepard, Jim Woody.

expression, especially of MHC class II. This was found in rheumatoid synovium by Klareskog and Wigzell (21) in Sweden and Janossy (47) in London, and in endocrine autoimmune tissue, thyroid, and pancreas by Franco Bottazzo and Ricardo Pujol-Borrell (20). Franco came to see me to discuss whether this upregulated class II expression had any immunological meaning. To someone like me, having worked for many years on mechanisms of T cell activation, including antigen presentation, the answer was obvious. 8

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But it seemed a bit too obvious. Could upregulation of antigen presentation, induced presumably by environmental events, be sufﬁcient to trigger autoimmunity in genetically susceptible individuals? The latter point is critical because of the importance of genetics, especially MHC, in regulation of the immune response, as Hugh McDevitt (48) ﬁrst showed. Experiments in both mice and humans had demonstrated that autoantigen-reactive T cells were present in nondiseased individuals. How might upregulated MHC be connected to autoimmunity? There seemed to be a clear scenario, based on Steeg and Oppenheim’s (49) ﬁnding that IFN-γ upregulated MHC class II expression. The pathway might run as follows: Local tissue infection, perhaps viral, or other local damage would release cytokines and autoantigens, activating local cells to augment their MHC class II and antigenpresenting function. The cytokines and autoantigens would then be able to activate nontolerant autoantigen-reactive T cells, which in turn would activate effector cells, B cells to generate autoantibody, and macrophages to produce cytokines and other mediators, together causing more tissue damage, cytokine release, and so the vicious cycle of an ongoing disease. With the possibility of abnormal suppressor or regulatory T cells, I could thus envision the pathogenesis of a chronic disease. I deﬁned this scenario in early 1983, while staying with my family in a holiday home we had just bought in Begur, on the Costa Brava. While there, I had the time and freedom to think critically and write this hypothesis. With coauthors who had generated the relevant data, it was published in the Lancet as an untested hypothesis, a format that now seems very antiquated (50). When was the last major concept published without any supporting data? Of course, that would raise the possibility that a rival group could scoop yours by generating the experimental evidence. Nevertheless, 25 years later, this hypothesis is still a reasonable approximation, and rereading it is not at all embarrassing.

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Testing this new concept experimentally was exciting. The necessary techniques were already available: Cell culture methods had progressed rapidly, and the understanding based on Kendall Smith’s work on T cell growth factor (35) and on antigen presentation permitted rapid evaluation of the new hypothesis. Very important in testing this hypothesis were two young colleagues, Jonathan Lamb (now a professor in Edinburgh, then a postdoc who had greatly improved human T cell cloning techniques while in Jim Woody’s lab) (51) and Marco Londei (an enthusiastic and bright medical graduate new to the lab but keen to make his mark in medical research). We passed the ﬁrst test swiftly: adherent thyroid cells, a population including many epithelial cells and some antigen-presenting cells, were able to restimulate, after inﬂuenza peptide incubation, MHCcompatible inﬂuenza-speciﬁc T cell clones (52). Other tests took a little longer. Cloning T cells from Graves’ disease samples was a challenge that Marco relished; he cultured the lymphocytic inﬁltrate ﬁrst with IL-2 to select for in vivo–activated T cells and then cloned them (53). Seeking cells that were restimulated by autologous adherent thyroid cells but not allogeneic thyroid epithelial cells was accomplished, and he obtained wonderful pictures of T cells stretched and adherent to epithelium, as well as more quantitative proliferative data. Subsequent work in collaboration with thyroid experts Basil Rapoport and Sandy McLachlan (54) and postdocs Sonia Quaratino and Colin Dayan identiﬁed the diversity of autoantigens recognized, thyroid-stimulating hormone receptor, thyroglobulin, and, most often, thyroid peroxidase (55). We analyzed the cytokines able to upregulate epithelial cell MHC expression and showed that IFN-γ and TNF were both important, varying with cell type. This work was driven by Ricardo Pujol-Borrell and my PhD student Ian Todd (56). So the cellular basis, the outline of the hypothesis, had been rapidly tested and substantiated between 1983 to 1986. Scientiﬁc interest in this concept was high; transgenesis was becoming an effective research tool, and so trans-

genic mice overproducing IFN-γ in the islet cells of the pancreas, driven by the insulin promoter, were generated by Nora Sarvetnick at Genentech, and these mice duly developed autoimmune diabetes (57). But of course many questions remained unsolved. Were the epithelial cells really the antigen-presenting cells initiating disease? This went against the dogma. Did the epithelial cells have a role in disease maintenance or in recruiting immune cells? To evaluate the medical signiﬁcance properly, we needed to identify the intercellular mediators involved, cytokines or others. But this was not possible with the operative samples of thyroid that could be obtained after the disease was quiescent enough to permit safe surgery. Furthermore, thyroid diseases have never been seen as major unmet needs because their treatments, while imperfect, have been good enough for a long time.

WONDERFUL COLLABORATIONS AND FRIENDS The ethos of effective collaboration—the pooling of diverse skills and resources to permit a more effective attack on a major challenge— had pervaded WEHI. Having learned the power of effective collaboration, I joined forces with Franco Bottazzo on my ﬁrst serious venture into autoimmunity, which made considerable progress. But personality differences limited this joint venture. I sought a more important autoimmune disease, like thyroid with a local site of disease that could be immunologically studied, and RA was an obvious choice. Nathan Zvaiﬂer, a leading U.S. rheumatologist, had come in the late 1970s to do a sabbatical with Av Mitchison, and I had the good fortune to be charged with looking after him. I suspect we both learned a lot from each other: Nat gained some insights into the ever increasing complexity of immunology, and I discovered that RA was a major immunological disease with many aspects not yet understood, which made it an important unmet need. When I subsequently rang to ask him who in London was www.annualreviews.org • Effective Therapy for Autoimmunity

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the best person to work with in this ﬁeld, he unhesitatingly said Ravinder (Tiny) Maini at the Kennedy Institute of Rheumatology (KIR), whom I duly rang. His enthusiasm for a potential new collaboration was evident, and he was in my ofﬁce accompanied by Lindsay, his laboratory technician, within two days. That was the start of a truly wonderful collaboration and friendship, which has transformed our careers and enabled the very difﬁcult task of translating laboratory science into effective therapy. It began with a detour, however. Systemic lupus erythematosus (SLE) was the major disease being studied by Maini’s group at the time, and so I explored whether the techniques used in Graves’ disease might be useful in studying SLE. With Tiny’s encouragement, I rapidly got involved in arthritis research, successfully applied for an Arthritis Research Campaign grant, and then was invited to see the new research center being built on the site of the Charing Cross Hospital, which had plenty of lab space compared with my base at the ICRF Tumor Immunology Unit at University College. Professors from Charing Cross and Westminster Medical School had gotten together to raise support for this new center, and Mary Glen-Haig, then chief administrator of the hospital, had found donors. Her friend Sir William Shapland, chief executive of the building ﬁrm Bernard Sunley & Sons, was the chairman of the Sunley Trust and of the group planning this research development, the Charing Cross Sunley Research Center. Because my work on autoimmunity was progressing well and because, being an optimist, I could envisage that it would eventually be tested in patients, I no longer felt it was appropriate to be personally supported by a cancer research organization, even if it was very rich and broad-minded, as the ICRF led by Walter Bodmer and Mike Crumpton was. Moving to an empty building (even if only four to ﬁve miles away and with only a few key staff ) to build up a team is traumatic and difﬁcult. One always underestimates the ﬁnancial and equipment needs. We had ambitious plans to discover the key molecular mediators in active RA synovium and develop new therapies

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aimed at interfering with them. I do not know the percentage of teams with such ambitious therapeutic goals that actually succeed, but it is certainly not high. We had certain key assets, including two leaders, one at the laboratory end and the other at the clinical end (Tiny), but we also had an appreciable overlap of understanding. Another asset was excellent team spirit, facilitated by the involvement of several fellows and students who had previously worked with one or the other of us. Working in the rapidly developing cytokine ﬁeld was enthralling, but its clinical importance had yet to be established or understood. It was a wonderful challenge, which was chieﬂy supported ﬁnancially by a variety of research charities. The Arthritis Research Campaign was the major one; it has had a large, long-term investment in KIR since its beginning in the 1960s and in my work at the Sunley Research Center from 1985, before I joined KIR (the Sunley Research Center became incorporated into the Kennedy Institute in 1992). This long-term funding made such risky research much more possible than funding on threeyear grants that The Wellcome Trust, Nufﬁeld Foundation, and Sunley Trust all contributed. Most importantly for the long-term challenge of this work was that Tiny and I were good friends. It is not clear that we would have worked so closely and effectively for over 20 years, overcoming various problems, had we not had the trust in each other that friendship brings. Joining me initially at the Sunley from University College were several key postdocs, including Marco Londei, whose work on Graves’ disease I mentioned above, and the late Glenn Buchan, from Otago, New Zealand. Glenn had begun successfully to use molecular biological techniques to study cytokine and cytokine receptor expression in synovium and was involved in reﬁning them to permit use with small human diseased tissue samples (58, 59). Regrettably, he died early in 2008 from cancer. Very important for the extensive grant writing that was needed was that my secretarial assistant Philippa Wells also decided to move with me.

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In retrospect, certain experimental choices were vital for understanding the role of cytokines in RA. First, we needed to focus on which mediators were actively synthesized at the site of disease, using mRNA analysis through the cloning of cytokine genes. Second, for studying regulation of cytokine production in rheumatoid synovium, we did not use the classical techniques of culturing and passaging rheumatoid synovial cells, a complex mixture of cells, until only adherent synovial ﬁbroblastlike cells are left. This technique made no sense to me, as 80–90% of the initial synovium, the hemopoietic-derived immune cells, were thus discarded, and, of course, after several passages, the environment of the remaining cells was very distinct from the initial environment (60). Hence, we used short-term cultures of the entire cell mixture in synovium (3–7 days maximum) to study synovial cytokine regulation. These experiments were carried out chieﬂy by postdoc Fionula Brennan (58, 61), now a professor at KIR. From her work emerged the ﬁrst evidence that TNF might be a therapeutic target. She demonstrated that in rheumatoid but not osteoarthritic mixed synovial cell cultures, anti-TNF antibodies dramatically reduced the production of IL-1 (61). Subsequently, this was extended to anti-TNF downregulating a range of other proinﬂammatory cytokines, GM-CSF, IL-6, and IL-8, which was all encouraging news (62–64). But it was worrying that anti-TNF also reduced the anti-inﬂammatory mediators, such as IL-10, IL-1 receptor antagonist, and soluble TNF receptors. This work is summarized by the TNF-dependent cytokine network concept, illustrated in Figure 4, which has proved a useful approximation to the truth. Richard Williams tested in animal models the hypothesis that TNF was a therapeutic target, using the collagen-induced arthritis model that he had already established in Tiny’s group (65). All animal models are imperfect; this one was less imperfect than others and was very useful in demonstrating the need for high concentrations of antibody for maximal efﬁcacy. It also showed clearly, by immunohistology, that leukocyte inﬁltration was markedly

Anti-inflammatory IL-10, IL-1ra, sTNF-R Immune system

TNF-α

IL-1 IL-6, IL-8, GM-CSF, etc. Pro-inflammatory

Figure 4 TNF-dependent cytokine cascade in rheumatoid arthritis (RA). This was an important component of the scientiﬁc rationale for anti-TNF therapy in RA.

reduced, and it demonstrated joint protection of both cartilage and bone. This joint protection was known in 1991 in the mouse but was not veriﬁed in humans with RA until 1999. In this work, we were greatly assisted by Robert Schreiber, who donated his anti-mouse TNF monoclonal antibody, unique at the time. He generously provided this hamster antibody in large amounts, without which we could not have done the work. It is of interest that coming from other vantage points, two other groups, of Thorbecke (66) and Piguet (67), concurrently demonstrated the beneﬁt of TNF blockade in mouse models of RA. Tiny Maini had started his research career at KIR with Dudley Dumonde. He had been involved in studying lymphocyte mitogenic factors in the late 1960s and had coined the term lymphokines (68) well before there was technology for identifying such rare molecules. So he was very aware of the importance and potency of such mediators, and exploring the role of cytokines in RA was thus for him also a natural progression. While I encouraged my colleagues to improve the sensitivity of techniques needed to quantitate cytokines, Tiny had the unenviable task of masterminding the collection of abundant samples of rheumatoid synovium needed and characterizing their clinical phenotype and biological marker proﬁle. To this day, I am puzzled that it is so difﬁcult to get the majority of surgeons to take the extra 1–2 minutes to ensure that the correct tissue www.annualreviews.org • Effective Therapy for Autoimmunity

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is collected in a sterile manner into the appropriate bottle of medium. The rare exceptions appear to be surgeons who understand the research process and the dependence of medical progress, indeed of all progress, on research. The cytokine ﬁeld was revolutionized by the molecular biology skills of the biotech industry. Scientists at a number of companies, e.g., David Goeddel, Pat Gray, and Axel Ullrich at Genentech and Craig Smith at Immunex, cloned cytokine genes, expressed the proteins, and generated antibodies. I made contacts with these companies, and they donated antibodies and cytokine reagents without the awful material transfer agreements that plague collaborative research today. Particularly important were antibodies to TNF, produced at Genentech, which had ﬁrst cloned it. At Genentech the extramural program manager then was Michael Shepard, who took a great interest in our work and helped us considerably to unravel the role of TNF in RA. He supplied all the cytokines, cDNAs, and antibodies to TNF and to LTα/TNF-β. After a few years, he returned to his own cancer research career, which was very productive, and he has found fame as the initiator, scientiﬁc champion, program manager, and developer of the anti-HER-2 antibody, trastuzumab, better known as Herceptin®, which has saved many thousands of lives of patients with breast cancer (69, 70). A personal reminiscence: When David Goeddel ﬁrst presented his group’s work on cloned TNF in 1984 at Schloss Elmau, I went to talk to him and was told that as the TNF project was a collaboration with a European company only they could supply European labs with TNF reagents. However, the European company was worried that my hypothesis that cytokines such as TNF might be involved in pathogenesis of disease would negatively inﬂuence the development of what they had wanted, which was a cytokine cancer cure. So they did not want to help me, but fortunately the Genentech research-driven culture did. James Woody, a U.S. Navy medically trained researcher, was a prot´eg´ee of Ken Sell who had beneﬁted from getting his PhD in the UK in

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three years, much quicker than was possible in the United States. Jim was rising rapidly in the U.S. Navy Medical Research Command, and Ken Sell, his chief, sent him to Av Mitchison in London to obtain his PhD. Somehow he ended up under my supervision. And being a very bright, diligent, well-organized scientist, he duly ﬁnished in the minimum time, often bringing his children to the lab at weekends in order to do so. He returned to Bethesda, jumping from being a PhD student to running a big laboratory for the U.S. Navy. From there, his career progressed in leaps and bounds, emulating his mentor Ken Sell to reach the top of the U.S. Navy Medical Research. While he was at the Navy, we kept in close touch, and he knew of our burgeoning work on TNF. The U.S. Navy funded some of this research. By the time Jim had ﬁnished his 20 years in the Navy, in 1991, and was considering pharmaceutical and biotech opportunities, he was aware that we were close to deﬁning TNF-α as a therapeutic target in RA. So it was very pleasing that he opted to join an emerging biotech company, Centocor, a pioneer in developing the monoclonal antibody ﬁeld. John Ghrayeb and his team at Centocor had grafted the human constant region to antibody variable genes from a murine anti-TNF hybridoma generated in Jan Vilcek’s laboratory (71), in response to Tony Cerami’s powerful arguments that blocking TNF-α might save thousands from death in sepsis (72). So in early 1991, before Jim had ofﬁcially started, I visited Centocor and presented our work leading up to the deﬁnition of TNF-α as a therapeutic target in RA. It received a warm reception, especially from Hubert Schoemaker, the chairman/CEO. Some of the company scientists were more skeptical, especially their only rheumatologist who somehow knew that anti-CD4 antibody would be much more effective for RA therapy than blocking TNF. Centocor was focused on sepsis, and in Europe, their IgM monoclonal antibody to LPS had been approved, on scant data. In the United States, it had not yet been approved, and so an interesting deal was set up, basically that I (and my colleagues) would help them to deﬁne

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the mechanism of action of their LPS antibody and how it protected despite its low afﬁnity, and they would help us with testing our TNF therapeutic target. Our academic-led project was not a normal clinical program. Jim Woody as chief scientist ran it instead of the clinical group, with the help of a number of Centocor staff, including Hanny Bijl, Dick McCluskey, Carrie Wagner, and Tom Schaible. The chimeric monoclonal antibody cA2 had already been administered in high dose to several dozen patients with sepsis. It failed to correct the septic shock, but importantly they did not get worse. This reassured us that its use in RA trials would not lead to overwhelming infections.

THE EXCITEMENT OF CLINICAL TRIALS The ﬁrst trial was an open study with no placebo controls, nonblinded owing to the unknown risks of blocking TNF in rheumatoid patients. Ten patients were initially planned to be treated with the high dose found necessary in mice. When the striking results of the ﬁrst patients were disclosed to the company, Centocor did not know exactly what to do, so it asked us to treat 10 more. Of course with 20 patients responding, it was easier to draw conclusions and publish than with only 10 (73). The results had matched or even exceeded our expectations. Over the slow (3 h) infusion of the antibody, many of the patients commented that they were already feeling better, less tired. Over the next day or two, reductions in stiffness and pain were noted. Large effusions in knee joints rapidly diminished. There had been concerns that blocking TNF, a host defense molecule, might promote infection, and so we had taken the precaution of starting the infusions slowly, with just one patient ﬁrst, treating them as inpatients, and we had our own nurse spend the night in their room in order to treat possible problems as rapidly and effectively as possible. It was a very thrilling time. All the patients we treated improved dramatically, despite having had long-standing active RA refractory

to current treatment. The ﬁrst two to three months were especially interesting because we did not know how long the beneﬁt would last. Patients returned to their normal activities, holidayed, played golf, etc., and were really happy. They thought the improvement might be long lasting. But it was not to be. There were 12 to 18 weeks of marked beneﬁt before relapse. There were no cures, but nevertheless there had been major improvement and a clear pointer for the future. I helped coorganize a conference in Arad, Israel, near the Dead Sea, with my friend David Naor. It was there, in midSeptember 1992, that Maini ﬁrst presented the dramatic results of the ﬁrst clinical trial. There were scientists from other companies, from Immunex, Genentech, Roche, etc., and the disclosure, probably premature owing to our naivet´e, started the race toward the clinic, as these companies had already produced TNF inhibitors for use in sepsis, based on Tony Cerami’s work (74). To establish what might happen with longerterm anti-TNF treatment, we sought ethical permission to retreat some of the patients from this ﬁrst trial after they had relapsed. Eight of 20 were retreated, up to three times. In each case, there was reintroduction of signiﬁcant clinical and biochemical beneﬁt (e.g., reduction in Creactive protein), suggesting that if TNF was blocked, other cytokines did not rapidly take over to drive the cytokine network (75). But this initial experiment was not a formal proof. There had been no controls or randomization, much needed in clinical trials with potentially high placebo responses. To do that, a formal double-blind (patients and clinicians), randomized, placebo-controlled trial was performed. Three European rheumatology friends, Joachim Kalden of Erlangen, Ferdinand Breedveld of Leiden, and Josef Smolen of Vienna, joined in with Tiny and me. There were issues to resolve, such as what to use as placebo. We chose human albumin to avoid immunizing patients to mouse antibodies, and the primary end point was limited to four weeks for ethical considerations and to reduce drop-outs in the placebo-controlled trial. Again, the results were www.annualreviews.org • Effective Therapy for Autoimmunity

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very clear and convincing. Both the high dose used previously and a tenth of the dose worked well, but the placebo infusion did not (76). We collected large samples—400 ml of blood from these patients—to do a very detailed analysis of the post-treatment events. The mechanism-ofaction studies were very informative, most importantly because they conﬁrmed that there was a very rapid diminution in other proinﬂammatory cytokines, for example IL-6. The reduction to baseline of a downstream cytokine in a few hours is evidence of a direct effect of anti-TNF (76, 77). The mechanism-of-action studies were performed in considerable detail, possible because it was an academic-led study, with the blood samples under our control. Few other clinical programs to date have been analyzed in such detail. We also looked at cellular changes in the blood: They were less informative than we had hoped but showed a rapid reduction in circulating neutrophils and monocytes. More interesting was a rapid increase in lymphocyte counts, which tend to be low in active RA, with more activated cells in the blood. The rapidity of the change suggested that there was a change of trafﬁcking, probably an exit of T lymphocytes from the joints. We were able to explore the hematology in more detail, and important pathogenic clues emerged. There was an increase in the hemoglobin concentration (usually low) in the patients within the four weeks of the trial. The high platelet counts in RA and high ﬁbrinogen, both potentially linked to accelerated atherosclerosis, tended to normalize (78). This was a clue that the abnormal cardiovascular outcomes in RA might be improved, but it took a long time for other groups to establish this, in large post-registration registries in the UK that Alan Silman, David Isenberg, and their colleagues have ably run (79, 80), as well as those in Sweden (81, 82) and other countries. Cell inﬁltration in the joints is a hallmark of chronic RA. The reduction in joint swelling suggested that fewer cells persisted after therapy. Biopsy studies clearly showed that to be the case, with reductions in lymphocyte and

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macrophage numbers and a thinner lining layer. How did this occur? Attempts to show increased apoptosis were not successful, but we did ﬁnd reductions in markers of cell recruitment. Thus, endothelial-speciﬁc E-selectin was reduced, both as detected in synovium by immunohistology and in serial serum samples as soluble form. Also reduced were intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, both involved in cell recruitment to tissues. The more quantitative serum soluble adhesion molecule assays were consistent with the less quantiﬁable histologic reductions. In a similar way, we also found that numerous chemokines are reduced in the synovium as well as in the blood (83, 84). Markers of tissue destruction were also diminished. Serum matrix metalloproteinase precursors are elevated in active RA and were reduced after anti-TNF therapy (85). Of course, serum assays fail to demonstrate what is active in the joint, but they do reﬂect the biosynthesis during active disease. Rheumatoid joints are very cellular and have often been described as resembling tumors. To sustain this augmented mass, new vessels are needed, and so angiogenesis is readily apparent. Ewa Paleolog and colleagues observed that angiogenic factors are also augmented in RA, and it was of great interest that the most potent of these, vascular endothelial growth factor (VEGF) (86), was rapidly but partly diminished after anti-TNF therapy. However, it took a lot of subsequent immunohistological analysis by Peter Taylor and colleagues to demonstrate a reduction in blood vessels (87). It was remarkable how much molecular work could be performed from one small clinical trial: We obtained many of longitudinal samples but still had many unanswered questions. The results all pointed toward normalization of the pathological processes, and while they did not show that TNF causes arthritis, they showed that TNF is a very important driver of active disease. In the mouse, the elegant work of Kollias and his colleagues has shown that transgenic mice overexpressing TNF does cause an erosive polyarthritis, even in mice lacking T and

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B cells (88, 89). We had learned much about disease mechanisms from the detailed work on the ﬁrst placebo-controlled trial. But there was a subsequent disaster. The freezer holding most of the samples from the next trial, a longer phase II, over six months, defrosted. The consternation, angst, frustration was awful; it was physically painful to think of the major scientiﬁc opportunities lost. In the phase III trial, Centocor controlled the samples to speed up the trial process and the hoped-for drug approval; hence, far fewer samples were collected, and the opportunity of investigating biological markers in greater detail was lost. By 1993, there was clear evidence of clinical beneﬁt, even in the most disease-active patients, but no cures. Retreatment was successful, but we were concerned about immunogenicity of this antibody, which, while chimerized (2 faces, 3/4 human constant region), still had the mouse variable regions that were likely to be immunogenic (71). Whether it was a drug that could be used for long-term therapy was unclear, so research was planned to learn how to augment the beneﬁt and reduce immunogenicity. As is the case for all major diseases (e.g., cancer, hypertension), combination treatment is necessary to optimize clinical beneﬁt. So we used the mouse model of collagen-induced arthritis to pilot potential approaches to augment beneﬁt. It was not difﬁcult to produce anti-TNF nonresponder mice. We needed to reduce the treatment dose to 50 μg/twice per week instead of the efﬁcacious 300 μg (65). In this model, using suboptimal doses of anti-TNF antibody, a range of additional T cell–directed therapies were tested, and cyclosporine, antiCD4, and CTLA4-Ig (90–92) were all effective, suggesting that there was enhancement of the clinical beneﬁt if T cell function was also reduced. In these experiments, there was synergy, as the effects of anti-CD4, cyclosporine, and CTLA4-Ig as monotherapy after disease onset were rather modest, if present at all (91). So from these animal model studies, especially the anti-CD4 experiment, the clinical trial design evolved in which patients with an inade-

quate response to methotrexate (MTX) were treated with various concentrations of antiTNF (by that time known as cA2, later inﬂiximab, later Remicade®), in order to augment their response. MTX is effective in a signiﬁcant proportion of rheumatoid patients, as demonstrated and championed by Michael Weinblatt in Boston (93, 94). In the 1990s, its impact was growing, and it was becoming recognized as the most effective disease-modifying antirheumatic drug. As it would not be possible to use two unlicensed drugs together (anti-CD4 is unlicensed), Tiny Maini and I chose to use MTX, which had been reported, among its legion of effects, to inhibit T cell function, promote apoptosis, and reduce IFN-γ production (95, 96), effects that resembled those of anti-CD4. Patients who had an inadequate response to MTX were abundant, and so our trial was designed to ﬁll an important clinical need. But an issue was the risk, especially of infection, in the combination. So a very low dose of MTX was chosen, 7.5 mg/week. However, at this time Centocor was struggling ﬁnancially, and the long-term clinical trial we had envisaged was shortened to 24 weeks, 12 weeks on therapy and 12 weeks further follow-up. Nevertheless, the results were very interesting and have been inﬂuential. Lowerdose cA2, 1 mg/kg at weeks 0, 2, 4, 8, and 12, was effective, with about 25–30% of patients showing 50% beneﬁt [using the American College of Rheumatology (ACR) 50 criteria], only up to week 4 if used alone. But with low-dose MTX, there was clear synergy, with 60–70% ACR 50 up to week 24 (97, 98). The results using higher doses of cA2, 3 mg/kg and 10 mg/kg, also showed the added beneﬁt of the addition of cA2 to MTX. It is now the combination most extensively used in routine practice, with about 70% of patients given the existing three anti-TNF drugs also being given MTX because of the increased efﬁcacy (99, 100). After Tiny had ﬁrst presented the exciting initial clinical results to Centocor management, Jim Woody made cA2 available to Sander Van Deventer, an enterprising gastroenterologist in Amsterdam, who successfully treated a Crohn’s disease www.annualreviews.org • Effective Therapy for Autoimmunity

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patient with ﬁstulas (101). Centocor then prioritized resources to Crohn’s clinical trials, to our dismay. This became the ﬁrst approved indication for cA2, now known as inﬂiximab or Remicade®. Despite its clinical priorities and cash limitations, Centocor agreed to fund an important imaging mechanism-of-action trial to investigate leukocyte trafﬁcking to joints before and after anti-TNF therapy. This was performed by Peter Taylor, who now ably leads the Kennedy clinical trials group, together with A.M. Peters, a leukocyte imaging expert at Hammersmith Hospital. This trial demonstrated that antiTNF reduces leukocyte trafﬁc to joints (84). This was an important clinical trial because reduced recruitment of inﬂammatory cells to disease sites probably accounts for the ability of anti-TNF to ameliorate so many diseases. With the success of the phase II trial in which MTX was supplemented with cA2, the multinational phase III was planned and eventually successfully executed with Peter Lipsky (a friend from the 1970s from his time as a postdoc at NIH with Alan Rosenthal) as the U.S. trial leader and Tiny as the European leader. The complexities and grind of phase III trials made this a nonexciting and stressful, though necessary, experience compared with the earlier trials. But through the whole process, working with Tiny was an enjoyable, educational experience, as we blended his rheumatological and other clinical skills with my immunology and cytokinology and entered ﬁelds new to us, where success rates had been dauntingly low.

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PROS AND CONS OF WORKING WITH INDUSTRY Academia and industry often do not mix well. Having studied medicine, I was interested in the practical application of immunological research. My industrial interactions started while I was still a postdoc. James Howard worked on immunity to polysaccharides (102), a ﬁeld analogous to one of my research topics, immunity to polymerized ﬂagellin (103). Both were repeated polymers that induced thymus-independent an16

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tibody responses. He invited me to consult with his group at Wellcome Research Labs, a small pharmaceutical company, known locally at the time as University of Beckenham for its academic bent. It had recruited Nobel Prize winners John Vane and James Black to run its research. This was a wonderful start to the pros. There was much to learn, and one was paid extra, which is very appreciated early in one’s career, with growing children and increasing bills to pay! My second exposure was with ICI Pharmaceuticals, the precursor of Zeneca, now AstraZeneca, when my PhD student Eric Culbert joined them: I have had a number of longterm consulting relationships, helping friends and colleagues. For example, I consulted with ed David Webb, ﬁrst at Syntex, then OSI, Syrrx, and now Celgene; Michael Moore; Jim Woody at Centocor, then Roche. I advised Michael Shepard, ﬁrst at Genentech, on the Herceptin project, then adenoviral gene therapy while he was at Canji, then targeted cancer therapy at Newbiotics, and now at Receptor Biologix. These long-term relationships were in many ways very educational. Thus, our work on adenoviral inhibitors for studying cytokine and other intracellular signaling pathways developed from an awareness of the utility of adenoviruses developed while helping Canji. But the work with Centocor was on quite a different scale, and despite its many frustrations at times, it was very beneﬁcial and helped drive anti-TNF therapy forward. Like all the best interactions, mutual beneﬁt is essential. In 1992, Centocor was a rapidly growing biotech company that thought it was going to be the one to capitalize on treating sepsis with monoclonal antibodies. It had an IgM (Centoxin) anti-LPS monoclonal antibody approved on limited data in Europe and was looking forward to new data and approval in the United States. When Jim Woody joined them as chief scientist, he was keen that we help them to ﬁll a gap, to understand how Centoxin mediated its beneﬁt (104). As this research would involve our ﬁeld of expertise, cytokines, it was logical. In return, it would be easier for him to encourage his

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colleagues to invest in and become interested in our untested and not yet accepted concept of TNF as the therapeutic target for RA. My colleague Peter Katsikis (105) duly found a novel mechanism by which complement may assist in endotoxin clearance, and Jim succeeded in getting Centocor to provide ﬁnancial and antibody resources to test anti-TNF therapy. The success of this interaction led to a promising novel therapy for a major unmet need and, in my opinion, eventually saved Centocor from extinction, as the endotoxin project failed and its share price crashed in 1992. Centocor had charismatic leaders Hubert Schoemaker and Michael Wall, and it tenaciously survived its problems with sepsis therapy and went on to be acquired for almost $5 billion by Johnson & Johnson in 1999. The early trials with Jim Woody at the helm and Harlan Weissman, a key player, were run harmoniously, as were the ﬁrst placebo-controlled trial and the six-month phase II trial. But then as the likelihood increased that clinical success would translate into commercial success, the management of the trials became focused on commercial speed of completion, and thus were run very differently. By this time, Jim Woody had left Centocor to become president of Roche in Palo Alto, California, and Hubert Schoemaker had been weakened by a brain tumor and its very aggressive therapy. We were left to manage the Centocor-KIR relationship with no internal champions. It was not congenial: Agreements and promises were reneged; stress ensued. Matters important to academics, such as publication and presentation rights, were challenged and arbitrarily overturned; issues of the nationality of presenters of key data were raised. It is almost unbelievable that it was felt in the late 1990s that an American was a more credible presenter of data than a European. Agreements about authorship were subsequently ignored by the company. Even worse is what happened after the drug was approved and started to sell and the other two TNF inhibitors also were sold. The respect scientists have for each other’s discoveries is often not shared by industry and its

lawyers. In Bob Dylan’s words, “money doesn’t talk, it swears.” I regret that anti-TNF is yet another of the British inventions that was not commercialized in Britain, but rather in the United States. This happened despite very extensive discussion in the late 1980s/early 1990s with the UK’s leading monoclonal antibody company. But this company missed the golden opportunity taken up by Centocor and its U.S. rivals, Immunex and Abbott, that has resulted in approximately $11 billion in sales of anti-TNFs in 2007. But overall, I am an enthusiastic supporter of working with the biotech and pharmaceutical industry. Many of the skills needed to get new treatments into the patient population are possessed by industry: medicinal chemistry, pharmacology, and especially the ﬁnancial resources for major clinical trials. These are complimentary with academia, and if these complimentary skills were harnessed more appropriately, society would undoubtedly beneﬁt. My actions with many ongoing industrial relationships probably speak louder than any words on this topic, and the practical outcome of the interaction between academia and industry is that there are now no more RA patients in wheelchairs.

PROMOTING TRANSLATIONAL RESEARCH What is translational research? There is no agreed deﬁnition, and that is part of the problem, but by conventional usage it is research designed to further human health: to bridge new discoveries in basic research and the applied research in clinical trials of therapeutic products. A major challenge in research has always been which experiment to perform, among the countless possibilities. Sir Peter Medawar has elegantly expounded on this, and “The Art of the Soluble” is not an exact science (106). How high to aim at any one time can result in major disappointment if the effort fails, but if it succeeds, wonderful gains can be achieved. So every scientist expresses their unique personality in their choice of projects and approach. I was acutely aware that despite the major www.annualreviews.org • Effective Therapy for Autoimmunity

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advances in cellular and molecular understanding of immunology, its key practical applications, vaccines, date back 200 years to the time before immunology was a major science. Why had there been so little progress, despite stellar advances such as the discovery of monoclonal antibodies by Kohler & Milstein (107)?

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Communication remains a problem in all complex organizations and societies, and scientiﬁc meetings have an important role in science. I ﬁrst became interested in organizing scientiﬁc meetings in 1983, wanting to promote the relatively new techniques of T cell cloning and their potential for helping unravel disease pathogenesis. This meeting took place in 1984 (108), after I had worked out how to raise funds for these small, intense, focused meetings. Critical to the venture was James Woody, when he was in the U.S. Navy, who convinced the Navy to support meetings. As he has progressed in his career, via Centocor and Roche, he has for many years been the major funder of many scientiﬁc meetings and made the meeting organization much less stressful for me. A key beneﬁt of organizing these meetings has been that my colleagues and I learned, made contacts and friends, and initiated important collaborations, with the help of discussions on the lawns and at the bar during these conferences, chieﬂy held at Trinity College, Oxford (109, 110). Many experiments were hatched at these meetings, which have been variously named T Cell Activation in Health and Disease; T Cells and Cytokines in Health and Disease; From Laboratory to the Clinic. I have held 20 of these meetings, coorganized with Andrew McMichael, a fellow at Trinity, again with the skilled enthusiasm of my long-term personal assistant, Philippa Wells, who has now capably helped me for 27 years, all ﬁnanced through friends and acquaintances in the biotech and pharmaceutical industries.

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But while translational research is now a hugely popular theme, from NIH with Dr. Zerhouni’s roadmap to the newly revamped Medical Research Council under Sir Leszek Borysiewicz, it is still very ill deﬁned, and many fail to understand its basic principles. Translation is the apex of the pyramid, an activity suitable for only a minority of projects. It depends ﬁrst on excellent quality of science, but second on science that reveals an important rate-limiting step in the complex biology that occurs in vivo. Not only that, the complex biology must be relevant to humans and their diseases, and not just to mice. I paraphrase the late Judah Folkman, who said in the late 1990s, when there was hype about blocking angiogenesis curing cancer: If you are a mouse with cancer, we can help you, but if you are human, it may take another 20 years. Humans differ from mice, most obviously in their longevity. With longevity there is a need for more robustly regulated biological systems for the many years before reproduction. So while mouse systems need to function for 10–12 weeks and are subject to Darwinian evolutionary selection pressure only for this period, for humans the selection process is more than 100 times longer, for over 20 years. We expect differences in complexity to emerge, so mice are not likely always to be an accurate model for human pathophysiology. It is thus puzzling and a continual challenge that many medical journals still fail to appropriately prioritize and encourage research performed with rare human disease material, often because all the controls cannot be performed as well as in mice. The latter is the current band wagon, as much progress is based on elegant genetically engineered experiments. But many failures in successful translation of laboratory research into disease relevance are likely due to technical issues such as the overuse of reductionist systems, interspecies differences, or the use of transformed cell lines, with their many mutations, as surrogates for normal human cells.

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Directing the Kennedy Institute of Rheumatology Directing KIR has been a major focus of my activity for the past six years since Tiny Maini retired as director. The KIR is the largest research institute dedicated to rheumatology and has been supported effectively by renewable long-term (ﬁve-year) major grants from the Arthritis Research Campaign in the UK covering 40–50% of the total budget and increasingly by the Kennedy Institute of Rheumatology Trust. The director’s role is to evolve strategy, recruit the best talent, provide them with the best resources, and let them get on with their research. Leading by example works better for most scientists than direction. KIR’s focus is translational research, from laboratory to clinic and back again, and it has a wide range of expertise from molecular science, proteomics, molecular modeling, etc., through cytokine biology, immunology, inﬂammation, matrix biology, and signaling to clinical research and trials. KIR has long been a global resource for research and training in rheumatology and related disciplines. My challenge is to leave it in an even better state than I found it when my friend Tiny transferred it to my care. It has been a privilege to work there long term with many talented colleagues, almost 25 years with Sir Ravinder Maini, 20 years with Fionula Brennan, almost that long with Brian Foxwell, and 10 years with the burgeoning osteoarthritis team leaders, Jeremy Saklatvala and Hideaki Nagase. The team spirit, mutual support, enthusiasm, and intellectual challenges make it a pleasure. Interacting with the Faculty of Medicine, Imperial College, which the KIR joined in 2000, opened up new avenues and access to many multidisciplinary colleagues in other branches of medicine, engineering, chemistry, etc. Taking part in the creation of the UK’s ﬁrst Academic Health Sciences Centre led by Stephen Smith, from the merger of Hammersmith, Charing Cross, and St. Mary’s hospitals, has been educational. The administrative issues at KIR, in a constantly changing scientiﬁc environment, are a challenge and are less

entertaining than the science, but the prospect of helping to deliver the fruits of research more effectively for our patients makes it worthwhile.

CONCLUSIONS Maintaining Life/Work Balance Science is fun, and should be fun. It is the ultimate experience in solving puzzles, puzzles that no one has previously solved, and you are even paid to solve them. If you are not able to enjoy the excitement and thrill of science, to enjoy the roller-coaster ride, and to shrug off the inevitable frustrations of failed experiments, malfunctioning equipment and colleagues, and rejected papers and grants, then a career in science will be more pain than pleasure and perhaps is not a wise choice for you. But with the fun and excitement comes the inevitable huge work load, and maintaining a life/work balance is a challenge that few can successfully manage. For those working in Europe, at least there is the hallowed tradition that long holidays are beneﬁcial, but my U.S. colleagues seem to take far fewer holidays. They spend more time in the lab, but does that add up to greater productivity? Having enjoyed outdoor activities and sports while growing up in Australia, I know long holidays provide not only an opportunity to enjoy family, friends, and the splendor of our planet, but also time to think creatively and strategically. Some of my best ideas emerged thousands of miles from the laboratory. A challenge for all scientists is to optimize their productivity; my warning is that more time in the lab might not be the best way. Eventually, we all learn that time is life’s most precious commodity.

Do We Value Practical Research Contributions? All of life is inﬂuenced by fad and fashion, and science is no exception. The term “blue sky research” (for pure basic research) clearly implies basic research’s desirability, whereas in contrast applied research implies sweat rather than

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inspiration. But is this really true? I could make an argument for the reverse. Thus, much basic research is not inspired, and, in the past quarter of a century, it has evolved from many projects following a preset pattern chieﬂy purifying a protein, to cloning the mRNA for a gene, to making transgenics and knockouts. Clearly, as techniques become better deﬁned, much diligence is necessary, but how much really new, creative, inspirational research takes place per project? I suspect that what really matters in research is its quality and imagination, and both are always needed for the best, pure, basic, blue sky, applied, or translational research to succeed at the highest level. My own experience is that society does indeed value research contributions of a practical or applied nature. If it is practical or applied, the effects of the research are easier to measure than they are with basic research. However, there appears to be greater delay before success in practical research is recognized, which is inevitably frustrating. The frustration stems in part from the delay of being recognized or rewarded only by objective concrete delivery, rather than by subjective promise or potential. In due course my work with my many collaborators has resulted in much personal recognition, including election to the National Academies of Science in the UK and Australia, honors such as the European Inventor of the Year award in the Lifetime achievement category, the Curtin Medal of Australian National University, and the award, together with Ravinder Maini, of a series of prestigious international prizes for medical research such as the Crafoord Prize of the Royal Swedish Academy of Science and the Albert Lasker Award for Clinical Medical Research. But the greatest reward of practical contributions, added to the respect of one’s peers, is the heartwarming acknowledgment by patients of the positive impact on their lives. It is an unexpected pleasure.

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what might happen next. What I have helped to achieve, ﬁrst, is that a cytokine, TNF, is now recognized as a very good therapeutic target for a cluster of chronic inﬂammatory diseases, including RA, juvenile RA, Crohn’s disease, ankylosing spondylitis, psoriatic arthritis, psoriasis, and ulcerative colitis (111). This raises the question of how important TNF is as a ﬁre alarm for noxious signals bringing in the ﬁre ﬁghters, leukocytes, and whether most conditions currently treated by corticosteroids might be treatable by anti-TNF. Second, my colleagues and I have helped to demonstrate that biological therapeutics, that is, monoclonal antibodies and antibody-like fusion proteins, can be used for chronic diseases in the long term, now very long term (up to 10 years and running). This has inevitably inﬂuenced the pharmaceutical industry, and now a very signiﬁcant percentage of new therapeutics entering trials are of this type. There are, of course, major beneﬁts. An important one is that biologics, with a large surface of interaction with their target, are more speciﬁc and selective than the small molecular, organic chemicals traditionally favored by the pharmaceutical industry. Hence, their side effects are more predictable because they are mechanism related. The unfortunate TeGenero disaster, in which an activating anti-CD28 monoclonal antibody was used to try to stimulate regulatory T cells, is worth noting. Some believe it was unexpected or unpredictable. However, most human immunologists like myself, who are aware of the variable toxicity of OKT3, an anti-CD3 antibody, which polyclonally activates T cells, believe it was extremely predictable (112). This disaster and the ensuing publicity have markedly inﬂuenced clinical trial capacity in the UK. There have been a number of subsequent successes for monoclonal antibodies and cytokine blockade. Anti-CD20 antibody, developed for lymphoma based on Ron Levy’s work, has been very successful (113) and was introduced to rheumatology by Jo Edwards (114). IL-1 blockade with IL-1 receptor antagonist has been approved but, because it

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has been less effective than TNF blockade, has not been widely used in RA (115). It has been very useful in, for example, MuckleWells syndrome. There is clear effectiveness of blocking the IL-6 receptor with an antibody developed by Tadamitsu Kishimoto (116, 117), and there is great anticipation for the utility of blocking RANK ligand for bone disorders (118). So an interesting possibility emerges. Are all diseases and unmet medical needs treatable by cytokine blockade? As an optimist I believe that will be close to the truth. The 100 or so cytokines (a term that I use to encompass interleukins, growth factors, IFNs, chemokines, members of the TNF family, etc.) are involved in all key biological processes, for example cell proliferation, cell motility, inﬂammation, immunology, angiogenesis, ﬁbrosis, etc. Hence, all diseases involve alterations in cytokine expression, and many are upregulated. These are potential therapeutic targets. My colleagues and I are pursuing important new therapeutic endeavors that might be treatable by cytokine blockade. For example, with Claudia Monaco, we are studying treatment of atherosclerosis; with Mervyn Maze, we are studying post-operative cognitive dysfunction; and with Tracy Hussell, Brian Foxwell, and Kendall Smith, we are studying acute respira-

tory distress induced by avian ﬂu. Only time will tell if these endeavors will succeed, but inevitably the ﬁeld of cytokine blockade or anticytokine medicine will ﬂourish in many more directions. Why should one bother to read semihistorical personal reviews? I am not sure, but there may be lessons for the less experienced. If so, one is that we now have wonderful technologies for permitting scientiﬁc progress in the ﬁeld of disease pathogenesis and therapy. These can unravel molecular mechanisms of diseases and permit the discovery, design, and development of new treatments that impact millions of lives. But this will not work for most projects, as most projects and hypotheses fail. But it will succeed for some. So there are enormous opportunities remaining to use science for the beneﬁt of human health and welfare. But the hurdles are tough and the risks high. I encourage as many clinicians as possible to spend the time and training, as I did, to merge both science and medicine, as there is a critical shortage of individuals, for example physician-scientists, who can synthesize these components to bring the translational discoveries to patients. Perhaps a summary of an exciting adventure that has beneﬁted many patients might encourage and challenge you to venture into that arena and see what you might achieve.

DISCLOSURE STATEMENT Over the years, I have interacted extensively with companies and so have been a paid consultant or scientiﬁc advisory board member to many companies involved in arthritis and cytokine work: Amgen, Astra-Zeneca, Abbott, Centocor, Glaxo, Celgene, Immunex, Merck, Roche, Wyeth, Novo Nordisk, Schering Plough, Boehringer-Ingelheim, Synovis Ltd., Xenova plc, Hydra Biosciences, Receptor BioLogix, Inc., Nuon Therapeutics, Canji, Inc., Trillium Therapeutics, Inc., Sandoz (now Novartis), Alza, Inc., Almirall Prodesfarma, Ferring AS, and Calyx Therapeutics. I or close colleagues have received grants in the past three years from Roche, Wyeth, Novo Nordisk, Celgene, Nuon Therapeutics, and Receptor Biologix, Inc. I have patents in the anti-TNF therapy ﬁeld and many others (I was European Inventor of the Year in 2007). I have signiﬁcant ﬁnancial holdings in Johnson & Johnson and Schering Plough.

LITERATURE CITED 1. Mishell RI, Dutton RW. 1966. Immunization of normal mouse spleen cell suspensions in vitro. Science 153:1004–6 www.annualreviews.org • Effective Therapy for Autoimmunity

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2. Marbrook J. 1967. Primary immune response in cultures of spleen cells. Lancet 2:1279–81 3. Diener E, Armstrong WD. 1967. Induction of antibody formation and tolerance in vitro to a puriﬁed protein antigen. Lancet 2:1281–85 4. Ada GL, Nossal GJ. 1987. The clonal-selection theory. Sci. Am. 257:62–69 5. Diener E. 1968. A new method for the enumeration of single antibody-producing cells. J. Immunol. 100:1062–70 6. Jerne NK, Nordin AA. 1963. Plaque formation in agar by single antibody-producing cells. Science 140:405 7. Feldmann M. 1971. Induction of immunity and tolerance to the dinitrophenyl determinant in vitro. Nat. New Biol. 231:21–23 8. Lennon V, Feldmann M. 1972. The detection of autoantibody-forming cells. I. An assay for plaqueforming cells to the basic protein of myelin in guinea-pigs. Int. Arch. Allergy Appl. Immunol. 42:627–40 9. Miller JF, Mitchell GF. 1968. Cell to cell interaction in the immune response. I. Hemolysin-forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct lymphocytes. J. Exp. Med. 128:801–20 10. 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 11. Nossal GJ, Cunningham A, Mitchell GF, Miller JF. 1968. Cell to cell interaction in the immune response. 3. Chromosomal marker analysis of single antibody-forming cells in reconstituted, irradiated, or thymectomized mice. J. Exp. Med. 128:839–53 12. Roitt IM, Greaves MF, Torrigiani G, Brostoff J, Playfair JH. 1969. The cellular basis of immunological responses. A synthesis of some current views. Lancet 2:367–71 13. Greaves MF, Roitt IM, Rose ME. 1968. Effect of bursectomy and thymectomy on the responses of chicken peripheral blood lymphocytes to phytohaemagglutinin. Nature 220:293–95 14. Feldmann M, Basten A. 1971. The relationship between antigenic structure and the requirement for thymus-derived cells in the immune response. J. Exp. Med. 134:103–19 15. Feldmann M, Basten A. 1972. Cell interactions in the immune response in vitro. 3. Speciﬁc collaboration across a cell impermeable membrane. J. Exp. Med. 136:49–67 16. Feldmann M, Basten A. 1972. Speciﬁc collaboration between T and B lymphocytes across a cell impermeable membrane in vitro. Nat. New Biol. 237:13–15 17. Schimpl A, Wecker E. 1972. Replacement of T-cell function by a T-cell product. Nat. New Biol. 237:15– 17 18. Feldmann M. 1972. Cell interaction in the immune response in vitro. II. The requirement for macrophages in lymphoid cell collaboration. J. Exp. Med. 135:1049–58 19. Lennon V, Feldmann M, Crawford M. 1972. The detection of autoantibody-forming cells. II. Cells in lymph nodes and central nervous system containing antibody to myelin basic protein. Int. Arch. Allergy Appl. Immunol. 43:749–58 20. Hanafusa T, Pujol-Borrell R, Chiovato L, Russell RC, Doniach D, Bottazzo GF. 1983. Aberrant expression of HLA-DR antigen on thyrocytes in Graves’ disease: relevance for autoimmunity. Lancet 2:1111– 15 21. Klareskog L, Forsum U, Scheynius A, Kabelitz D, Wigzell H. 1982. Evidence in support of a selfperpetuating HLA-DR-dependent delayed-type cell reaction in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 79:3632–36 22. Bussard AE, Lurie M. 1967. Primary antibody response in vitro in peritoneal cells. J. Exp. Med. 125:873– 92 23. Schrader JW, Feldmann M. 1973. The mechanism of antigenic competition. I. The macrophage as a site of a reversible block of T-B lymphocyte collaboration. Eur. J. Immunol. 3:711–17 24. Wagner H, Feldmann M. 1972. Cell-mediated immune response in vitro. I. A new in vitro system for the generation of cell-mediated cytotoxic activity. Cell. Immunol. 3:405–20 25. Wunderlich JR, Canty TG. 1970. Cell mediated immunity induced in vitro. Nature 228:62–63 26. Mitchison NA. 1971. The carrier effect in the secondary response to hapten-protein conjugates. I. Measurement of the effect with transferred cells and objections to the local environment hypothesis. Eur. J. Immunol. 1:10–17

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116. Nishimoto N, Kishimoto T. 2006. Interleukin 6: from bench to bedside. Nat. Clin. Pract. Rheumatol. 2:619–26 117. Maini RN, Taylor PC, Szechinski J, Pavelka K, Broll J, et al. 2006. Double-blind randomized controlled clinical trial of the interleukin-6 receptor antagonist, tocilizumab, in European patients with rheumatoid arthritis who had an incomplete response to methotrexate. Arthritis Rheum. 54:2817–29 118. Miller PD, Bolognese MA, Lewiecki EM, McClung MR, Ding B, et al. 2008. Effect of denosumab on bone density and turnover in postmenopausal women with low bone mass after long-term continued, discontinued, and restarting of therapy: a randomized blinded phase 2 clinical trial. Bone 43:222–29

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Structural Biology of Shared Cytokine Receptors Xinquan Wang, Patrick Lupardus, Sherry L. La Porte, and K. Christopher Garcia Howard Hughes Medical Institute, Stanford University School of Medicine, Departments of Molecular and Cellular Physiology, and Structural Biology, Stanford, California 94305; email: [email protected], [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2009. 27:2.1–2.32

Key Words

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

interleukin, signaling, structure

This article’s doi: 10.1146/annurev.immunol.24.021605.090616

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0001$20.00

Recent structural information for complexes of cytokine receptor ectodomains bound to their ligands has signiﬁcantly expanded our understanding of the macromolecular topology and ligand recognition mechanisms used by our three principal shared cytokine signaling receptors—gp130, γc , and βc . The gp130 family receptors intricately coordinate three structurally unique cytokine-binding sites on their four-helix bundle cytokine ligands to assemble multimeric signaling complexes. These organizing principles serve as topological blueprints for the entire gp130 family of cytokines. Novel structures of γc and βc complexes show us new twists, such as the use of a nonstandard sushitype α receptors for IL-2 and IL-15 in assembling quaternary γc signaling complexes and an antiparallel interlocked dimer in the GM-CSF signaling complex with βc . Unlike gp130, which appears to recognize vastly different cytokine surfaces in chemically unique fashions for each ligand, the γc -dependent cytokines appear to seek out some semblance of a knobs-in-holes shape recognition code in order to engage γc in related fashions. We discuss the structural similarities and differences between these three shared cytokine receptors, as well as the implications for transmembrane signaling.

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INTRODUCTION

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The interaction of cell surface receptor extracellular domains with secreted ligands is essential to most types of cell signaling and cellcell communication. This initial step and the subsequent activation of membrane-proximal and -distal intracellular signaling cascades lead to speciﬁc, although often redundant, cellular responses that control cell proliferation, differentiation, maturation, and survival. Cell surface receptors usually bind their ligands through highly speciﬁc molecular interactions to provide the tight regulation necessary for control of physiological responses. However, researchers increasingly appreciate that many receptor systems exhibit, to a greater or lesser extent, cross-reactivity with a spectrum of different ligands. There are many examples of degenerate, shared receptors with central roles in signaling (1). In neurobiology, the p75 neurotrophin receptor can recognize a family of neurotrophic factors, including nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 (2). The glial cell line–derived neurotrophic factor family ligands, which include glial cell line–derived neurotrophic factor, neurturin, artemin, and persephin, share the RET receptor as the signaling subunit in their receptor complexes (3). In the immune system, shared receptors exist in both adaptive (T cell receptors, costimulatory molecules B7/CD28) (4) and innate immunity (NKG2D natural killer receptor, scavenger, and pattern-recognition receptors such as RAGE and Toll) (5). However, the most widespread roles for shared receptors are found for cytokines (6–9), which are secreted growth factors that control cell growth and proliferation primarily in the immune and hematopoietic systems. There are three major shared receptors in the class I cytokine receptor family: the common gamma chain (γc ), gp130, and the common beta chain (βc ), which participate in the formation of receptor complexes for nearly 20 different cytokines (Figure 1). Important insights into the mechanisms for cross-reactivity of shared cytokine receptors

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have emerged from structural studies of complexes between cytokines and extracellular domains of their receptors (10). During the past several years, crystal structures have been determined for gp130, γc , and βc complexes with cytokines, including gp130 bound to human herpes virus (HHV)-8 IL-6 (11), IL-6 (12), and leukemia inhibitory factor (LIF) (13); LIF complexed with LIF receptor (14); γc bound to IL-2 (15, 16) and IL-4 (17); and more recently βc bound to GM-CSF (18). Gp130, γc , and βc share a rudimentary core structural blueprint for the assembly of the extracellular cytokinereceptor signaling complexes; however, there are many important deviations between these three systems that result in substantially different signaling complex topologies. Collectively, these structures allow us to delineate common and unique structural features for both ligand recognition and assembly of signaling complexes by these three major shared cytokine receptors in the class I family, which is the focus of this review. Owing to space constraints, we could not cite all contributors to this ﬁeld. We refer the reader to excellent treatises on various aspects of cytokine structure, receptor interaction, and signaling; see References 10, 19–35.

THE CLASS I CYTOKINE RECEPTORS Cytokines represent a diverse group of small soluble proteins that when secreted by one cell can act on the same cell, in an autocrine fashion, or on another cell, in a paracrine fashion (36). Through binding to speciﬁc cell surface receptors, they initiate signals that are critical to a diverse spectrum of functions, including induction of immune responses, cell proliferation, differentiation, and apoptosis. Structural analysis has allowed the grouping of cytokines into different structural classes, including the helical cytokines (37), the trimeric tumor necrosis factor (TNF) family (38), the cysteine knot growth factors (39), and the β-trefoil growth factors (40). Cytokines can also be classiﬁed according to the type of receptor that they engage.

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IL-2

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Figure 1 Diversity of shared cytokine-receptor interactions. Shared cytokine receptors γc (a), gp130 (b), and βc (c) are represented schematically on a cell membrane. The respective interacting cytokines with known three-dimensional structures are shown with cylinder representations of the four-helix bundles. (Abbreviations: LIF, leukemia inhibitory factor; OSM, oncostatin-M; CNTF, cillary neurotrophic factor; CLC, cardiotrophin-like cytokine; CHR, cytokine-binding homology region; HHV-8, human herpes virus; GM-CSF, granulocyte-macrophage colony-stimulating factor.)

On the basis of common structural features, the cytokine receptors are grouped into six major families: class I cytokine receptors, class II cytokine receptors, TNF receptors, IL-1 receptors, tyrosine kinase receptors, and chemokine receptors (36, 41, 42). The class I cytokine receptors, also known as the hematopoietin receptors, constitute the largest group among the cytokine receptor family (41, 43, 44). These are type I membrane proteins with an N-terminal extracellular and C-terminal intracellular orientation. The extracellular segments of the class I cytokine receptors show a modular architecture, which is characterized by a ∼200 residue–long cytokinebinding homology region (CHR) (45) possessing the classical binding motif for cytokines, as structurally delineated in the human growth hormone (hGH) receptor complex (46). The CHR module consists of two ﬁbronectin typeIII (FNIII) domains connected by a linker, and it represents the signature recognition module for helical cytokines that is present on every type I cytokine receptor (Figure 1). The upper, N-terminal domain contains four (47) con-

served cysteine residues that form interstrand disulﬁde bonds. The lower, C-terminal domain has a conserved Trp-Ser-X-Trp-Ser motif (45, 46). Mutagenesis studies have shown an essential structural role for these amino acids in maintaining the tertiary structure of the protein, but they are not involved in cytokine interaction (48). These signature sequence and structural features have been used to identify novel cytokine receptors in several genomes (49–51). The cytokine-binding site for most CHR modules is at the apex of the elbow region, consisting mainly of the interstrand loops connecting the β-strands from both N- and Cterminal domains (46). The basic CHR module is present in every class I cytokine receptor, and for some, such as the receptors for hGH (46, 52) and erythropoietin (EPO), a single CHR is sufﬁcient to mediate ligand binding and receptor homodimerization. However, many other class I cytokine receptors require additional domains, such as the Ig-like domain and additional membrane-proximal ﬁbronectin domains, found in the gp130 family, to function and respond to cytokines (11, 12). The α

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receptors of IL-2 and IL-15 of the γc family are atypical cytokine receptors in that they do not contain CHR, but rather sushi domains (discussed below) (53, 54). The deﬁning structural feature of the cytokines recognized by the class I cytokine receptors is a four-helix bundle motif (43, 55), which is composed of four amphipathic helices having solvent-facing hydrophilic sides and hydrophobic sides that form the core of the helical bundle. These four helices are oriented into a unique up-up-down-down topology that is only found in the helical cytokines (43, 55). Structural predictions, later conﬁrmed by several crystal and NMR structures, indicated that these cytokines could be further subclassiﬁed on the basis of the length of the helices (19, 37). Short-chain cytokines, represented by IL-2 and IL-4, have helices of 8–10 residues. The longchain cytokines, such as gp130 family cytokines, hGH, and EPO, have helices of 10–20 residues. Finally, some cytokines, such as IL-5 and interferon (IFN)-γ, have two four-helix bundles forming an eight-helix architecture (55–57). Cytokine binding induces receptor oligomerization that leads to the juxtaposition of the intracellular domains of the signaling subunits. Unlike receptors for many growth factors (e.g., insulin, epidermal growth factor) that have intracellular domains possessing tyrosine kinase activity on the same polypeptide chain, the class I cytokine receptors have no intrinsic enzymatic activity. Rather, the intracellular domains of the class I cytokine receptors are constitutively associated with tyrosine kinases of the Janus kinase ( JAK) family, and to a more restricted degree the TYK kinases (26, 31, 32, 34). After the JAK/TYK kinases are activated by ligand-induced receptor oligomerization, they phosphorylate themselves and the intracellular domains of the receptors. The phosphorylated tyrosine residues in the receptors then serve as the docking sites for a second family of proteins, the signal transducer and activator of transcription (STATs). Binding of STATs to the intracellular domains of the receptors leads to their tyrosine phosphorylation and subsequent dissociation

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from the receptors. The phosphorylated STATs form dimers and translocate into the nucleus, where they bind to DNA recognition sequences and act as transcription factors (33, 34, 58). Besides the major JAK-STAT signaling pathway, the class I cytokine receptors also use other signaling mechanisms such as the RAS-RAF-MAP kinase pathway (59), PI3 kinase (60), and insulin receptor substrate (61). The structural aspects of JAK-STAT communication during receptor signaling are poorly understood and represent a major future frontier in cytokine receptor structural biology.

SHARED RECEPTORS IN THE CLASS I CYTOKINE RECEPTOR FAMILY Although the original structural paradigm for cytokine receptor complexes was derived from the homodimeric hGH system (21, 46), most of the class I cytokine receptors do not signal through homodimerization (Figure 2). In fact, most form heterodimers (e.g., IL-4, IL-7, etc.) (17), and some even form heterotrimers (IL-2 and IL-15 receptors) (15, 16), tetramers (viral IL-6/gp130, G-CSF/G-CSFR) (11, 62), hexamers (human IL-6/IL-6Rα/gp130) (12), and even dodecamers (GM-CSF/GM-CSFRα/βc ) (18). A signiﬁcant feature of these heterooligomeric receptor complexes is the use of a common, shared receptor subunit as a signaltransducing chain together with a cytokinespeciﬁc chain. When subgrouped by shared receptors, there are three major classes of heteroreceptor complexes in the class I cytokine receptor family: those that use βc , those that use gp130, and those that use γc (Figure 2). In most cases, the shared receptors do not show appreciable afﬁnity for cytokines, but in the presence of cytokine-speciﬁc α receptors they can form high-afﬁnity cytokine receptor complexes that are capable of initiating intracellular signaling cascades. Such a characteristic afﬁnity-conversion effect is used by shared receptors as a means of imposing tissue speciﬁcity (8). Shared receptors also must recognize different cytokines that have relatively low

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sequence identities, requiring that they be polyspeciﬁc for different ligand surface structures and chemistries, yet speciﬁc enough not to cross-react with inappropriate cytokines. In this respect, the shared receptors may teach us much about the basic structural and chemical mechanisms of protein-protein cross-reactivity.

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Gp130 is the founding member of the tall cytokine receptors and is the common signaltransducing receptor component for the gp130, or IL-6/IL-12, family of cytokines (Figure 1b and Figure 2b) that exhibit highly pleiotropic biological activities (23, 63). There are currently ten members in the gp130 family of cytokines: IL-6, IL-11, LIF, cillary neurotrophic factor (CNTF), oncostatin M (OSM), cardiotrophin 1 (CT-1), NNT-1/BSF3 [also known as cardiotrophin-like cytokine (CLC)], and IL-27. There are two viral homologs of IL6, one from HHV-8 IL-6 and another from the Rhesus macaque rhadinovirus (Rm IL-6) (Figure 1b). Except for the viral IL-6 homologs that bind directly to gp130 alone (11, 64), the signaling functions of gp130 cytokines are mediated through a set of receptor complexes that are formed by combining gp130 with other receptors (Figure 2b) (63). The association of gp130 with cytokine-speciﬁc, nonsignaling receptor IL-6Rα (65) or IL-11Rα executes the activities of IL-6 or IL-11, respectively. Other signaling receptors such as LIF receptor (LIFR) and OSM receptor (OSMR) can also participate in signaling complexes with gp130. The nonsignaling CNTF α receptor (CNTFRα) can recognize the cytokines CNTF, CT-1, and CLC as part of a quaternary signaling complex with gp130 and LIFR (66–68). Thus, CNTFRα exhibits the ability to bind three different cytokines, which is a rare example of degeneracy by an α receptor. Gp130 can also engage the recently identiﬁed heterodimeric cytokine IL-27 (p28/EBI3) in conjunction with the signaling receptor TCCR (T cell cytokine receptor, also known as WSX-1) (69). A major distinguishing feature of gp130 cytokines is that they possess

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Figure 2 Diversity of shared receptor-receptor interactions. Shared cytokine receptors βc (a), gp130 (b), and γc (c) and their various receptor partners are depicted. These complexes are formed by the combination of ligand-speciﬁc α and/or β receptors with shared cytokine receptors. (Abbreviations: TCCR, T cell cytokine receptor; TSLPR, thymic stromal-derived lymphopoietin receptor.)

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the unique site III receptor-binding site at the tip of the cytokine that is necessary for gp130 activation (12, 70, 71). The extracellular part of gp130 is composed of six contiguous β-sandwich domains with a single Ig domain at the top (D1), followed by one CHR module (D2 and D3), and three ﬁbronectin III-like domains (D4–D6) leading to the cell membrane (Figure 1b). Both the CHR and Ig domains are necessary for full activation. There are three known crystal structures of complexes involving gp130: the IL6/IL-6Rα/gp130-D1D2D3 hexamer (12), the HHV-8 IL-6/gp130-D1D2D3 tetramer (11), and LIF/gp130-D2D3 (13). In addition, there are two low-resolution, electron microscopic (EM) three-dimensional reconstructions of the entire extracellular complexes of IL-6 (72) and IL-11 (73). The crystal structures established that gp130 cytokines use the canonical sites I and II to engage the elbow regions of the α receptor and gp130 CHR, respectively. Site III in the cytokine engages the Ig domain of gp130 so that each gp130 contacts two different cytokines in an antiparallel fashion (Figure 3a). This basic assembly template is used by all gp130 cytokine receptor family members, including the nonshared members of the tall receptor family such as G-CSF, leptin, and OSMR. Originally, G-CSF was crystallized with only the CHR of its receptor (74). Subsequently, a mutational study of G-CSF based on the viral IL-6/gp130 complex structure (which revealed the ﬁrst site III) determined that GCSF contains a site III (75). Recently, the full complex of the G-CSFR Ig domain plus CHR has been solved with G-CSF (62), and this complex is almost identical in structure to that of the viral IL-6/gp130 complex and other gp130 complexes in the use of site III (11, 12). Several recent advances have established the site III paradigm for heterodimeric gp130/LIFR signaling complexes. The 4.0 A˚

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structure of the D1–D5 domains of LIFR in complex with LIF conﬁrmed that LIFR uses a high-afﬁnity site III interaction in which the interhelical loops at one tip of LIF engage the D3 Ig domain of LIFR in an almost orthogo2.6

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nal orientation (Figure 3b) (14). Binding measurements have also conﬁrmed that LIFR and CNTF alone interact via a high-afﬁnity site III that is presumably analogous to that of the LIF/LIFR interaction. A recent single-particle EM analysis of full-length gp130 and LIFR in complex with CNTFRα and CNTF (76) has conﬁrmed the architecture of the quaternary gp130/LIFR/CNTFRα/CNTF complex proposed in Boulanger et al. (13) and for the trimeric LIF/LIFR/gp130 complex modeled in Huyton et al. (14) (Figure 3c). The structure of an intact gp130/LIFR heterodimeric complex is an important milestone for research on this class of receptors, as there has been some controversy about the functionally active domains of LIFR involved in cytokine-mediated complex formation with gp130. With the basic site II/III architecture of the heterodimeric gp130/LIFR signaling complex predicted from a variety of structural and biochemical data, it is now clear that other gp130related heterodimeric cytokines such as IL-12 (p35/p40) (77), IL-23 (p19/p40) (78), and IL27 (p28/EBI3) (79) also engage their cognate receptors in some variation of this basic organizing principle (69, 80, 81). Interestingly, in the case of these heterodimeric cytokines (which consist of a four-helix bundle cytokine in complex with a soluble α receptor), the gp130like receptors IL-12Rβ1 (IL-12 and -23) and TCCR/WSX-1 (IL-27) lack an N-terminal Iglike domain and hence engage site II via their CHR domains. Site III is then free to interact with the second signaling receptor IL-12Rβ2, IL-23R, or gp130, respectively, all of which contain the top-mounted Ig domain required for site III interaction. In this fashion, the presence of the Ig domain serves as a structural beacon for the receptor that engages in site III interaction. IL-12 and IL-23 also represent unique examples of two cytokines sharing both an α receptor (p40) and a signaling receptor (IL-12Rβ1), while gaining speciﬁcity by using different site III receptors (IL-12Rβ2 and IL-23R). An additional characteristic feature of the gp130 family receptors is that that they are

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IL-6Rα gp130 Figure 3 Structures of human IL-6/IL-6Rα/gp130 hexameric complex (a), mouse LIFR in complex with human LIF (b), and human CNTF/ CNTFRα/LIFR/gp130 (c) assembled from known crystallographic, biochemical, and electron microscopic data. In (a) the model shown was derived from the crystal structure of the IL-6 hexamer headpiece (12) together with the single-particle reconstruction of the entire extracellular complex (72). IL-6 signaling is mediated through homodimerization of gp130 in a symmetric hexameric arrangement with a nonsignaling IL-6Rα receptor. In (b) the model shown derives from the 4 A˚ crystal structure of the LIF/LIFR complex (14) missing the membrane-proximal domains that are depicted as cartoons. In (c), the quaternary LIFR/gp130/CNTF/ CNTFRα complex is derived from a combination of the crystal structures of LIF/gp130 (13), CNTF (163), and a single-particle reconstruction of the entire quaternary complex (76). CNTF signals through the asymmetric heterodimerization of gp130 and LIFR and the nonsignaling CNTFα receptor. In panel (d ), the assembly pathway for IL-6 signaling is depicted as elucidated from References 12, 164. IL-6 ﬁrst engages IL-6Rα through a site I interaction to form a composite interface (site II) that recruits gp130. This trimeric structure can then engage a second trimer through two site III interfaces to form a productive signaling complex.

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taller than other cytokine receptors, by virtue of three additional membrane-proximal domains. A three-dimensional reconstruction of negatively stained six-domain gp130 complexed with IL-6 and the IL-6Rα receptor indicated that the gp130 membrane-proximal legs are bent back toward one another through the ﬂexing of a hinge between the D3 and D4 domains (73). This leg closure has also been observed in a cryoelectron microscopic analysis of the hexameric gp130/IL-11/IL-11Rα complex (74). Thus, although the FNIII leg domains have retained conformational ﬂexibility to allow for the close apposition of the intracellular domains required for intracellular signaling, it remains unclear whether the unliganded gp130 exists in this bent conformation or if the engagement of the shorter cytokine/Rα binary complex forces gp130 to bend in order to accommodate the height differences.

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THE COMMON BETA CHAIN: βc βc is a type I transmembrane protein that serves as a shared signaling subunit for the receptors of IL-3, IL-5, and GM-CSF (Figure 1c and Figure 2a), which are related cytokines involved in the regulation of hematopoiesis and inﬂammation (82–84). Although βc does not measurably bind any of the ligands alone, its coexpression with cytokine-speciﬁc α receptors enhances the afﬁnity of cytokine binding. The activated receptor complex, consisting of the cytokine ligand plus the α and βc receptors, initiates the intracellular signaling pathway mainly through JAK2 associated with the cytoplasmic domain of βc receptors (85). The extracellular part of βc has four ﬁbronectin domains, forming two contiguous CHR modules (Figure 1c), with features conserved among the class I cytokine receptors. The crystal structure of the unliganded extracellular domain of βc shows it to exist as an unusual, intertwined, strand-swapped, antiparallel homodimer (86). This structure, together with mutagenesis studies (87), led to the proposition that the possible cytokine-binding site is composed of D1 of one chain and D4 of another chain in the βc homod2.8

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imer that join in an antiparallel fashion. Excitingly, the recent crystal structure of the βc in complex with the GM-CSFα receptor (GMRα) and GM-CSF conﬁrms that βc engages the cytokine via a composite D1/D4 interface similar to site II in gp130 and γc , while the accessory receptor GMRα engages the cytokine via a site I–like interface (Figure 4) (18). It has been reported that GMRα does not engage a JAK kinase (85), leaving the JAK2-bound βc as the sole carrier of the signal-transducing kinase. The asymmetric unit of the βc /GMRα/GMCSF complex consists of a 2:2:2 hexamer with the C termini of βc ∼ 140 A˚ apart, therefore making it hard to reconcile how JAK kinases bound to βc subunits of a single βc dimer could be activated. Importantly, crystallographic contacts between βc D4 domains of two separate βc /GMRα/GM-CSF hexamers suggested βc signaling may be mediated by two hexamers dimerized into a dodecameric structure (18). Site-directed mutagenesis of this interface abrogated GM-CSF-induced signaling; thus, it appears that a second βc dimer in complex with GMRα and GM-CSF is necessary to complete the active signaling unit. These studies clarify what turns out to be a highly interesting deviation from the typical cytokine receptor signaling paradigm and can likely be extrapolated to explain the activation mechanisms of the other βc -family cytokines IL-3 and IL-5.

THE COMMON GAMMA CHAIN: γc γc serves as a shared signaling receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (Figure 1a and Figure 2c) (8). The biological importance of γc is illustrated by the fact that mutations in either γc or the associated JAK3 kinase can abolish the function of all γc -dependent cytokines and cause X-linked severe combined immunodeﬁciency diseases (X-SCID) (88, 89). One interesting twist in the γc family is that the IL-2 receptor (IL-2R) and IL-15R signaling complexes are heterotrimers composed of structurally unique α subunits and shared IL2Rβ and γc subunits (Figure 2c). This is in

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GM-CSF GMRα βc Figure 4 The 2:2:2 GM-CSF/GMRα/βc complex viewed from the side (a) and from the top (b) (18). GMRα engages the cytokine GM-CSF via a canonical site I interaction, whereas the βc receptor engages site II on GM-CSF by using a composite cytokine-binding homology region (CHR) interface generated by domain 1 (D1) of one βc subunit and domain 4 (D4) of the second βc .

contrast to type I IL-4R, IL-7R, IL-9R, and IL21R, which heterodimerize cytokine-speciﬁc α subunits and γc (Figure 2c). Another interesting twist in the γc family is limited sharing of several α receptors including IL-2Rβ, IL-4Rα, and IL-7Rα to recognize different cytokines as well as different receptors (Figure 2c). IL-2Rβ serves as a receptor for both IL-2 and IL-15. IL-4Rα heterodimerizes with γc to form type I IL-4R, and with IL-13Rα1 to form type II IL4R. Type II IL-4R is also the functional recep-

tor of IL-13 (discussed below) (25, 90, 91). IL7Rα can also form a receptor heterodimer with TSLPR (thymic stromal-derived lymphopoietin receptor) to recognize TSLP (92, 93).

STRUCTURAL STUDIES OF CYTOKINE-RECEPTOR COMPLEXES IN IL-2 AND IL-4/IL-13 SYSTEMS The cytokine IL-2 is a prototype member of cytokines and has pleiotropic actions in

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the immune system (28, 94, 95). Produced mainly by activated T cells, IL-2 promotes the proliferation, differentiation, and survival of mature T and B cells and the cytolytic activity of natural killer (NK) cells (95). There are three receptor chains for IL-2: IL-2Rα, IL-2Rβ, and γc , which form three different receptor complexes on different target cells. Isolated IL-2Rα has been termed the low-afﬁnity IL-2 receptor (Kd ≈ 10 nM) and is not currently believed to be involved in signal transduction (96). IL-2Rβ and γc form the intermediateafﬁnity complex (Kd ≈ 1 nM) expressed on NK cells, macrophage, and resting T cells (95), although IL-2Rβ alone has very low afﬁnity (Kd ≈ 100 nM) and γc alone has no detectable binding afﬁnity for IL-2 (97, 98). The heterodimerization of IL-2Rβ and γc in the presence of IL-2 is necessary and sufﬁcient for effective signaling through the activation of JAK1 and JAK3 kinases associated with the intracellular domains of the IL-2Rβ and γc , respectively (99, 100). A complex with three subunits, IL-2Rα, IL-2β, and γc , is the highafﬁnity complex (Kd ≈ 10 pM) for IL-2 and is the receptor form on activated T cells (101). The high-afﬁnity receptor complex mediates most biological effects of IL-2 in vivo (102). Prior to structural analysis, the Ciardelli group published a series of elegant papers measuring the various rate constants and afﬁnities for soluble forms of the different compositions of complexes (103–107). A similar analysis of the soluble complex assembly has also been conducted using thermodynamic measurements instead of kinetic studies (97). Therefore, the IL-2 receptor system has been one of the most rigorously characterized receptor systems using both cellular and biochemical approaches. IL-4 is another principal regulatory cytokine during the immune response and is crucially important in allergy and asthma (90). Once resting T cells are antigen activated and expand in response to IL-2, the fate decision of Th1 versus Th2 is inﬂuenced by IL-4. Th2 cells secrete IL4, which both stimulates Th2 in an autocrine fashion and acts as a potent B cell growth factor to promote humoral immunity (90). There

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are two types of receptor complexes for IL-4 (Figure 2c) (61, 91). Type I IL-4R is predominantly expressed on the surface of hematopoietic cells and consists of IL-4Rα and γc . Type II IL-4R consists of IL-4Rα and IL-13Rα1 and is predominantly expressed on the surface of nonhematopoietic cells, and this receptor complex is also the functional receptor of IL-13 (108– 110). The crystal structures of the high-afﬁnity IL-2/IL-2Rα/IL-2Rβ/γc quaternary complex and IL-4/IL-4Rα/γc , IL-4/IL-4Rα/IL13Rα1, and IL-13/IL-4Rα/IL-13Rα1 ternary complexes have all been determined (Figure 5) (15, 17). The structural comparison between IL-2/IL-2Rα/IL-2Rβ/γc and IL-4/IL-4Rα/γc complexes allows us to probe the basis by which γc can recognize six distinct cytokines. For the convenience of comparison between these two structures, and to eliminate redundancy in the chapter, we describe them in parallel throughout the following sections.

OVERALL STRUCTURE The ﬁrst complex structure of γc to be solved was the IL-2/IL-2Rα/IL-2Rβ/γc quaternary complex (15, 16) (Figure 5a), which is composed of one copy each of IL-2, IL-2Rα, IL2Rβ, and γc . Viewed from the perspective of the cell membrane, IL-2Rα sits on top of IL-2, and receptors IL-2Rβ and γc form a Y shape in which IL-2 sits in the fork (Figure 5a). The overall structural organization of the IL4/IL-4Rα/γc ternary complex is very similar to that of the IL-2 quaternary complex with a 1:1:1 stoichiometry (Figure 5b) (17), except for the absence of a top-mounted IL-2Rα. In the IL-4/IL-4Rα/γc ternary complex, receptor IL-4Rα and γc form a Y-shape heterodimer that binds to IL-4 in the classical site I/site II paradigm (Figure 5b).

BINARY COMPLEX OF IL-2Rα/IL-2 After resting T cells are activated by antigen, expression of IL-2Rα is upregulated in order to sensitize the T cells to low concentrations

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www.annualreviews.org • Structural Biology of Shared Cytokine Receptors

Extracellular complex structures of IL-2/IL2Rα/IL-2Rβ/γc (a) (15), IL-4/IL4Rα/γc (b), IL-4/IL4Rα/IL-13Rα1 (c), and IL-13/IL-4Rα/IL13Rα1 (d ) (17) extracellular signaling complexes depicted on a cell membrane.

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of IL-2, which is required for clonal expansion. The human IL-2Rα receptor chain, also known as Tac antigen or CD25, is a ∼55-kDa polypeptide consisting of an extracellular domain of 219 residues, a transmembrane domain of 19 residues, and a short cytoplasmic tail containing 19 residues (111–113). The short cytoplasmic tail does not participate in the signal transduction, although it is highly conserved between mice and humans, suggesting some important functional roles that remain unknown (100). From sequence analysis, IL2Rα also clearly lacks the signature features of class I cytokine receptors such as IL-2Rβ and γc . With the goal of targeting IL-2Rα with therapeutics for immunosuppression, the crystallization of the IL-2/IL-2Rα was an important benchmark in the ﬁeld (114). In fact, monoclonal antibody anti-Tac identiﬁes IL-2Rα and blocks the interaction of IL-2 with IL-2Rα (115, 116). The humanized form of anti-Tac (daclizumab, or Zenapax®) has been approved by the FDA for use in preventing renal transplant rejection (117). In several other clinical trials, daclizumab also provided a reduction of rejection in patients receiving liver, cardiac, and pancreatic islet transplants (118–120). In 2005, the structure of a recombinant soluble form of IL-2Rα complexed with IL-2 was solved to 2.8 A˚ (53) and revealed a very unusual structure for both the IL-2Rα as well as its mode of interaction with IL-2. The globular part of the IL-2Rα extracellular region is composed of two domain-swapped sushi modules (D1 and D2). Strands A and B from one sushi domain (A-B on C-D-E) are exchanged with strands F and G from another sushi domain (F-G on H-I-J), so the two domain-swapped sushi modules in IL-2Rα now become F on G-C-D-E (D1 domain) and A on B-H-I-J (D2 domain) (Figure 6a). As a result of this domain swap, IL-2Rα uses a composite surface to dock into a groove on IL-2 between the A and B helices, the same surface that serves as a binding site for antagonist drugs (121). This can be considered the dorsal surface of the cytokine with respect to the membrane, poised to present IL-2 to the side-oncoming IL-2Rβ and

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γc (Figures 5a, 6c). The IL-2/IL-2Rα binding interface (site a) has a total buried surface of 1670 A˚ 2 and is dominated by two hydrophobic clusters with a surrounding polar periphery, which may contribute to IL-2Rα’s high-afﬁnity binding and exquisite speciﬁcity. A recent mutational study identiﬁed IL-2 hot-spot residues Glu-62, Tyr-45, and Phe-42 as the most energetically critical residues in the receptor/IL-2 interface (122). Interestingly, these residues also serve as the hot-spots for several small molecule drugs, suggesting that the small molecule and receptor use the same energetic mechanism for binding. The implications are that residue contact footprints in a protein/protein interface may, in some cases, serve as a useful surrogate scaffold for design of small molecules. The IL-2/IL-2Rα complex represents the initiating step for formation of the quaternary signaling complex, so many anticipated that IL2Rα’s role was simply to capture free IL-2 and present it to IL-2Rβ and γc (Figure 7a and Table 1). Moreover, the expression level of IL-2Rα is 10- to 20-fold higher than that of IL-2Rβ on activated T cells (123). Thus, the excess of IL-2Rα molecules and relatively highafﬁnity binding to IL-2 would facilitate efﬁcient capture of free IL-2 and its delivery to IL-2Rβ through a restricted two-dimensional handoff on the same cell membrane. That IL2Rα did not appear to make any contact with either IL-2Rβ or γc in the quaternary signaling complex was a surprise (Figure 5a, 6c) (15). The linker connecting the globular domains of IL-2Rα to the cell membrane, which is disordered in the structure, does not appear capable of forming receptor-receptor contact with IL2Rβ even if fully extended (Figure 6c). This is a rather surprising ﬁnding given the longstanding observation that the coexpression of IL-2Rα and IL-2Rβ forms the pseudo-highafﬁnity complex that can bind to IL-2 with a Kd of ∼30 pM, much higher than IL-2 binding to IL-2Rβ alone (Kd ≈ 100 nM) (124). Isothermal titration calorimetry (ITC) experiments with the soluble receptor ectodomains also showed a twofold afﬁnity increase between IL-2 and IL-2Rβ in the presence of IL-2Rα

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a

b D1

C-ter

C-ter

H

G E D C

B

F

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I J

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H I

J IL-15

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trans

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IL-2Rβ γc

IL-2Rβ cis

cis

γc

Figure 6 IL-2/IL-2Rα and IL-15/IL-15Rα binary complexes. (a) IL-2Rα is composed of two sushi modules (D1 and D2) that swap the β-strands, forming a noncanonical sushi fold topology (53). (b) IL-2Rα and IL-15Rα contact the dorsal surface of IL-2 and IL-15, respectively (130, 131). (c) IL-2 quaternary complex and modeled IL-15 quaternary complexes. IL-2Rα presents IL-2 in cis, whereas IL-15Rα presents IL-15 in cis or trans. www.annualreviews.org • Structural Biology of Shared Cytokine Receptors

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IL-2Rβ IL-2

IL-2Rα

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IL-2

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IL-13 IL-13

IL-13

Site I

Site II + III IL-4Rα

IL-13Rα1

IL-13Rα1

IL-13Rα1

Figure 7 The sequential assembly pathways of the IL-2/IL-2Rα/IL-2Rβ/γc quaternary (a), IL-4/IL-4Rα/γc (b), IL-4/IL-4Rα/IL-13Rα1 (c), and IL-13/IL-4Rα/IL-13Rα1 (d ) ternary complexes. See Table 1 for the interaction afﬁnity and thermodynamic parameters of each binding site.

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Table 1 Interaction affinity and thermodynamic parameters of binding sites in IL-2/IL-2Rα/IL-2Rβ/γc quaternary complex, IL-4/IL-4Rα/γc , IL-4/IL-4Rα/IL-13Rα1, and IL-4/IL-4Rα/IL-13Rα1 ternary complexesa Complex IL-2/IL-2Rα/Rβ/γ

IL-4/IL-4Rα/γc

Kd (nM)

ΔH (Kcal/mol)

Site a

Binding site

10

−5.2

18.4

−10.5

Site I

63

−6.9

8.9

−9.5

Site II

12

−10.3

0.72

−10.4

Site I

1

−11.2

3.5

−12.2

559

−11.7

−10.5

−8.6

1

−11.2

3.5

−12.2

Site II IL-4/IL-4Rα/IL-13Rα1

Site I

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a

ΔG (Kcal/mol)

487

−4.8

13.0

−8.7

Site I

30

−15.0

−15.6

−10.3

Site II+III

20

−5.8

16.0

−10.6

Site II+III IL-13/IL-4Rα/IL-13Rα1

ΔS (cal/molK)

Data compiled from References 17 and 97.

(97). Kinetic studies of both membrane-bound and soluble ectodomains have shown that the on rate of IL-2 for IL-2Rβ is 3 to 20 times faster in the presence of IL-2Rα (96, 98). A simple mechanistic explanation for these afﬁnity data would be the presence of a composite binding surface for IL-2 contributed by both IL-2Rα and IL-2Rβ, which is not seen in the structure (Figure 5a). So what is the basis of the cooperativity? One explanation is that the afﬁnity-enhancing role of IL-2Rα is independent of structural effects and is achieved by simply capturing and concentrating free IL-2 at the cell surface, as mentioned above. The other possibility is an IL-2Rα-induced conformational change in IL-2 that favors the binding to IL-2Rβ. After comparison of the IL-2 structures in the quaternary complex, binary complex, and unbound states, one local conformational adjustment on IL-2 upon IL-2Rα binding was found at the beginning of the helix C, where several turns of the helix are slightly unwound and translated forward by approximately 1.0 A˚ toward IL-2Rβ. This local conformational change enables the movement of IL-2 residue Asn-88 into hydrogen-bonding proximity to IL-2Rβ residue Arg-42 (Figure 8a). Consistent with this, mutation of Asn-88 in IL2 ablates binding to IL-2Rβ (125). We take this to suggest that IL-2Rα may induce and stabilize a favorable IL-2Rβ-binding conformation of the IL-2 C helix in IL-2, in addition to its roles in capture and delivery.

IL-15 is the only other cytokine that uses a speciﬁc sushi-domain α receptor (IL-15Rα) (126, 127). In addition, IL-15 uses IL-2Rβ and γc as its signaling components in the receptor heterotrimer (128, 129). Because IL-15Rα has only one sushi domain in the extracellular part, there is no possibility for a strand exchange as observed in IL-2Rα, which has been conﬁrmed by a NMR structure of the IL-15Rα sushi domain and two complex crystal structures of IL15Rα sushi domain with IL-15 from human and mouse (54, 130, 131). In these structures, the IL-15Rα sushi domain shows a canonical sushi fold topology (Figure 6b). The IL-15/IL-15Rα and IL-2/IL-2Rα binary complexes have similar cytokine-receptor docking modes: The α receptor sushi domain binds to the dorsal surface of the cytokine (Figure 6b), and the cytokinereceptor interaction footprints of IL-15Rα on IL-15 and IL-2Rα on IL-2 also have substantial overlap (130–132), but IL-15Rα has an approximately 1000-fold higher binding afﬁnity to IL15 than that of IL-2Rα to IL-2 (126). Compared to the dominant hydrophobic patches, with a surrounding polar periphery seen in the IL-2/IL-2Rα binding interface, the interaction area between IL-15 and IL-15Rα is dominated by salt bridges and hydrogen bonds with superior shape complementarity (130, 131). The charge-charge interactions may cause the low Koff value between IL-15 and IL-15Rα that is responsible for the high afﬁnity. Similar to IL-2/IL-2Rα, the presence of the IL-15Rα

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Binding site I in IL-2Rβ quaternary complex

a

Binding site I in IL-4Rα ternary complex

Helix C

CC'1

Helix B Helix A

Helix C R42

Helix A N88 D20 R88

CC1'

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EF1

H133

Y134

E9

D72 Y13

EF1 BC2

Y182 AB1

Binding site IIa in IL-2 quaternary complex

b

BC2

FG2

Binding site IIa in the IL-4 ternary complex Helix C

CC1'

Helix D CC1'

Helix A EF1

E122 R121

Q126 Y103

Helix A

Y124

C209 FG2

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Y182

C160

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BC2

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C160 BC2

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γc

E122

S127

Y103 Q126 C209 C160

T118

S125

S130 I129

EF1

T123

Y124

Y103 C209

R121

C160

Figure 8 Speciﬁc versus degenerate binding sites I and IIa in the IL-2/IL-2Rα/IL-2Rβ/γc and IL-4/IL-4Rα/γc complexes. (a) Left panel shows the binding site I between IL-2 helices A and C and IL-2Rβ loops in the elbow region. The corresponding binding site I in the IL-4 ternary complex is shown in right panel. (b) Binding site IIa in the IL-2 quaternary complex (γc /IL-2) and in the IL-4 ternary complex (γc /IL-4) are shown in left and right panels, respectively. 2.16

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endows IL-15 with a much higher afﬁnity for IL-2Rβ and γc (30, 133). One possible explanation for this is the fact that IL-15Rα is known to present IL-15 in trans, from cell to cell, to the IL-2Rβ/γc complex (134) (Figure 6c). The disconnection of IL-15Rα from lying in the same membrane as the IL-2Rβ and γc components may require a more profound allostery for IL-15Rα to serve effectively as an afﬁnity converter for IL-15. In other words, the trans-signaling role of IL-15Rα necessitates that it enhance IL-15 binding to IL-2Rβ through space rather than simply through surface capture on the same cell membrane. Although trans-presentation is currently believed to be the major mechanism by which IL-15 exerts its biological effects in vivo, the assembly of a cis IL-15/IL-15Rα/IL-2Rβ/γc quaternary complex on the surface of the same cell is also used (131, 135) (Figure 6c).

IL-2Rβ/IL-2 AND IL-4Rα/IL-4 INTERFACES IL-2Rβ and IL-4Rα are functional and structural counterparts in their respective signaling complexes, and both form one of the two major signaling subunits in their γc receptor complexes. The ∼75-kDa human IL-2Rβ chain is composed of an extracellular domain of 214 residues, a transmembrane domain of 25 residues, and an intracellular domain of 286 residues (136). The ∼140-kDa human IL-4Rα chain has 207 residues in the extracellular domain, 24 in the transmembrane region, and 569 in the intracellular domain (137). Although the nomenclature is confusing, IL-2Rβ is analogous to the α receptors for other γc cytokines, but because IL-2 has the additional nonstandard initiating receptor, IL-2Rα, IL-2Rβ is then referred to as the β receptor on the basis of the sequence of interactions with IL-2. The intracellular domains of IL-2Rβ and IL4Rα possess the box 1 and box 2 motifs at the membrane-proximal region that constitute the binding sites for JAK1 (34). The cytokine binding–induced association of IL-2Rβ or IL4Rα with γc will bring their intracellular do-

mains into close proximity, inducing the activation of the JAK kinases (Figure 5a,b). After the capture of IL-2 by IL-2Rα, the delivery of IL-2 to IL-2Rβ in cis represents the second step in the formation of the quaternary IL-2 receptor complex (Figure 7a and Table 1). The binding interface between IL-2 and IL-2Rβ (site I) buries ∼1350 A˚ 2 and is formed by residues from helices A and C in IL-2 and loops CC 1, EF1, BC2, and FG2 in IL-2Rβ (Figure 8a). The interface is highly polar, with eight hydrogen bonds directly between IL-2 and IL-2Rβ residues and seven buried water molecules mediating the interactions between IL-2 and IL-2Rβ by forming hydrogen bonds with protein atoms (Figure 8a). Solvent exchange with the layer of water molecules between IL-2Rβ and IL-2 could explain the fast on and off rates and the weak afﬁnity of the IL-2/IL-2Rβ binary complex. Two residues of IL-2 that have been shown by mutagenesis to be critical for IL-2Rβ binding, Asp-20 (138) and Asn-88 (125), are involved in hydrogen bonding networks to both water molecules and side chains on IL-2Rβ. There is excellent knob-inhole shape complementarity between IL-2Rβ and IL-2 (Figure 8a). As mentioned, IL-2Rβ is also used by IL-15 to form a quaternary complex along with the IL-15Rα and γc (30, 133) (Figure 6c). IL-15 has limited sequence homology (19%) with IL-2, so its contacts with IL-2Rβ are almost certainly through a unique set of interactions. The apparently central role that water molecules play in bridging hydrogen bonds between IL-2Rβ and IL-2 would contribute to the ability of IL-2Rβ to cross-react through remodeling of this hydration layer to accommodate the IL-15 residues. The ﬁrst step in the formation of the IL-4/IL-4Rα/γc complex is the binding of IL-4 with IL-4Rα receptor (139, 140) (Figure 7b and Table 1). These interactions were ﬁrst elucidated in the IL-4/IL-4Rα binary complex (141). The comparison of IL-4/IL-4Rα binary and IL-4/IL-4Rα/γc ternary complex structures reveals that the engagement of γc does not cause substantial conformational changes

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in the mode of interaction with IL-4Rα. Minor differences in peripheral interface contacts between the IL-4 type I binary and ternary complexes are likely due to the differing resolutions of the structures and crystal packing forces. The interacting residues from helices A and C in IL-4 and loops CC 1, EF1, AB1, BC2, and FG2 in IL-4Rα, bury a total surface area of ∼1520 A˚ 2 (site I) (Figure 8a). The chemical nature of the IL-4/IL-4Rα interface is also highly polar, similar to that of the IL-2/IL-2Rβ interface. The interacting residues in the IL-4/IL-4Rα interface can be grouped into two major clusters centered at Glu-9 (IL-4) to Tyr-134 (IL-4Rα) and Arg-88 (IL-4) to Asp-72 (IL-4Rα), respectively, each containing an inner polar core surrounded by outer hydrophobic residues (Figure 8a). Considering there are only two bridging water molecules in the IL-4/IL-4Rα interface, the buried polar and charged interactions contribute to the rapid on rate (Kon ≈ 1.3 × 106 M−1 s−1 ) and slow off rate (Koff ≈ 2.1 × 10−3 s−1 ) that result in high-afﬁnity binding between IL-4 and IL-4Rα (142). An exhaustive double-mutant analysis of the IL-4-IL-4Rα interaction has been carried out by the Sebald group (143), and we refer the reader to this paper for a rigorous, energetic deconvolution of the cytokine-receptor interface. In summary, both the IL-2 and IL-4 interactions with their respective α chains are characterized by polar and charged contacts. This is consistent with the high degree of speciﬁcity these receptors show for their cytokine, in contrast to γc ’s degeneracy. Although not completely generalizable, polar and charged contacts primarily mediate speciﬁcity in proteinprotein interactions because their energetic content is greatly affected by the precise structural context within an interface (144). Van der Waals and hydrophobic interactions are more suitable to a promiscuous binding surface because these are less structure-selective interactions that are a result more of water exclusion than of speciﬁc structural context and pairwise atomic contacts (144).

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RECRUITMENT OF γc BY IL-2/IL-2Rαβ AND IL-4/IL-4Rα The interaction of the IL-2/IL-2Rα/IL-2Rβ ternary complex with γc is the last step in formation of the IL-2 signaling complex on activated T cells (Figure 7a and Table 1). IL2 has very low afﬁnity for γc alone (Kd ≈ 700 uM) (97, 98), requiring precomplexation with IL-2Rα/IL-2Rβ (in the high-afﬁnity complex) or IL-2Rβ alone (in the intermediateafﬁnity complex) to bind γc with Kd in the low nM range. IL-2Rβ and γc do not have a measurable afﬁnity for one another (97). The two weak interactions, IL-2 with γc and IL-2Rβ with γc , combine to produce a high afﬁnity for γc . In the complex structure, two contact surfaces, a small one between IL-2 and γc and a larger one between IL-2Rβ and γc , form the interaction surface between IL-2/IL-2Rαβ and γc (Figure 5a). The IL-2/γc binding surface (site IIa) reﬂects its degenerate recognition capabilities, showing a remarkable ﬂatness and almost tangential contact with IL-2 (Figure 8b). The IL-2/γc interface is the smallest of the four protein/protein interfaces in the complex, burying ∼970 A˚ 2 of surface area. In contrast to the IL-2Rβ interface, the γc binding surface is rather devoid of extended side chain–speciﬁc interactions with IL-2 and exhibits primarily main chain contact (Figure 8b). Unlike the IL-2/IL-2Rβ interface that has a broad array of speciﬁc polar interactions, the IL-2/γc interface is composed of small contact patches (Figure 8b). The ﬁrst one is composed of residue Tyr-103 from γc and residues Ser127 and Ser-130 from IL-2. In γc , the Tyr-103 side chain does not protrude outward toward IL-2 but is instead pinned back via a hydrogen bond with the Cys-209 main chain and is therefore positioned so that its aromatic ring packs ﬂat against the side chains of Ser-127 and Ser-130 in IL-2 (Figure 8b). The second contact patch is around residue Gln-126 in IL-2, whose side chain is ﬂattened and almost parallel to the surface formed by main chain atoms

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of residues Pro-207 to Ser-211 in the FG2 loop of γc (Figure 8b). The second part of the composite interface between IL-2/IL-2Rαβ and γc is formed by extensive interactions between the D2 domains of IL-2Rβ and γc (site IIb) (Figure 5a). The residues in the IL-2Rβ/γc interface bury over 1750 A˚ 2 of surface area, which is the most extensive seen so far between receptor domains in cytokine-receptor complexes. The IL-2Rβ/γc interface is predominantly composed of polar interactions, with a total of 17 hydrogen bonds surrounding a small hydrophobic stripe. After the initial binding of IL-4 with the IL-4Rα receptor, the association of γc is also mediated through a composite interface: IL4/γc and IL-4Rα/γc (Figure 7b and Table 1). The IL-4/γc interface buries a total surface of ∼1020 A˚ 2 , which is slightly larger than that of the IL-2/γc interface but still the smallest in the IL-4/IL-4Rα/γc complex (site IIa) (Figure 8b). The Tyr-103 residue and the FG2 loop ﬁxed by the Cys-160 to Cys-209 disulﬁde bond in γc interact with IL-4 in a binding mode similar to that of γc within the IL-2 receptor complex (Figure 8b). There is low sequence identity between IL-2 and IL-4, and so the contacting residues in IL-4 with Tyr-103 are Glu-122 and Ser-125, which still contact the aromatic ring of Tyr-103 through van der Waals contacts instead of forming speciﬁc polar interactions (Figure 8b). In the IL-4/γc binding interface, the Arg-121 residue from IL4, which replaces the critical Asn-126 position in IL-2, contacts with main chain atoms of residues Pro-207 to Ser-211 in γc through its long methylene side chain (Figure 8b). While maintaining these two binding epitopes that are also observed in the IL-2/γc interface, the IL-4/γc interface has an additional hydrophobic patch consisting of Tyr-124 in IL-4 and the interacting Leu-208 and Tyr-182 in γc (Figure 8b). Also contributing to the IL-4Rα/γc interface are extensive receptor-receptor contacts between the D2 domains of the respective CHR (site IIb) (Figure 5b). However, the packing between IL-4Rα/γc is less intimate than that

of the IL-2Rβ/γc interface, as evidenced by its much smaller buried surface (∼1200 A˚ 2 versus ∼1750 A˚ 2 ). The IL-4Rα/γc interface also has extensive polar interactions surrounding a small hydrophobic stripe in the center, dominated by Tyr-154 from IL-4Rα and Phe-186 from γc . The IL-4Rα-γc interaction is much weaker (Kd ∼ uM) (17) in the IL-4 ternary complex than that between IL-2Rβ and γc in the IL-2 quaternary complex (Table 1). The fact that receptor-receptor contacts seen in the respective complexes are less extensive explains much of this afﬁnity difference.

DEGENERATE CYTOKINE RECOGNITION BY γc Protein-protein interactions and, in fact, most receptor-ligand interactions are usually characterized by speciﬁcity for one ligand. Therefore, the molecular basis for γc recognition of six different cytokines has been not only a fundamental question in understanding cytokine recognition, but also a basic problem in physical chemistry. As a shared signaling subunit, the engagement of γc is the last step in the formation of functional signaling complexes (Figure 7a,b). On the basis of the structural information from the IL-2 quaternary complex and the IL-4 ternary complex, we can conclude that the preformed complexes of cytokines with the α receptors provide a composite binding site for γc that is composed of two interaction interfaces: cytokine/γc and α receptor/γc . The small buried surface area (∼970 A˚ 2 between IL-2 and γc and ∼1020 A˚ 2 between IL-4 and γc ) and the formation of relatively nonspeciﬁc atomic interactions characterize the cytokine/γc binding interface. In contrast, the α receptor/γc binding interface has a large buried surface area (∼1750 A˚ 2 between IL-2Rβ and γc and ∼1200 A˚ 2 between IL-4Rα and γc ) composed predominantly of speciﬁc polar interactions. Extensive mutagenesis work has identiﬁed Tyr-103, Cys-160, and Cys-209 in γc as critical residues for the engagement of all γc dependent cytokines (145–147). Because certain residues around these hot-spot positions

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were exclusively implicated in the binding of different cytokines, such as Ile-100 and Leu102 for IL-4 binding and Asn-44, Leu-161, and Glu-162 for IL-21 binding, the γc -binding sites for different cytokines overlap but are not identical (146, 147). The side chain of Tyr-103 does not form speciﬁc side chain interactions with residues in IL-2 and IL-4, but it presents its aromatic ring for contact with residues Ser127 and Ser-130 in IL-2 and Glu-122 and Ser125 in IL-4 (Figure 8b). Residues Cys-160 and Cys-209 form a unique disulﬁde bond that connects loops FG2 and BC2 in the D2 domain of γc (Figure 8b). This disulﬁde bond ﬁxes the bent conformation of loop FG2, where the main chain atoms from residues Ser-207 to Pro211 directly contact the methylene side chain of residue Gln-126 in IL-2 or Arg-121 in IL4 (Figure 8b). The critical role of these hotspot residues in γc is also illustrated by the fact that some of their mutations have been found in the human γc gene of X-SCID patients (88). Although the cytokines recognized by γc have an average 19% sequence identity among them, helix D is the most conserved region, which is also the major contact area used by γc dependent cytokines to bind γc . Phe-114 and Ile-115 are two strictly conserved positions at the N terminus of helix D in IL-2, but they are not involved in γc binding, and their hydrophobic side chains point into the helical core. Residue 126 in IL-2 helix D is a conserved Asn in the γc -dependent cytokines IL-9, IL-15, and IL-21, while IL-4 and IL-7 contain Arg121 and Lys-139 at this position. The Gln-126 in IL-2 and Arg-121 at this position in IL-4 both contribute to binding with γc in their respective complexes (Figure 8b). Although this position serves as a common contact point for γc , its importance in binding with γc can vary with different cytokines. In IL-2, Gln-126 is the major γc -binding determinant, and mutation of this site greatly reduces binding afﬁnity (148). In IL-4, Arg-121 is only one of the minor γc binding determinants, and the nearby residues Ile-11, Asn-15, and Tyr-124 serve as the major determinants (146).

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In the absence of obvious conserved hotspot residues in γc -dependent cytokines, shape complementarity appears to play a dominant role. This assertion is supported by the observation that both IL-2 and IL-4 provide a shallow groove that accommodates the protruding hot-spot binding residues in γc . In IL-2, the bulky side chains from residues Thr-123, Qln126, Ser-127, Ile-129, and Ser-130 form the walls of a canyon, which receives a protruding ridge on γc composed of Tyr-103 and the Cys-160 to Cys-209 disulﬁde bond (Figure 8b). The groove in IL-4 is surrounded by residues Thr-118, Arg-121, Glu-122, Tyr-124, and Ser125, which are in the same positions with those of IL-2 (Figure 8b). The formation of the groove for the placement of hot-spot residues in γc would only require that residues have side chains with similar volume and shape, which can be achieved without the need for strictly conserved residues. We expect that other γc dependent cytokines, IL-7, IL-9, IL-15, and IL-21, will also form a similar shallow groove on helix D that serves as the docking site for the protruding binding site on γc . The functional role of this groove appears to facilitate complex formation with γc by guiding a perfect geometrical alignment of the D2 domains of the cytokine-speciﬁc α receptor and γc , resulting in the numerous interatomic contacts (H-bonds, van der Waals, etc.) in the D2/D2 interface between α receptor and γc . In other words, the knob-in-hole shape complementarity between the groove on the cytokine and the protruding γc -binding site acts as a guide to align the receptor D2 domains for the intimate interaction that they exhibit.

RECRUITMENT OF IL-13Rα1 IN TYPE II IL-4 AND IL-13 RECEPTOR COMPLEXES As previously mentioned, IL-4Rα represents an important subfamily of γc -cytokine receptors in that it serves as a shared signaling receptor within three different cell surface complexes. IL-4Rα and IL-13Rα1 form receptor heterodimers on cells of nonhematopoietic

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stem cell origin, functioning as the type II receptor complex for both IL-4 and IL-13. IL-13Rα1 is derived from the same ancestral subgroup as γc (149), and its divergence derives from its extra N-terminal Ig-like domain that has contact with the dorsal surfaces of both IL-4 and IL-13 (site III) in IL-4/IL4Rα/IL-13Rα1 and IL-13/IL-4Rα/IL-13Rα1 complexes (Figure 5c,d ). The IL-13Rα1 D1 domain–binding sites on IL-4 and IL-13 have extensive overlap with the respective α receptor–binding sites on IL-2 and IL-15. In site III of both complexes, the C strand of IL13Rα1 interacts with the C-D strand of IL-4 and IL-13, forming an antiparallel beta sheet. The residues Trp-65 and Ile-78 on the C strand of IL-13Rα1 form a hydrophobic patch that opposes complementary hydrophobic residues in IL-13—these interactions are missing between IL-4 and IL-13Rα1 (Figure 5c,d ). Consistent with this structural data, mutational studies have shown that after deleting the D1 domain, the IL-13Rα1 D2D3 CHR module does not detectably bind to IL-13, but it can still form a ternary complex with IL-4 and IL-4Rα (17). This difference in the energetics of site III interactions between the respective cytokines likely explains the requirement of IL-13Rα1 D1 domain for signaling in the IL-13 type II complex, in contrast to the IL-4 type II complex (150). Although IL-4 and IL-13 use the same IL4Rα/IL-13Rα1 receptor heterodimer for signaling, the type II IL-4 and IL-13 complexes assemble in the reverse cooperative sequences (Figure 7c,d ). Similar to the type I IL-4 complex, type II IL-4 complex is formed by the initial high-afﬁnity binding of IL-4 with IL4Rα (site I) with a subnanomolar Kd , followed by the recruitment of IL-13Rα1 (site II and site III) with a much lower afﬁnity of 487 nM (Figure 7c and Table 1). In contrast, for the type II IL-13 complex, IL-13 ﬁrst binds to IL-13Rα1 with an afﬁnity of 30 nM, and the IL-13/IL-13Rα1 binary complex then recruits IL-4Rα with an afﬁnity of 20 nM (Figure 7d and Table 1). The driver (receptor for initial cytokine interaction) and trigger (receptor recruited for

signaling) terminology was previously proposed for the assembly of γc heterodimeric complexes (151). The type II IL-4 and IL-13 complexes have switched the driver and trigger in their respective assembly pathways. The recruitment of the trigger IL-4Rα in the type II IL-13 complex is more energetically favorable (Kd ≈ 20 nM) than the recruitment of the trigger IL-13Rα1 by the type II IL-4 complex (Kd ≈ 487 nM). Measurement of STAT6 phosphorylation induced by IL-4 and IL-13 in the human epithelial carcinoma cell line A549 (expressing IL-4Rα and IL-13Rα1, but not γc ) revealed that IL-4 is more potent (17). In this A549 cell line, the expression level of IL-13Rα1 is higher than that of IL-4Rα. In other cell lines where IL-13Rα1 expression level is limiting, IL-13 can become more potent than IL-4 in stimulating signaling (152). It was therefore proposed that when IL-13Rα1 is abundant, the highafﬁnity binding of IL-4 with its driver IL-4Rα would determine the signaling potency. When IL-13Rα1 is limiting, the IL-13 and IL-13Rα1 binding afﬁnity (Kd ≈ 30 nM) would still allow the efﬁcient formation of IL-13/IL-13Rα1 binary complex, and the subsequent relatively high-afﬁnity binding (Kd ≈ 20 nM) with trigger IL-4Rα could favor the formation of the type II IL-13 complex, resulting in more potent IL-13-induced signaling. These studies showed that membrane-proximal signaling events induced by a cytokine could be collectively inﬂuenced by many factors: the structural aspects of extracellular cytokine receptor interactions (e.g., receptor orientation and conformation), the concentration of cytokine, receptor expression level, the sequence of receptor assembly, and cytokine receptor binding afﬁnity. Each of these factors could potentially be manipulated to effect a therapeutic endpoint in this system that has obvious clinical importance for asthma.

COMPARISON OF γc WITH gp130 With a raft of structures of both γc and gp130 complexes with cytokines, we can now assess the similarities and differences between the structural mechanisms by which these

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a IL-2/γC

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Figure 9 Cross-reactivity of the cytokine-binding homology region (CHR) of γc and gp130. (a) Contact surface of γc and gp130 when bound to IL-2, IL-4, HHV-8 IL-6, IL-6, and LIF. Hydrophobic residues are colored as blue surface area and hydrophilic residues as red surface area. The helices and the contacting residues for each of the cytokines are shown docked onto the surface of the receptor. (b) Surface representations showing the contact surface of each cytokine. Note that IL-2 and IL-4 have a similar distribution of hydrophobic and hydrophilic residues in the contact area. For gp130 family cytokines, HHV-8 IL-6 has primarily hydrophobic contact surface area. The contact area on IL-6 is more polar, and LIF has the most signiﬁcant hydrophilic contact surface. (c) The packing environment of the common binding epitope residues Tyr-103 (γc ) and Phe-169 (gp130) against the cytokines.

shared receptors cross-react (Figure 9). The ectodomain of γc is composed of one CHR domain, whose elbow region at the interdomain boundary is the contact area for six different short-chain cytokines. While the ectodomain of gp130 is taller than that of γc and consists of one top-mounted Ig domain, one CHR module, and three extra membrane-proximal ﬁbronectin domains (Figure 3), the main gateway entry point for all long-chain gp130 family cytokines is also the elbow of the CHR module, analogous to γc (12). The structural analogies between the CHR modules on γc and gp130 are, then, very clear. Their cytokinebinding surfaces are similar in the distribu2.22

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tion of hydrophobic and hydrophilic residues, with a largely hydrophobic core shared region and discontinuous peripheral polar patches, but gp130 has more buried surface area within the cytokine-receptor interface compared with γc , consistent with gp130 engaging the larger longchain cytokines (Figure 9a). The buried surface area on γc contributing to IL-2 and IL-4 binding is ∼500 A˚ 2 , whereas the buried surface area on gp130 upon binding human IL6, human LIF, and viral IL-6 is 710, 700, and 610 A˚ 2 , respectively. Gp130 also has a larger core hydrophobic region of its interfaces. This can possibly be understood from the standpoint that gp130 is a shared receptor capable

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of binding to some cytokines, such as LIF and OSM, in the absence of an α receptor, therefore not requiring the receptor-receptor contact necessary in the γc complexes. γc , in contrast, requires an α receptor for all cytokine interactions and therefore has a smaller binding interface with cytokines, as the energetics are distributed across both α receptor and cytokine. In the hydrophobic core of the interfaces of both γc complexes, Tyr-103 is the hot-spot residue that contributes about 20% of the buried surface area upon binding with IL-2 and IL-4 (Figure 9c). Gp130 has Phe-169, an analog of Tyr-103 in γc , in the center of the hydrophobic core of its binding site, that contributes the largest fraction of buried surface area and is critical for ligand engagement of all cytokines (71, 153–155) (Figure 9c). Another noteworthy observation is the rigidity of the binding surfaces on γc and gp130. A comparison between the unliganded (153) and liganded forms of gp130 shows almost no rotameric ﬂexibility in the side chains of interacting residues. Although we do not have the structure of unliganded γc , the superimposition of γc structures onto the complex of IL-2 and IL-4 shows a similar rigidity of the side chains of interacting residues. Thus, conformational plasticity is most likely not used by either shared receptor as a means of cross-reactivity. More generally, the idea that conformational plasticity will be a mechanism to enable cross-reactivity in protein-ligand interactions has largely been supplanted by the observation that degeneracy can be provided simply through enthalpyentropy compensation of rigid interacting surfaces (13, 156). The binding epitopes on γc -dependent cytokines for γc are on helices A and D, whereas the binding epitopes for gp130 on the gp130 family cytokines are on helices A and C. The binding surfaces on IL-2 and IL-4 for γc are quite similar in the distribution of hydrophobic and hydrophilic residues (Figure 8b). Structurally nonidentical but positionally analogous residues on the respective γc cytokines form the grooves similar in topology and chemical nature for the docking of γc residue Tyr-103

and the loop formed by the Cys-160 to Cys209 disulﬁde bond (Figure 8b). In contrast, the surfaces contributing to the binding to gp130 in HHV-8 IL-6, LIF, and IL-6 are very different in the distribution of hydrophilic and hydrophobic residues (Figure 8b). The gp130-binding surface on HHV-8 IL-6 has the largest hydrophobic area, and the binding surface on IL-6 is signiﬁcantly more polar. The contact surface on LIF is the most polar, consistent with four well-deﬁned water molecules that participate in an intermolecular hydrogen bond network observed in the crystal structure of LIF in complex with gp130 (13). The topology and chemical nature of the observed grooves in gp130 family cytokines for the docking of Phe-169 from gp130 vary greatly between different cytokines (Figure 8c). The groove on HHV-8 IL-6 represents one extreme with a deep pocket. The surface grooves on human IL-6 and LIF that accommodate Phe-169 are more similar in overall topology, but are not as deep as observed in HHV-8 IL-6. In human IL-6, the Phe-169 does not sit deeply in the pocket, but instead packs directly against the side chains from IL-6. The gp130-binding groove on LIF is the most polar of all three cytokines. The polar head groups of hydrophilic residues from LIF form the walls of the pocket and direct the aromatic ring of Phe169 to pack against LIF in a similar fashion to the packing of Phe-169 against human IL-6. Analysis of the binding surfaces on γc and gp130 indicates that they use chemically inert complementary surfaces to bind to different cytokines, as opposed to adjusting their main chain and/or side chain conformations to interact speciﬁcally with divergent cytokine residues. This observation contrasts with notions of receptor promiscuity through binding site ﬂexibility (157). Because structural adaptation is not used by γc or gp130 as a means of cross-reactivity, the basis for degenerate recognition lies in the unique chemistry of the CHR epitope. Extensive thermodynamic studies of the binding between gp130 and different cytokines by ITC have revealed that their interactions are all primarily entropy driven, presumably because of desolvation of the

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interacting surfaces (LIF: 5 cal/molK; human IL-6: 45 cal/molK; OSM: 30 cal/molK; and CNTF: 62 cal/molK), albeit to varying extents commensurate with the surface polarity of the cytokine (10). We interpret these data to mean that gp130 uses desolvation as a structurally insensitive means of cross-reacting with structurally unique surfaces. The multiple solventexposed aromatic residues are likely covered with immobilized water clathrates in the unbound state, so that expulsion of the water into bulk solvent would be extremely entropically favorable. ITC analysis of γc interactions with the preformed IL-2/IL-2Rβ and IL2/IL-2Rα/IL-2Rβ complexes shows that the entropic contribution is smaller than that of gp130 (IL-2/IL-2Rβ: 4.85 cal/molK; IL-2/IL2Rα/IL-2Rβ: 0.72 cal/molK) (97). We still lack comparative data for the binding of γc with other γc -dependent cytokines, but we expect that γc uses similar thermodynamic solutions for the recognition of different cytokines. The entropy contribution may be smaller than that of gp130 because γc has a smaller contact area. Also, compared with gp130, the entropy contribution by the elbow region (site II) of γc to binding likely plays less of a role in cytokine recognition because of the extensive D2-D2 interactions between γc and the α receptors. The D2-D2 contacts are more polar in nature than are the cytokine-elbow region contacts and would presumably be more enthalpically driven, which would offset the entropy-driven recognition of the cytokine. Thus, as for gp130, γc uses enthalpy-entropy compensation to modulate its binding afﬁnity for diverse surface chemistries.

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CONCLUSIONS AND FUTURE PERSPECTIVES Gp130, βc , and γc —our principal shared cytokine receptors—appear to use a variety of both distinct and similar mechanisms to recognize diverse cytokines and ultimately to assemble into productive signaling complexes. The complexes exhibit the basic core template in which the CHR of the cytokine-speciﬁc α re2.24

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ceptors and the shared receptors engage the sides of the four-helix bundle in the typical site I/site II manner seen in homodimeric receptor complexes. The presence of an additional site III interacting Ig domain distinguishes gp130 from γc , and the antiparallel βc dimer demarks the most obvious signature of the βc complexes. In gp130, the heterodimers between gp130 and α receptors are nonproductive because in most cases the α receptors do not contain intracellular signaling motifs. Thus, site III is necessary to dimerize gp130, each of which is bound to a JAK, and signal. This stands in contrast to the γc family, in which the α receptors do contain intracellular signaling domains, and so the γc /α receptor heterodimers are productive: γc does not need to be dimerized. Another major difference is the extensive receptor-receptor contact seen between γc and the α receptors. The relatively energetic role of this contact versus the cytokine/γc contact remains to be determined, but it may be that the receptor-receptor contact between the D2 domains of the CHRs is the primary driving force for heterodimerization and that the role of the cytokines is to tip the energetic balance toward heterodimer formation. Finally, the chemical basis of degeneracy is also nicely paralleled in both receptors. Gp130 and γc are structurally rigid and present relatively ﬂat, hydrophobic binding sites for cytokine engagement that are rather devoid of highly charged and speciﬁc polar contacts. There are several important future questions that remain regarding the extracellular structures of these shared receptors. For gp130, several cytokines such as CNTF, OSM, and LIF heterodimerize gp130 with LIFR. Now that the basic template for dimerization of gp130 and LIFR has been elucidated by EM and structural studies, a high-resolution structure of the intact heterodimer remains an exciting puzzle to solve. In the γc family, with only two complex structures so far, there is some indication of the presence of a possible recognition code between cytokines and γc . This would be very exciting and different from gp130 cytokines, which do not appear to share any sequence or structural motifs necessary for gp130 engagement. The

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mechanism by which βc cross-reacts with IL-3 and IL-5 awaits additional complex structures for comparison with GM-CSF. However, the major structural frontier for cytokine receptors remains to obtain a better picture of the intracellular machinery. So far, we have no idea about the tertiary structure of any cytokine receptor intracellular domain. These regions may be unstructured unless bound to adaptors JAK and STAT. For STAT molecules, there are several structures now bound to DNA (158, 159) and also recently bound to phosphorylated peptides from

cytokine receptors (160). However, we do not have any full-length JAK structures. The crystal structures of kinase domains of JAKs are a step forward (161, 162), but ultimately we need to know how the N- and C-terminal ends of JAK communicate, as well as how the cytokine receptors box1 and box2 bind to JAK proteins. To fulﬁll these goals, higher-order imaging techniques such as EM and tomography will likely be used to obtain snapshots of entire cytokine receptors in lipid environments to preserve the integrity of the transmembrane regions.

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

ACKNOWLEDGMENTS The authors acknowledge the contribution of Figure 4 from Michael Parker and Jack KingScott prior to publication. We also acknowledge helpful discussions with Warren Leonard, Nicole Hanick, Mathis Rickert, and Marty Boulanger. This work was supported by NIH (RO1-AI51321), Keck Foundation, Sandler Foundation, Damon Runyon Cancer Research Foundation, and the Howard Hughes Medical Institute. LITERATURE CITED 1. Garcia KC, ed. 2004. Advances in Protein Chemistry, Vol. 68. London/San Diego: Academic. 506 pp. 2. Dechant G, Barde YA. 2002. The neurotrophin receptor p75(NTR): novel functions and implications for diseases of the nervous system. Nat. Neurosci. 5:1131–36 3. Airaksinen MS, Saarma M. 2002. The GDNF family: signalling, biological functions and therapeutic value. Nat. Rev. Neurosci. 3:383–94 4. Rudolph MG, Stanﬁeld RL, Wilson IA. 2006. How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24:419–66 5. Strong RK. 2002. Asymmetric ligand recognition by the activating natural killer cell receptor NKG2D, a symmetric homodimer. Mol. Immunol. 38:1029–37 6. Nicola NA. 1994. Cytokine pleiotropy and redundancy: a view from the receptor. Stem Cells 12(Suppl. 1):3–14 7. Taga T, Kishimoto T. 1995. Signaling mechanisms through cytokine receptors that share signal transducing receptor components. Curr. Opin. Immunol. 7:17–23 8. Ozaki K, Leonard WJ. 2002. Cytokine and cytokine receptor pleiotropy and redundancy. J. Biol. Chem. 277:29355–58 9. Donnelly RP, Sheikh F, Kotenko SV, Dickensheets H. 2004. The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain. J. Leukoc. Biol. 76:314–21 10. Boulanger MJ, Garcia KC. 2004. Shared cytokine signaling receptors: structural insights from the gp130 system. Adv. Protein Chem. 68:107–46 11. Chow D, He X, Snow AL, Rose-John S, Garcia KC. 2001. Structure of an extracellular gp130 cytokine receptor signaling complex. Science 291:2150–55 www.annualreviews.org • Structural Biology of Shared Cytokine Receptors

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Immunity to Respiratory Viruses Jacob E. Kohlmeier and David L. Woodland Trudeau Institute, Saranac Lake, New York 12983; email: [email protected]

Annu. Rev. Immunol. 2009. 27:3.1–3.22

Key Words

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

lung, T cell, memory, inﬂuenza

This article’s doi: 10.1146/annurev.immunol.021908.132625

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0001$20.00

The respiratory tract is characterized by an extensive surface area that is in direct contact with the environment, posing a signiﬁcant problem for effective immune surveillance. Yet most respiratory pathogens are quickly recognized and controlled by a coordinated response involving the innate and adaptive arms of the immune system. The investigation of pulmonary immunity to respiratory viruses during a primary infection has demonstrated that multiple innate and adaptive immune mechanisms are necessary for efﬁcient antiviral responses, and the inhibition of any single mechanism can have disastrous consequences for the host. Furthermore, the investigation of recall responses in the lung has shown that protection from a secondary challenge infection is a complex and elegant process that occurs in distinct stages. In this review, we discuss recent advances that describe the roles of individual components during primary and secondary responses to respiratory virus infections and how these discoveries have added to our understanding of antiviral immunity in the lung.

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INTRODUCTION RSV: respiratory syncytial virus

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PRR: patternrecognition receptor Chemokines: a family of chemoattractant cytokines important for inﬂammation and immune system homeostasis

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The mucosal surfaces of the respiratory, intestinal, and genital tracts are the primary portals of entry for a wide range of pathogens. The lung in particular is in direct and continuous contact with the surrounding environment, sampling nearly 10 liters of air each minute. Despite this continual exposure to potential antigens, the lung is generally maintained in a quiescent, noninﬂamed state. However, once a pathogen establishes a productive infection in the lung, an orchestrated process of pathogen recognition, inﬂammatory cytokine production, and cell migration leads to the generation of robust adaptive immune responses that eradicate the invading organism while limiting collateral damage to the lung tissue. Respiratory viruses, such as inﬂuenza virus, parainﬂuenza virus, respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus (SARS-CoV), rhinovirus, and adenovirus, are important human pathogens that establish acute infections usually localized to the upper respiratory tract. Despite the presence of these viruses in the population at endemic levels, it has taken the emerging threat of potential pandemics to thrust this class of pathogens into the public spotlight. For instance, the emergence of the highly pathogenic H5N1 inﬂuenza virus in 1997 has generated substantial public health concern owing to its extreme virulence and its potential to spread rapidly through the human population (1). Currently, there is considerable effort to develop improved vaccines capable of providing broad protection against these different types of viruses. However, achieving the goal of developing safe and effective vaccines to these pathogens has been complicated by our incomplete knowledge of how the immune system recognizes, contains, and eradicates respiratory viruses. An increasing number of reports have taken advantage of respiratory virus infection models in an attempt to develop a better understanding of mucosal immune responses in general and to provide new insight into speciﬁc pathogens that are major causes of morbidity

Kohlmeier

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and mortality worldwide. The use of small animal models to investigate immunity to these pathogens has generated a wealth of information regarding the dynamics of immune responses in the lung and has identiﬁed many individual components of the response necessary for the successful resolution of infection. Perhaps the best-characterized models are mouseadapted strains of inﬂuenza and murine parainﬂuenza viruses, which elicit robust T cell and B cell responses similar to infections in humans. The power of these models for understanding antiviral immunity in the lung has been enhanced by the advent of technologies for identifying antigen-speciﬁc responses and by the availability of a wide array of genetic tools. Here, we discuss recent advances in our understanding of antiviral immunity in the lung, from the initiation of innate and adaptive responses following primary virus infection to the recall of antigen-speciﬁc T cells during a secondary response, with a focus on lessons learned from murine models of inﬂuenza and parainﬂuenza virus infections.

INITIATION OF IMMUNE RESPONSES IN THE LUNG Innate Recognition of Infection A common feature of respiratory virus infections is that the initial infection is established in epithelial cells lining the respiratory tract. Epithelial cells, as well as alveolar macrophages and dendritic cells (DCs), continually sample the constituents of the airway lumen and detect the presence of an invading virus through pattern-recognition receptors (PRRs) (Figure 1) (2). The recognition of pathogen-associated molecular patterns by these receptors initiates a cascade of signals that results in the production of cytokines and chemokines. The release of these inﬂammatory mediators into the surrounding environment alerts the innate immune system to the presence of infection and establishes a localized antiviral state. In addition, chemokines provide the necessary signals for the recruitment

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Lung airways

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Lung parenchyma

Lung-draining lymph node

Respiratory dendritic cell

Naive T cell

Alveolar macrophage

Naive B cell

Red blood cell

Memory T cell

Lymph node dendritic cell

Virus-speciﬁc B cell

Figure 1 The resting pulmonary immune system. The lung airways, lung parenchyma, and lung-draining lymph nodes are three key sites of the antiviral immune response to respiratory viruses. In the absence of infection, alveolar macrophages and dendritic cells sample the constituents of the airway lumen for the presence of invading pathogens. Memory T cells from prior respiratory virus infections are localized to each of these sites, with large numbers of cells present in the airways and parenchyma for several months postinfection. Virus-speciﬁc B cells are also localized to each of these sites, with resting memory B cells widely distributed while long-lived plasma cells are localized primarily to lymphoid tissue associated with the respiratory tract and the bone marrow.

of circulating leukocytes to the site of infection. Finally, the combination of inﬂammatory cytokines and PRRs initiates the process of DC maturation and trafﬁcking that is re-

quired for the induction of adaptive immune responses. The best described of the PRRs are those of the Toll-like receptor (TLR) family, which in www.annualreviews.org • Immunity to Respiratory Viruses

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Type I interferons: a family of inﬂammatory cytokines, including IFN-α and IFN-β, produced in response to PRR signaling

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NK cell: natural killer cell

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mammals is composed of up to 15 unique receptors that are expressed by numerous cell types and recognize a wide range of microbial proteins, lipids, and nucleic acids (3). With respect to respiratory viruses, TLR3, 7, and 9 recognize various products of viral replication (dsRNA, ssRNA, and unmethylated CpG DNA, respectively) (4–7), whereas TLR4 recognizes the fusion (F) protein of RSV (8). TLRs that recognize nucleic acids are located in late endosomes. This location optimizes the TLRs’ ability to interact with viral nucleic acids while limiting their access to host-derived nucleic acids (9, 10). Although TLRs expressed on the cell surface (TLR4) or within the cell (TLR3, 7, 8, and 9) utilize different signaling pathways, each of these receptors can activate the transcription of interferon (IFN)-inducible genes (11). In addition, several recent reports have demonstrated that viral RNA is also recognized by several RNA helicases. Retinoic acid-inducible gene I (RIG-I) interacts with 5 -triphosphate RNA and is important for early cytokine production in response to numerous RNA viruses (12–15). Melanoma differentiation-associated gene 5 (MDA5) is a related helicase that recognizes polyinosinic polycytidylic acid and is crucial for innate recognition of picornaviruses (16). Similar to signaling through TLRs, the pathways utilized by RNA helicases ultimately trigger IFN regulatory factor (IRF) and nuclear factor-κB (NF-κB) activation (17). The key difference between these molecules and TLRs is that the RNA helicases are localized throughout the cytosol, rather than being regulated to intracellular compartments. Thus, viruses that infect cells by direct membrane fusion and do not enter endosomes can nevertheless trigger innate immune responses via RNA helicases.

The Early Inflammatory Response The innate recognition of viral components through PRRs described above leads to a program of gene expression that promotes a localized antiviral state and elicits the recruitment of inﬂammatory cells to the site of infection. 3.4

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Foremost among the early cytokines produced following infection are the pleiotropic antiviral cytokines of the type I interferon family, of which IFN-α and IFN-β are most commonly associated with early antiviral responses in the lung. Although nearly all cell types are capable of producing type I IFNs, numerous studies in mice and humans have shown that plasmacytoid DCs (pDCs) are the primary source of IFN-α and IFN-β following infection with a systemic virus (18). With respect to respiratory viruses, a recent study has provided in vivo evidence that alveolar macrophages are the primary producers of IFN-α during a parainﬂuenza virus infection and necessary for efﬁcient virus clearance (19). Notably, this study demonstrated that although IFN-α production by pDCs was largely TLR-dependent, alveolar macrophages required RIG-I signaling for optimal IFN-α production. However, the importance of alveolar macrophage-derived IFNα remains uncertain, as subsequent work has demonstrated that alveolar macrophage depletion had no effect on virus clearance during RSV infection (20). Therefore, type I IFN production in the lung appears to employ a level of redundancy, with alveolar macrophages or pDCs predominating depending on the type of virus infection. Type I IFNs produced following respiratory virus infections form a feedback loop by signaling through the IFN-α/β receptor and act in concert with PRR signaling to promote sustained production of proinﬂammatory cytokines such as TNF-α, IL-1, and IL-6 from lung-resident innate immune cells (21, 22). These proinﬂammatory cytokines and PRR-mediated signals also prompt alveolar macrophages, DCs, and epithelial cells to initiate a coordinated program of chemokine production following virus infection. DCs secrete successive waves of chemokines following inﬂuenza virus infection, beginning with those capable of recruiting inﬂammatory cells such as neutrophils and NK cells, and followed by chemokines associated with the recruitment of monocytes and memory T cells (23). Epithelial cells and alveolar macrophages also contribute

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to early chemokine production following infection and/or inﬂammation, particularly those chemokines capable of recruiting monocytes and memory T cells (24, 25). The chemokines produced during the innate immune response in the lung are capable of recruiting a wide range of innate and adaptive immune cell types. However, during a primary respiratory virus infection, the innate immune cells recruited to the lung are predominantly neutrophils and NK cells (Figure 2). The role that neutrophils play in respiratory virus clearance has not been well deﬁned, despite the large number of these cells present in the lung following infection. In fact, it has recently been shown that inhibition of neutrophil recruitment to the lung following inﬂuenza virus infection had no effect on the course of infection, suggesting that these cells do not play an essential role in virus clearance (26). In contrast, NK cells directly recognize inﬂuenza virus– and parainﬂuenza virus–infected cells through the interaction of the activating receptor NCR1 (NKp46 in humans) with hemagglutinin glycoprotein (27). NK cell recognition of virus-infected cells in coordination with proinﬂammatory cytokines resulted in enhanced cytolysis and IFN-γ production by NK cells (28–30). A recent deﬁnitive study investigating the importance of NK cells for protection against inﬂuenza virus showed a signiﬁcant increase in the number of NK cells in the lung beginning around day 3 postinfection. Importantly, this study also demonstrated that inﬂuenza virus infection was lethal in mice lacking the NK cell–activating receptor NCR1 (31). In addition to establishing a localized inﬂammatory environment and recruiting innate immune cells, PRR- and cytokine-mediated signals are important for the maturation and trafﬁcking of DCs to the draining lymph nodes preceding the initiation of the adaptive immune response. Under steady-state (i.e., noninﬂammatory) conditions, the trafﬁcking of DCs from the lung compartment to the draining lymph nodes is a continuous process that is dependent on the chemokine receptor CCR7 (32). The DCs that enter the lymph nodes under these

conditions are not fully mature, and it is believed that this process plays a role in the establishment of immune tolerance in the lung. Following inﬂuenza virus infection, lung-resident DCs increase expression of molecules involved in antigen presentation such as MHC class II, CD80, CD86, and CD40. Beginning as early as 6 h postinfection, there is an increase in the trafﬁcking of DCs from the lung to the draining lymph nodes that is maintained for several days (33, 34). In addition, the trafﬁcking of respiratory DCs to the lymphoid tissues is essential for the generation of adaptive immunity, as blocking DC migration abrogates antigen-speciﬁc T cell responses. Curiously, the trafﬁcking of DCs to the draining lymph nodes during both steady-state and inﬂammatory conditions is dependent on CCR7, suggesting that the accelerated recruitment following infection is not mediated by different chemokine receptors. However, investigators (35, 36) recently demonstrated that the interaction of the chemokine CCL5 with its receptor CCR5 is indirectly responsible for this enhanced trafﬁcking following infection by increasing the expression of CCR7 expression on DCs and enhancing migration across high endothelial venules. Thus, the innate recognition of infection leading to inﬂammatory cytokine and chemokine expression results in both the maturation and accelerated trafﬁcking of DCs, enabling more efﬁcient antigen presentation to T cells in the draining lymph nodes. A unique aspect of antiviral immunity in the lung is the potential for adaptive responses to be generated in local lymphoid structures such as nasal-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) (37). These structures exhibit similar organization to encapsulated lymph nodes with distinct T and B cell zones, high endothelial venules, and the expression of homeostatic chemokines important for DC and naive T cell migration (38). Importantly, these structures can signiﬁcantly contribute to the antiviral response, as mice devoid of secondary lymphoid tissues are able to mount effective, albeit delayed, virusspeciﬁc T cell and B cell responses that are www.annualreviews.org • Immunity to Respiratory Viruses

BALT: bronchusassociated lymphoid tissue

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Innate response (3-6 days p.i.)

Adaptive response (7-10 days p.i.)

Lung airways

Lung airways

Lung parenchyma

Lung parenchyma

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Lung-draining lymph node

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Virus-speciﬁc B cell

Alveolar macrophage

Neutrophil

Red blood cell

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Lymph node dendritic cell

Eﬀector T cell

Naive T cell

Virus

Naive B cell Figure 2 Innate and adaptive immune responses during a primary respiratory virus infection. During the innate response, virus detection in the lung by PRRs initiates a program of cytokine and chemokine production that leads to the recruitment of neutrophils and NK cells from the circulation to the lung airways and lung parenchyma. The inﬂux of innate immune cells and the production of cytokines limits early virus replication prior to the adaptive response. Concurrently, activated, antigen-bearing DCs migrate to the lung-draining lymph node, where they interact with antigen-speciﬁc naive T cells and generate a population of differentiated effector T cells. During the adaptive response, effector CD4+ T cells provide help to virus-speciﬁc B cells within the lymph node, and effector CD4+ and CD8+ T cells exit the lymph node and migrate to the lung. Large numbers of effector T cells accumulate in the lung airways and lung parenchyma, and through the production of cytokines and the lysis of infected epithelial cells, virus is cleared around 10 days postinfection. It is important to note that virus-speciﬁc antibody, which is not illustrated, also plays a critical role in virus clearance. 3.6

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originated and propagated within the BALT (39). Although the contribution of these localized lymphoid structures to the overall immune response in mice with normal secondary lymphoid tissues is difﬁcult to dissect, the proximity of these structures to the site of virus replication, and their ability to support a robust and diverse adaptive response suggests that they may play an important role in antiviral immunity.

ADAPTIVE IMMUNITY DURING PRIMARY RESPONSES T Cell Responses Naive CD4+ and CD8+ T cells within lymphoid tissues continually scan the surface of DCs for the presence of cognate antigen/MHC complexes (40, 41). This dynamic process, combined with the protrusion of dendrites that increases the DC surface area and the circulation of naive T cells through the lymph node, enhances the probability that extremely small numbers of antigen-speciﬁc naive T cell precursors, which range from 20–1200 cells of a given speciﬁcity in mice, will come into contact with their cognate antigen and enter into a program of proliferation and differentiation (42–44). As antigen-bearing mature DCs enter the lung-draining lymph nodes following respiratory virus infection, naive T cells speciﬁc for that antigen form stable interactions with the DCs, and the signals delivered by antigen recognition through the T cell receptor in addition to accessory signals delivered through costimulatory molecules result in T cell priming (45, 46). The initial priming of naive antigenspeciﬁc CD4+ and CD8+ T cells occurs within 72 h following inﬂuenza virus infection and initiates a program of sustained proliferation resulting in the accumulation of large numbers of virus-speciﬁc effector T cells (47–49). The instructions delivered by DCs during this initial expansion phase can have a dramatic impact on the survival and function of the responding T cells. For example, expression of FasL on DCs following inﬂuenza infection has been shown to regulate the magnitude of the CD8+ T cell

response (50). In addition, factors such as TCR avidity, costimulation, and the local inﬂammatory milieu all contribute to the generation of differentiated effector T cells prior to their exit from the lymph node and subsequent trafﬁcking to the lung (51–54). The appearance of antigen-speciﬁc effector T cells at the site of virus infection (i.e., the lung airways and lung parenchyma) is ﬁrst observed around days 6–7 postinfection with inﬂuenza and parainﬂuenza viruses (Figure 2). Chemokines expressed in the lung are recognized by blood-borne effector T cells, leading to changes in integrin afﬁnity that allow for tight binding to the blood vessel wall and extravisation into the surrounding tissue (55, 56). Endothelial selectins are also important for this process, as mice lacking expression of the molecules or their receptors showed a dramatic decrease in the trafﬁcking of CD4+ and CD8+ T cells to the lung (57, 58). Having migrated from the circulation into the lung tissue, effector T cell–expressed adhesion molecules are important for movement and survival within the interstitial spaces and airways of the lung. In addition, the expression of β1 integrins has been shown to control the localization of effector T cells to distinct compartments of the lungs. For example, cells expressing the integrin α1β1 (VLA-1) are predominantly associated with collagen Type IV–rich areas surrounding the airways and blood vessels, whereas cells expressing α2β1 (VLA-2) are predominantly associated with the collagen Type I–rich areas of the interstitial spaces (59). Analyses of chemokine and chemokine receptor expression in the lung during the adaptive phase of the immune response have shown elevated expression of numerous molecules associated with effector T cell trafﬁcking (60, 61). Surprisingly, very few published reports have directly investigated the role of speciﬁc chemokine receptors in the trafﬁcking of effector T cells during acute respiratory virus infections. One reason for this gap may be that the presence of multiple chemotactic signals in the lung at this stage of infection and the redundant nature of the chemokine system have made www.annualreviews.org • Immunity to Respiratory Viruses

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dissecting the importance of individual receptors in effector T cell recruitment a difﬁcult task (62). Nevertheless, several studies have identiﬁed roles for speciﬁc chemokine receptors in effector T cell trafﬁcking to the lung under resting or inﬂammatory conditions using different (i.e., nonviral) models that may provide some insight for future studies employing respiratory virus infections. For example, effector CD8+ T cells require CCR5 for migration from the pulmonary vasculature into the lung parenchyma in naive, uninfected mice (63). Also, an analysis of CD4+ T cells to the lung airways in asthmatic humans has shown that CCR6 and CXCR3 may be important for their trafﬁcking to this site (64). With regard to respiratory viruses, the trafﬁcking of effector T cells during RSV infection is at least partially dependent on CX3CR1, suggesting a potential role for this chemokine receptor in other paramyxovirus infections (65). To elucidate a role for speciﬁc chemokine receptors during acute respiratory virus infections requires future studies focused on receptors that are known to contribute to recruitment during inﬂammation, combined with studies already conducted that have characterized the expression of chemokine receptors on respiratory virus–speciﬁc T cells (66, 67). The continual migration of effector T cells from lymphoid tissues during an acute infection results in a massive increase in the numbers of antigen-speciﬁc cells in the lung airways and lung parenchyma from days 7–10 postinfection (68). The arrival of effector T cells has an immediate and dramatic impact on the viral load through the expression of cytokines and the direct lysis of infected cells. Inﬂuenzaspeciﬁc CD4+ and CD8+ effector T cells in the lung predominantly produce IFN-γ and TNF-α, and CD4+ effector T cells also produce IL-2 and IL-10 (69–72). CD8+ effector T cells localize to the respiratory epithelium and induce apoptosis of infected epithelial cells through Fas-FasL interactions or the exocytosis of cytolytic granules containing perforin and granzymes (73, 74). Together, these effector mechanisms contribute to the rapid decline in viral load beginning around day 7 postinfec-

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tion and result in virus clearance around day 10 postinfection (75, 76).

B Cell Responses B cell responses and virus-speciﬁc antibody play an important role in the clearance of inﬂuenza virus during primary infection, especially during infection with a highly pathogenic virus (77). Studies with B cell–deﬁcient mice have shown that, although early virus control (days 3–6 postinfection) is not impaired, these mice fail to clear the virus and ultimately succumb to infection (78, 79). The protective effect of B cells in these studies appears to be mediated at least in part through the production of virus-speciﬁc IgM because mice lacking only this isotype had delayed virus clearance and increased mortality (80, 81). Also, virusspeciﬁc IgM has been shown to provide protection from inﬂuenza-induced pathology in the presence of T cells (82). Together, these studies clearly deﬁne a role for the early production of virus-speciﬁc IgM in B cell–mediated protection during inﬂuenza virus infection. However, it is important to note that a direct comparison of B cell–deﬁcient and IgM-deﬁcient mice found that B cell–deﬁcient mice were more susceptible to inﬂuenza virus infection (81). Therefore, the production of neutralizing isotype-switched, virus-speciﬁc antibody during the later stages of the primary response is required for optimal virus clearance and antibody-mediated protection (83). The conventional model of isotypeswitching involves direct contact between antigen-speciﬁc CD4+ T cells and antigenpresenting B cells in lymphoid tissues. This antigen-dependent interaction, in addition to CD40-CD40L interactions and cytokine signaling, drives B cell proliferation and antibody isotype switching (84). Although this model of B cell activation and antibody production is the primary mechanism for virus-speciﬁc antibody production during inﬂuenza virus infection, several studies have demonstrated that these interactions are not absolutely required for virus-speciﬁc IgA and IgG antibodies. For

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example, inﬂuenza virus–speciﬁc IgA production during the early stages of infection is CD4+ T cell dependent but does not require cognate T-B cell interactions (85). In addition, CD4 T cell–deﬁcient mice can generate virusspeciﬁc IgG, although the inﬂuenza-speciﬁc antibody titers are considerably decreased in these mice (79). Taken together, these studies demonstrate that both T cell–dependent and –independent mechanisms contribute to inﬂuenza-speciﬁc antibody production.

T CELL AND B CELL MEMORY TO RESPIRATORY VIRUSES T Cell Memory The peak of effector T cell numbers in the lung following inﬂuenza and parainﬂuenza virus infections generally occurs around 10 days postinfection and coincides with virus clearance. The resolution of infection and waning inﬂammation has a dramatic impact on the virus-speciﬁc T cell population, initiating a program of contraction in which 90–95% of effector T cells are deleted by apoptosis (86). The outcome of this process is the establishment of a stable pool of memory T cells that persists in both peripheral and lymphoid tissues (87–89). Considerable progress has been made in recent years identifying the cues that instruct antigenspeciﬁc effector T cells to develop into longlived memory T cells, the factors that maintain the memory T cell population over time, the anatomical location and trafﬁcking patterns of different memory T cell subsets, and the relationship between different memory T cell subsets and the efﬁcacy of the recall response. In the initial months following a respiratory virus infection, antigen-speciﬁc CD4+ and CD8+ T cells can be found throughout lymphoid and nonlymphoid tissues in mice and humans, with the highest frequency of these cells located in the lung airways and lung parenchyma (90–93). However, the number of antigen-speciﬁc T cells in the lung wanes over time, and by one year postinfection the frequency and number of antigen-speciﬁc T cells

is similar between the lung and other peripheral or lymphoid tissues (90, 94). Importantly, the decline in memory T cell numbers in the lung over time correlates with a decline in the ability of antigen-speciﬁc T cells to control viral load during a secondary challenge (95). The large number of antigen-speciﬁc T cells in the lung following respiratory virus infection is believed to be maintained by persistent depots of inﬂuenza antigens that are presented within the draining lymph nodes for several months following virus clearance (96, 97). This antigen depot is capable of inducing T cell activation (as measured by CD69 expression) and low levels of proliferation (as measured by CFSE dilution) for up to 60 days postinfection. However, the low level of proliferation supported by this antigen depot may be sufﬁcient to account for the higher number of antigen-speciﬁc T cells found in the lung for several months postinfection. In support of this hypothesis, the waning antigenspeciﬁc T cell numbers observed beginning several months postinfection coincides with the disappearance of prolonged antigen presentation. However, although antigen-speciﬁc T cell numbers at sites such as the lung airways decline for several months postinfection, this population of cells stabilizes at a low level and is maintained indeﬁnitely (94, 98, 99). Thus, once the depot of persistent antigen has been cleared, the low number of antigen-speciﬁc memory T cells found in the lung airways is maintained by a background level of recruitment from the circulation (100). Memory T cells generated by respiratory viruses are heterogeneous in terms of their phenotype and function. This heterogeneity has led to the classiﬁcation of memory T cells into two subsets based on their preferential migration of peripheral (effector memory T cells, TEM ) or lymphoid (central memory T cells, TCM ) tissues (101). These subsets can be delineated on the basis of expression of CD62L and CCR7, which direct entry into lymphoid tissues. The majority of virus-speciﬁc memory T cells present throughout the body 1– 6 months postinfection express a TEM phenotype (CD62Llo and CCR7− ) and preferentially www.annualreviews.org • Immunity to Respiratory Viruses

TEM : effector memory T cell TCM : central memory T cell

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migrate to nonlymphoid sites (67, 102). Over time, the systemic memory T cell pool undergoes a gradual conversion to a TCM phenotype (CD62Lhi and CCR7+ ) that results in their localization to the lymph nodes and also to the bone marrow (103–105). Although there is conﬂicting evidence regarding the relationship between the TEM and TCM lineages, current evidence suggests that these two subsets are distinct populations generated during the initial infection, and the outgrowth of the TCM population over time is due to increased homeostatic turnover (106, 107). Regardless of the relationship between TEM and TCM , the passage of time leads to a shift in the preferential localization of virus-speciﬁc memory T cells, and in turn alters the dynamics and efﬁcacy of the recall response. For several months following virus clearance, large numbers of TEM are present in the lung and are able to provide immediate antiviral effector functions upon secondary infection. Over time, however, the number of virus-speciﬁc TEM able to provide this immediate response in the lung dramatically declines. Instead, the majority of virus-speciﬁc cells exist as TCM present in lymphoid tissues capable of rapid proliferation and the generation of new effector T cells following a secondary challenge. Therefore, the ability of antigen-speciﬁc memory T cells to immediately recognize and respond to a secondary virus challenge at the site of infection is lost over time.

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B Cell Memory Similar to the kinetics of T cell memory, inﬂuenza-speciﬁc memory B cells are rapidly established in multiple tissues following virus clearance. However, it is evident that there are profound differences between T cell and B cell memory with regard to their generation, trafﬁcking, and maintenance. B cell memory is characterized by two distinct populations of cells: long-lived plasma cells that continually secrete antibody and memory B cells that persist in a quiescent state (108, 109). The generation of B cell memory, particularly the generation of long-lived plasma cells, is dependent on cog3.10

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nate T-B cell interactions and CD40 signaling that occurs in the germinal center (110, 111). In support of this ﬁnding, investigators demonstrated that the inﬂuenza virus–speciﬁc IgG response generated in CD40-deﬁcient mice rapidly wanes and is undetectable by 60 days postinfection (79). In addition, it has recently been demonstrated that invariant natural killer T cells can also provide B cell help and enhance IgG responses (112). Therefore, although the data demonstrate that contact-dependent interactions are required for the generation of longlived B cell memory, these interactions can involve different cell types. Following inﬂuenza virus clearance, plasma cells leave the germinal centers and migrate to the bone marrow, where they continue to secrete virus-speciﬁc antibody. In addition, longlived plasma cells secreting inﬂuenza virus– speciﬁc IgA are localized and maintained in lymphoid tissues lining the respiratory tract (113). In contrast, resting memory B cells are widely dispersed to many tissues, where they can remain for many months. Interestingly, these cells localize at a higher frequency in the lung tissue following inﬂuenza virus infection, suggesting that the nature of the infection may alter the migratory capacity of these cells (114). Although it is unclear whether speciﬁc adhesion molecules or chemokine receptors play a role in the tissue-speciﬁc migration of memory B cells, the localization of these cells to the lung would allow them to rapidly recognize and respond to a secondary inﬂuenza virus challenge.

RECALL RESPONSES TO SECONDARY CHALLENGE It is well accepted that neutralizing, virusspeciﬁc antibodies (humoral immunity) provide optimal protection against most respiratory viruses by blocking the ability of the virus to establish infection. However, many viruses have evolved mechanisms to circumvent antibody-mediated immunity, allowing for secondary infections of closely related virus strains. For example, variability in the coat proteins of inﬂuenza virus enables the virus to evade

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neutralizing antibody and allows yearly inﬂuenza epidemics (115). Therefore, antibodymediated protection alone, while effective against secondary infection with a homologous virus, does not protect against variant viruses that arise through mutation. In contrast, the internal proteins of inﬂuenza virus, where many T cell epitopes are located, are highly conserved across many strains. Therefore, memory T cells speciﬁc for internal virus antigens are able to mount recall responses against heterologous virus strains (cellular immunity) and can provide broader protection against secondary challenge. In addition, non-neutralizing antibody to conserved internal proteins can enhance the memory T cell response to an inﬂuenza challenge (116). This section focuses on the experimental evidence that has been generated describing the cellular immune response to secondary virus infection in the absence of neutralizing antibody.

Lung Conditioning Much has been learned regarding the factors that inﬂuence memory T cell recall responses in animal models. However, a key consideration when interpreting the data from animal models is that these studies are often performed on speciﬁc pathogen-free mice. Although this limits the number of variables that could impact the results of an experiment, the immune response in a completely naive lung may not reﬂect the normal situation (117, 118). For example, the lungs of naive mice are devoid of lymphoid structures, such as BALT, that are present after respiratory virus clearance (119). Another issue is that prior inﬂuenza virus infections alter the responses to unrelated pathogens (120). Infection history can dramatically alter the clinical outcome of new infections depending on both the type of pathogen and order in which different pathogens are encountered (121). These studies have suggested a role for prior infections or inﬂammation in modifying the lung environment, thereby altering the manner in which the innate immune system reacts to subsequent inﬂammatory cues (122).

T Cell Recall Responses in the Lung As stated previously, the resolution of a primary respiratory virus infection generates a substantial number of antigen-speciﬁc memory T cells that are localized to both lymphoid and peripheral sites, such as the lung airways and the lung parenchyma. Several seminal studies have demonstrated that these respiratory virus– speciﬁc memory T cells mediate accelerated virus clearance and enhance survival following secondary challenge with related viruses (95, 123). More recently, evidence has emerged that memory T cell responses are far more complex than previously appreciated and that distinct populations of memory T cells segregated by their anatomical location and migratory capacity mediate different stages of the recall response. Thus, the recall response can be divided temporally, based on when these populations encounter virus-infected cells in the lung, and functionally, based on the steps required for their accumulation at this site (Figure 3). Importantly, it is the combination of these stages (discussed below) that results in the enhanced speed and magnitude of the recall response. Virus-speciﬁc memory T cells in the lung airways are believed to provide an initial line of defense against a secondary infection because these cells would be the ﬁrst to encounter antigen (124). Virus-speciﬁc memory T cells are present in considerable numbers for at least several months postinfection, despite their proximity to the harsh external environment and presence of mucus and surfactants (94, 125, 126). However, likely owing to the harsh environment, this population of cells is highly dynamic and is maintained by a process of continual recruitment from the circulation (100). Although lung airway memory T cells lack effector functions such as cytolytic activity (127), these cells are able to produce cytokines in response to antigen or inﬂammatory cytokines (98). Importantly, a direct role for these cells in protective immunity was demonstrated by the transfer of antigen-speciﬁc memory T cells to the lung airways of naive mice, which resulted in signiﬁcantly reduced viral titers following infection (124). Therefore, the ﬁrst stage of the www.annualreviews.org • Immunity to Respiratory Viruses

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Early stage of recruitment (2-5 days p.i.)

Late stage of recruitment (4-7 days p.i.)

Lung airways

Lung airways

Lung parenchyma

Lung parenchyma

Lung-draining lymph node

Lung-draining lymph node

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Memory T cell

Alveolar macrophage

Virus-speciﬁc B cell

Red blood cell

Neutrophil

Lymph node dendritic cell

NK cell

Naive T cell

Eﬀector T cell

Naive B cell

Virus

Figure 3 Dynamics of the T cell recall response to secondary virus challenge. Similar to a primary response, neutrophils and NK cells are recruited to the lung in response to localized inﬂammation. In addition, circulating memory CD8+ T cells are also recruited from the circulation to the lung in a CCR5-dependent manner during the early phase of infection, thereby increasing the number of antigen-speciﬁc cells at the site of infection and limiting virus replication. Memory T cells in lymphoid tissues are activated by antigenbearing DCs from the lung, and, owing to their increased precursor frequency and activation status compared with naive T cells, they are able to rapidly generate large numbers of secondary effector T cells. Secondary effector T cells migrate to the lung airways and lung parenchyma and mediate rapid virus clearance. Although not illustrated, the generation of secondary adaptive immune responses can also occur in lymphoid tissues (such as BALT) within the lung. 3.12

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recall response involves cells present at the site of infection that limit viral replication during the ﬁrst several days of infection while antigenbearing DCs are trafﬁcking to lymphoid tissues. An established paradigm of secondary T cell responses is the more rapid generation of effector T cells compared with a primary T cell response. Despite this more rapid generation, in respiratory virus infections this still allows for approximately four days between initial virus infection and the appearance of secondary effector T cells in the lung. However, several groups have demonstrated a dramatic increase in the number of virus-speciﬁc CD8+ T cells in the lung airways prior to the appearance of secondary effector T cells (128, 129). This increase in virus-speciﬁc CD8+ T cells could not be accounted for by localized proliferation within the airways, as these cells had not recently divided and the increase in cell number did not require cognate antigen stimulation. Rather, these studies showed that increased numbers of virusspeciﬁc CD8+ T cells in the airways was due to the inﬂammation-dependent recruitment of nondividing memory T cells from the circulation (130). Thus, the recruitment of circulating memory CD8+ T cells to the lung airways in response to inﬂammation serves to increase the number of antigen-speciﬁc memory T cells at the site of virus replication prior to the secondary effector T cell response. Although these studies had clearly demonstrated that the recruitment of memory CD8+ T cells occurs in response to localized inﬂammation, the mechanism that directs circulating memory CD8+ T cells to the lung airways and the importance of this process during secondary respiratory virus challenge had not been determined. Recently, we demonstrated that CCR5 expressed on memory CD8+ T cells was required for their recruitment to the airways during the early stages of the recall response (131). The role for CCR5 in memory CD8+ T cell recruitment is unique for the lung airways, as no defect in recruitment was observed in the lung parenchyma. Importantly, inhibiting memory CD8+ T cell recruitment to the airways resulted in signiﬁcantly higher

virus titers during the early phase of infection. Therefore, the second stage of the recall response involves the CCR5-dependent recruitment of circulating memory CD8+ T cells to the lung airways, resulting in increased numbers of antigen-speciﬁc cells at the site of virus replication and a further decrease in viral load. The ﬁnal stage of the recall response involves the activation of memory T cells in the draining lymphoid tissue by antigen-bearing DCs, resulting in the proliferation and expansion of secondary effector T cells that can migrate to the lung and eradicate the infection. Although this process occurs in a similar manner during both primary and secondary infections, the reduced stimulatory requirements, more rapid acquisition of effector functions, and increased precursor frequency of memory T cells compared with naive T cells allows for the accelerated generation of effector T cells (132, 133). Despite the reduced activation requirements of memory T cells, professional antigenpresenting cells (i.e., DCs) are still required for the optimal generation of secondary effector T cells during a recall response (134). The importance of memory T cell priming by DCs during an inﬂuenza virus infection was shown when inﬂuenza-speciﬁc memory CD8+ T cells transferred into mice lacking bone marrow–derived DCs failed to provide protection from virus challenge (135). The heterogeneity of the memory T cell population can also have a substantial impact on the magnitude of the secondary effector T cell response. We have shown that the ability of memory CD8+ T cells to proliferate and generate effector T cells that accumulate in the lung following secondary virus infection improves over time, and this improvement in recall efﬁcacy was observed in both the TEM and TCM populations (67). However, there have been conﬂicting reports regarding the relative contributions of the TEM and TCM populations to recall responses (136–140). The discrepancy in these ﬁndings suggested that the division of memory CD8+ T cell subsets solely between effector and central memory cells was insufﬁcient to describe their recall potential. A more www.annualreviews.org • Immunity to Respiratory Viruses

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thorough examination of the TEM and TCM subsets revealed that both populations could be further delineated based on the expression of activation markers such as CD27, CD43, and killer cell lectin-like receptor G1 (KLRG1) (141). Similar to the expression of CD62L that deﬁnes the TEM and TCM subsets, the distribution of the memory CD8+ T cell pool deﬁned by these activation markers changes over time, so that by one year postinfection most cells display a resting (CD27hi CD43lo ) phenotype. Importantly, the memory T cell subsets deﬁned by activation marker expression showed substantial differences in their ability to mount recall responses to respiratory viruses. Memory CD8+ T cells with the most activated phenotype (CD27lo CD43lo ) were 5- to 20-fold less efﬁcient at generating secondary effector T cells that could accumulate in the lung than were memory CD8+ T cells with the most resting phenotype (CD27hi CD43lo ) (141). Therefore, the third and ﬁnal stage of the recall response depends on the stimulation of memory T cells by DCs in the lymphoid tissues that give rise to a new population of secondary effector T cells. Furthermore, the ability of memory T cells to mount a robust recall response depends on the activation status of these cells when they re-encounter antigen.

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CONCLUDING REMARKS Our understanding of the mechanisms that the immune system employs to identify and eliminate acute respiratory viruses has grown considerably over the past decade. The discovery of different PRRs and the role they play in the initiation of the immune response has illustrated how the innate immune system distinguishes between host and pathogen. The dissection of the inﬂammatory response into individual cytokines and chemokines has allowed us to determine the importance of these molecules for protective responses, and to determine the contribution of different cell types to antiviral immunity. The characterization of memory T cell and B cell subsets has demonstrated the importance of these populations for protection from a secondary challenge and shown how different subsets of these cells contribute to the recall response. However, it is apparent that the various facets of antiviral immunity in the lung are far more complex than originally thought, and subtleties discovered so far hint at additional issues that must be resolved. Future studies investigating the innate and adaptive immune responses in ever greater detail will be required to develop a comprehensive picture of antiviral immunity in the lung.

SUMMARY POINTS 1. Viral nucleic acids recognized by TLR3, 7, and 9 in the endosome, or by RNA helicases in the cytosol, initiate a signaling cascade that activates IRFs and results in the production of type I IFNs. 2. Inﬂammatory signals trigger the production of chemokines by epithelial cells, alveolar macrophages, and DCs that attract innate immune cells prior to the generation of adaptive immunity. The early recruitment of NK cells to the lung and their recognition of virus-infected cells by the activating receptor NCR1 are essential for protection from an inﬂuenza virus infection. 3. The appearance of virus-speciﬁc effector T cells expressing cytokines and cytolytic molecules in the lung and the production of virus-speciﬁc IgM and IgG by B cells in the lymphoid tissue combine to resolve acute respiratory virus infections within approximately 10 days postinfection. 4. Following virus clearance, memory T cells are established in lymphoid and peripheral tissues, including a large population of antigen-speciﬁc cells that is localized to the lung

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airways and lung parenchyma. Virus-speciﬁc memory B cells are localized to the lymphoid tissue associated with the respiratory tract (primarily IgA-secreting cells) and the bone marrow (primarily IgG-secreting cells).

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5. Changes in the systemic memory T cell pool from a predominantly TEM to TCM phenotype over time, coupled with the disappearance of residual antigen in the lymph nodes, results in the redistribution of virus-speciﬁc memory T cells from peripheral to lymphoid tissues. 6. The recall response of memory T cells to a secondary virus infection in the lung airways can be separated into three distinct stages. The ﬁrst phase involves virus-speciﬁc memory T cells located in the lung airways, the second phase involves the CCR5-dependent recruitment of circulating memory T cells to the airways, and the third stage involves the appearance of secondary effector T cells that were generated in lymphoid tissue.

FUTURE ISSUES 1. How do different DC subsets in the lung impact the innate response to virus and inﬂuence the quality of the adaptive immune response? 2. Which chemokine receptors, or combination of chemokine receptors, are required for the migration of effector T cells to the various compartments of the lung? 3. Does tissue-speciﬁc imprinting occur during a respiratory virus infection that directs the preferential migration of memory T cells and memory B cells to the lung? 4. How is T cell memory generated during a respiratory virus infection, and what are the relationships between memory T cells that express different activation markers. 5. How do previous infections alter the lung environment and what are the consequences of these alterations for innate immunity? 6. By what mechanisms do memory T cells in the lung airways limit early virus replication during a secondary infection, and do these cells inﬂuence the development of the secondary immune response by altering the course of infection?

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

ACKNOWLEDGMENTS We thank M. Blackman for a critical reading of the manuscript and A. Bernat for graphic design. This work was supported by funds from the Trudeau Institute and by NIH grants AI067967, AI076499, AG021600, and T32 AI49823 to D.L.W., and F32 AI071478 to J.E.K. LITERATURE CITED 1. Horimoto T, Kawaoka Y. 2001. Pandemic threat posed by avian inﬂuenza A viruses. Clin. Microbiol. Rev. 14:129–49 www.annualreviews.org • Immunity to Respiratory Viruses

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89. Joshi NS, Cui W, Chandele A, Lee HK, Urso DR, et al. 2007. Inﬂammation directs memory precursor and short-lived effector CD8+ T cell fates via the graded expression of T-bet transcription factor. Immunity 27:281–95 90. Marshall DR, Turner SJ, Belz GT, Wingo S, Andreansky S, et al. 2001. Measuring the diaspora for virus-speciﬁc CD8+ T cells. Proc. Nat. Acad. Sci. USA 98:6313–18 91. Cauley LS, Cookenham T, Miller TB, Adams PS, Vignali KM, et al. 2002. Cutting edge: virus-speciﬁc CD4+ memory T cells in nonlymphoid tissues express a highly activated phenotype. J. Immunol. 169:6655–58 92. Masopust D, Vezys V, Usherwood EJ, Cauley LS, Olson S, et al. 2004. Activated primary and memory CD8 T cells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin. J. Immunol. 172:4875–82 93. de Bree GJ, van Leeuwen EM, Out TA, Jansen HM, Jonkers RE, van Lier RA. 2005. Selective accumulation of differentiated CD8+ T cells speciﬁc for respiratory viruses in the human lung. J. Exp. Med. 202:1433–42 94. Hogan RJ, Usherwood EJ, Zhong W, Roberts AD, Dutton RW, et al. 2001. Activated antigen-speciﬁc CD8+ T cells persist in the lungs following recovery from respiratory virus infections. J. Immunol. 166:1813–22 95. Liang S, Mozdzanowska K, Palladino G, Gerhard W. 1994. Heterosubtypic immunity to inﬂuenza type A virus in mice. Effector mechanisms and their longevity. J. Immunol. 152:1653–61 96. Jelley-Gibbs DM, Brown DM, Dibble JP, Haynes L, Eaton SM, Swain SL. 2005. Unexpected prolonged presentation of inﬂuenza antigens promotes CD4 T cell memory generation. J. Exp. Med. 202:697–706 97. Zammit DJ, Turner DL, Klonowski KD, Lefrancois L, Cauley LS. 2006. Residual antigen presentation after inﬂuenza virus infection affects CD8 T cell activation and migration. Immunity 24:439–49 98. Marsland BJ, Harris NL, Camberis M, Kopf M, Hook SM, Le Gros G. 2004. Bystander suppression of allergic airway inﬂammation by lung resident memory CD8+ T cells. Proc. Nat. Acad. Sci. USA 101:6116–21 99. Kohlmeier JE, Miller SC, Woodland DL. 2007. Cutting edge: antigen is not required for the activation and maintenance of virus-speciﬁc memory CD8+ T cells in the lung airways. J. Immunol. 178:4721–25 100. Ely KH, Cookenham T, Roberts AD, Woodland DL. 2006. Memory T cell populations in the lung airways are maintained by continual recruitment. J. Immunol. 176:537–43 101. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708–12 102. Masopust D, Vezys V, Marzo AL, Lefrancois L. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291:2413–17 103. Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, et al. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 3:225–34 104. Mazo IB, Honczarenko M, Leung H, Cavanagh LL, Bonasio R, et al. 2005. Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells. Immunity 22:259–70 105. Becker TC, Coley SM, Wherry EJ, Ahmed R. 2005. Bone marrow is a preferred site for homeostatic proliferation of memory CD8 T cells. J. Immunol. 174:1269–73 106. Marzo AL, Klonowski KD, Le Bon A, Borrow P, Tough DF, Lefrancois L. 2005. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat. Immunol. 6:793–99 107. Moulton VR, Bushar ND, Leeser DB, Patke DS, Farber DL. 2006. Divergent generation of heterogeneous memory CD4 T cells. J. Immunol. 177:869–76 108. Bachmann MF, Kundig TM, Odermatt B, Hengartner H, Zinkernagel RM. 1994. Free recirculation of memory B cells versus antigen-dependent differentiation to antibody-forming cells. J. Immunol. 153:3386–97 109. Slifka MK, Antia R, Whitmire JK, Ahmed R. 1998. Humoral immunity due to long-lived plasma cells. Immunity 8:363–72 110. Noelle RJ, Roy M, Shepherd DM, Stamenkovic I, Ledbetter JA, Aruffo A. 1992. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc. Nat. Acad. Sci. USA 89:6550–54

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111. Lee BO, Moyron-Quiroz J, Rangel-Moreno J, Kusser KL, Hartson L, et al. 2003. CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses. J. Immunol. 171:5707–17 112. Galli G, Pittoni P, Tonti E, Malzone C, Uematsu Y, et al. 2007. Invariant NKT cells sustain speciﬁc B cell responses and memory. Proc. Nat. Acad. Sci. USA 104:3984–89 113. Liang B, Hyland L, Hou S. 2001. Nasal-associated lymphoid tissue is a site of long-term virus-speciﬁc antibody production following respiratory virus infection of mice. J. Virol. 75:5416–20 114. Joo HM, He Y, Sangster MY. 2008. Broad dispersion and lung localization of virus-speciﬁc memory B cells induced by inﬂuenza pneumonia. Proc. Nat. Acad. Sci. USA 105:3485–90 115. Stamboulian D, Bonvehi PE, Nacinovich FM, Cox N. 2000. Inﬂuenza. Infect. Dis. Clin. N. Am. 14:141–66 116. Rangel-Moreno J, Carragher DM, Misra RS, Kusser K, Hartson L, et al. 2008. B cells promote resistance to heterosubtypic strains of inﬂuenza via multiple mechanisms. J. Immunol. 180:454–63 117. Goulding J, Snelgrove R, Saldana J, Didierlaurent A, Cavanagh M, et al. 2007. Respiratory infections: Do we ever recover? Proc. Am. Thorac. Soc. 4:618–25 118. Kim EY, Battaile JT, Patel AC, You Y, Agapov E, et al. 2008. Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease. Nat. Med. 14:633–40 119. Moyron-Quiroz JE, Rangel-Moreno J, Hartson L, Kusser K, Tighe MP, et al. 2006. Persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs. Immunity 25:643– 54 120. Walzl G, Tafuro S, Moss P, Openshaw PJ, Hussell T. 2000. Inﬂuenza virus lung infection protects from respiratory syncytial virus-induced immunopathology. J. Exp. Med. 192:1317–26 121. Chen HD, Fraire AE, Joris I, Welsh RM, Selin LK. 2003. Speciﬁc history of heterologous virus infections determines antiviral immunity and immunopathology in the lung. Am. J. Pathol. 163:1341–55 122. Williams AE, Edwards L, Humphreys IR, Snelgrove R, Rae A, et al. 2004. Innate imprinting by the modiﬁed heat-labile toxin of Escherichia coli (LTK63) provides generic protection against lung infectious disease. J. Immunol. 173:7435–43 123. Cerwenka A, Morgan TM, Dutton RW. 1999. Naive, effector, and memory CD8 T cells in protection against pulmonary inﬂuenza virus infection: homing properties rather than initial frequencies are crucial. J. Immunol. 163:5535–43 124. Hogan RJ, Zhong W, Usherwood EJ, Cookenham T, Roberts AD, Woodland DL. 2001. Protection from respiratory virus infections can be mediated by antigen-speciﬁc CD4+ T cells that persist in the lungs. J. Exp. Med. 193:981–86 125. Ostler T, Hussell T, Surh CD, Openshaw P, Ehl S. 2001. Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus. Eur. J. Immunol. 31:2574–82 126. Wiley JA, Hogan RJ, Woodland DL, Harmsen AG. 2001. Antigen-speciﬁc CD8+ T cells persist in the upper respiratory tract following inﬂuenza virus infection. J. Immunol. 167:3293–99 127. Vallbracht S, Unsold H, Ehl S. 2006. Functional impairment of cytotoxic T cells in the lung airways following respiratory virus infections. Eur. J. Immunol. 36:1434–42 128. Chen HD, Fraire AE, Joris I, Brehm MA, Welsh RM, Selin LK. 2001. Memory CD8+ T cells in heterologous antiviral immunity and immunopathology in the lung. Nat. Immunol. 2:1067–76 129. Topham DJ, Castrucci MR, Wingo FS, Belz GT, Doherty PC. 2001. The role of antigen in the localization of naive, acutely activated, and memory CD8+ T cells to the lung during inﬂuenza pneumonia. J. Immunol. 167:6983–90 130. Ely KH, Cauley LS, Roberts AD, Brennan JW, Cookenham T, Woodland DL. 2003. Nonspeciﬁc recruitment of memory CD8+ T cells to the lung airways during respiratory virus infections. J. Immunol. 170:1423–29 131. Kohlmeier JE, Miller SC, Smith J, Lu B, Gerard C, et al. 2008. The chemokine receptor CCR5 plays a key role in the early memory CD8+ T cell response to respiratory virus infections. Immunity 29:101–13 132. Seder RA, Ahmed R. 2003. Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat. Immunol. 4:835–42 133. Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, et al. 2003. Duration of antiviral immunity after smallpox vaccination. Nat. Med. 9:1131–37 134. Zammit DJ, Cauley LS, Pham QM, Lefrancois L. 2005. Dendritic cells maximize the memory CD8 T cell response to infection. Immunity 22:561–70 www.annualreviews.org • Immunity to Respiratory Viruses

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135. Castiglioni P, Hall de S, Jacovetty EL, Ingulli E, Zanetti M. 2008. Protection against inﬂuenza A virus by memory CD8 T cells requires reactivation by bone marrow-derived dendritic cells. J. Immunol. 180:4956–64 136. Roberts AD, Woodland DL. 2004. Cutting edge: effector memory CD8+ T cells play a prominent role in recall responses to secondary viral infection in the lung. J. Immunol. 172:6533–37 137. Vaccari M, Trindade CJ, Venzon D, Zanetti M, Franchini G. 2005. Vaccine-induced CD8+ central memory T cells in protection from simian AIDS. J. Immunol. 175:3502–7 138. Bachmann MF, Wolint P, Schwarz K, Oxenius A. 2005. Recall proliferation potential of memory CD8+ T cells and antiviral protection. J. Immunol. 175:4677–85 139. Stock AT, Jones CM, Heath WR, Carbone FR. 2006. Cutting edge: central memory T cells do not show accelerated proliferation or tissue inﬁltration in response to localized herpes simplex virus-1 infection. J. Immunol. 177:1411–15 140. Klonowski KD, Marzo AL, Williams KJ, Lee SJ, Pham QM, Lefrancois L. 2006. CD8 T cell recall responses are regulated by the tissue tropism of the memory cell and pathogen. J. Immunol. 177:6738–46 141. Hikono H, Kohlmeier JE, Takamura S, Wittmer ST, Roberts AD, Woodland DL. 2007. Activation phenotype, rather than central- or effector-memory phenotype, predicts the recall efﬁcacy of memory CD8+ T cells. J. Exp. Med. 204:1625–36

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Review in Advance first posted online on November 13, 2008. (Minor changes may still occur before final publication online and in print.)

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Immune Therapy for Cancer Michael Dougan and Glenn Dranoff

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Department of Medical Oncology and Cancer Vaccine Center, Dana-Farber Cancer Institute and Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; email: glenn [email protected]

Annu. Rev. Immunol. 2009. 27:4.1–4.35

Key Words

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

monoclonal (antibody), inﬂammation, vaccine, adjuvants, immunotherapy

This article’s doi: 10.1146/annurev.immunol.021908.132544 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0001$20.00

Abstract Over the past decade, immune therapy has become a standard treatment for a variety of cancers. Monoclonal antibodies, immune adjuvants, and vaccines against oncogenic viruses are now well-established cancer therapies. Immune modulation is a principal element of supportive care for many high-dose chemotherapy regimens. In addition, immune activation is now appreciated as central to the therapeutic mechanism of bone marrow transplantation for hematologic malignancies. Advances in our understanding of the molecular interactions between tumors and the immune system have led to many novel investigational therapies and continue to inform efforts for devising more potent therapeutics. Novel approaches to immune-based cancer treatment strive to augment antitumor immune responses by expanding tumor-reactive T cells, providing exogenous immune-activating stimuli, and antagonizing regulatory pathways that induce immune tolerance. The future of immune therapy for cancer is likely to combine many of these approaches to generate more effective treatments.

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INTRODUCTION

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Immune therapy is already established as a central component of many cancer treatment regimens (Table 1). Tumors express a wide variety of proteins that can be recognized by the immune system. In addition to microbial proteins, mutated proteins, and fusion proteins, the immune system can recognize developmentally and tissue-restricted proteins, as well as proteins that are highly overexpressed by cancer cells. Established therapies employ a variety of manipulations to activate antitumor immunity. These include passive immunization Table 1

with monoclonal antibodies, the introduction of adjuvants into the tumor microenvironment, and the systemic delivery of cytokines. Immune therapy can ameliorate the toxic effects of standard chemotherapy and is an essential element in the curative mechanism of bone marrow transplantation for hematologic malignancies. Vaccination against and treatment for microbial infections can effect sterilizing immunity against cancer-promoting microorganisms, acting as tumor prophylaxis. Investigational immune therapies for cancer seek to build upon established treatment regimens to devise more efﬁcacious and less toxic cancer therapy. A wide variety of novel

Approved immune therapies for cancera

Established therapies

Indication

References

Monoclonal antibodies Rituximab Ibritumomab tiuxetan Tositumomab Alemtuzumab Gentuzumab Trastuzumab Cetuximab Panitumumab Bevacizumab

NHL, CLL NHL NHL CLL AML Breast cancer Colorectal cancer Colorectal cancer Colorectal, lung

11, 14 12 13 9 10 18 20 21 24–26

Immune adjuvants BCG Imiquimod

Superﬁcial bladder cancer Basal cell carcinoma, VIN, actinic keratosis

39–42 45, 46

Cytokines IFN-α IL-2 TNF-α

melanoma, RCC melanoma, RCC Soft tissue sarcoma, melanoma

49, 50 47, 48 54, 55

Supportive therapy G(M)-CSF Leucovorin

Myelosuppressive chemotherapy MTX rescue

56–58 59

Prophylactic immune therapy HBV vaccine HPV vaccine Antibiotics (H. pylori ) NSAIDs (FAP, ulcerative colitis)

Hepatocellular carcinoma Cervical cancer Gastric cancer, MALT lymphoma Colorectal cancer

61–62 63 69–71 72–75

Bone marrow transplantation Allogeneic DLI

Hematologic malignancies Hematologic malignancies

77–81 82

a Abbreviations: AML, acute myelogenous leukemia; BCG, bacilli Calmette-Gu´erin; CLL, chronic lymphocytic leukemia; DLI, donor lymphocyte infusion; FAP, familial adenomatous polyposis; HBV, hepatitis B virus; HPV, human papilloma virus; MALT, mucosal-associated lymphoid tissue; MTX, methotrexate; NHL, non-Hodgkin’s lymphoma; NSAID, nonsteroidal anti-inﬂammatory drug; RCC, renal cell carcinoma; VIN, vulvar intraepithelial neoplasia.

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strategies have been developed based on fundamental advances in our understanding of the interactions between tumors and the immune system. Collectively, these strategies attempt to augment protective antitumor immunity and to disrupt the immune-regulatory circuits that are critical for maintaining tumor tolerance.

CURRENT IMMUNE THERAPIES

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Monoclonal Antibodies Antibodies play an essential role in providing protective immunity to microorganisms, and the administration of tumor-targeting monoclonal antibodies has proven to be one of the most successful forms of immune therapy for cancer. The infusion of manufactured monoclonal antibodies can generate an immediate immune response while bypassing many of the limitations that impede endogenous immunity. The target speciﬁcity and optimal afﬁnity of manufactured antibodies can be carefully selected, and the quantity of antibody infused can be set to achieve biologically active antibody titers rapidly. Because therapeutic antibodies are initially produced in animals, even self-proteins to which the immune system is generally tolerant can be targeted, a signiﬁcant advantage given that most tumor proteins are also expressed on nonmalignant tissues. Monoclonal antibody therapies are typically not as toxic as conventional cytotoxic cancer chemotherapy, although binding to nonmalignant cells can, in some cases, precipitate significant adverse reactions (1–4). The animal origin of monoclonal antibodies can also lead to treatment-limiting hypersensitivity reactions; however, these reactions have been minimized by substituting human IgG1 sequences outside of the antibody-binding domain (5–7). Mice that exclusively express human antibody genes have also been used to produce fully humanized antibodies (8). Nine monoclonal antibodies, targeting six tumor-associated proteins, are clinically approved for the treatment of cancer. Five of these antibodies bind surface proteins that

are highly expressed on hematologic tumors: CD52 in chronic lymphocytic leukemia (CLL) (alemtuzumab), CD33 in acute myelogenous leukemia (AML) (gentuzumab), and CD20 in non-Hodgkin’s lymphoma (NHL) and CLL (rituximab, ibritumomab tiuxetan, and tositumomab) (9–13). Of these, rituximab is the most widely used and has now been added to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) as part of the standard treatment for NHL; in combination with CHOP, rituximab induces 76% complete remissions, contrasted with 63% for CHOP therapy alone (11, 14). Four monoclonal antibodies (trastuzumab, cetuximab, panitumumab, and bevacizumab) have been approved for the treatment of solid tumors, although none of these has yet shown efﬁcacy comparable with rituximab in NHL (15–22). Trastuzumab, cetuximab, and panitumumab bind proteins of the epidermal growth factor receptor (EGFR) family, either targeting EGFR itself (cetuximab and panitumumab) or targeting the related protein HER2/neu (transtuzumab). EGFR proteins play an important role in transmitting growth signals to a variety of epithelial tumors and have also been targeted by small molecule signal transduction inhibitors (23). Both EGFR-targeting antibodies have been approved for the treatment of metastatic colorectal cancer in patients who have previously failed standard chemotherapy (17, 19–21). In this patient population, cetuximab and panitumumab increase progressionfree survival and are associated with 10–20% and 10% response rates, respectively (20, 21). Trastuzumab was the second monoclonal antibody approved for cancer therapy and is used, either alone or in combination with paclitaxel, for the treatment of invasive, HER2/neupositive breast cancer, which represents approximately 20–30% of invasive breast cancers. Treatment with trastuzumab is associated with variable response rates, ranging from 11% to 26% when used as monotherapy to 50% when used in combination with chemotherapy. In comparison, chemotherapy alone has a response rate of 32% (18). www.annualreviews.org • Immune Therapy for Cancer

CLL: chronic lymphocytic leukemia AML: acute myelogenous leukemia NHL: non-Hodgkin’s lymphoma

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NSCLC: non-small cell lung cancer

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ADCC: antibodydependent cellular cytotoxicity

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Bevacizumab is the only monoclonal antibody with anticancer activity that does not directly target malignant cells. Instead, bevacizumab binds vascular endothelial growth factor (VEGF), a critical mediator of tumor angiogenesis. Inhibiting angiogenesis slows the delivery of nutrients and oxygen to tumors, inhibiting growth without severely compromising normal tissue function (15, 16). Bevacizumab has shown some efﬁcacy in a range of solid tumors, including nonsquamous nonsmall cell lung cancer (NSCLC), metastatic colon cancer, metastatic HER2/neu-negative breast cancer, renal cancer, and pancreatic cancer (15, 16, 24–26). By binding to their targets, antibodies exercise their functions through several effector mechanisms, including steric inhibition and neutralization, complement activation, and activation of cell-mediated cytotoxicity. Each of these mechanisms may play a role in the antitumor activity of monoclonal antibodies; however, at present the relative importance of these mechanisms is not completely clear. Several monoclonal antibodies are known to inhibit signaling downstream of their targets. Cetuximab and pantitumumab are potent inhibitors of EGFR signaling, acting both to block interactions with epidermal growth factor and to prevent conformational changes in the receptor that are required for dimerization and signaling (27–29); similarly, rituximab and trastuzumab alter signaling downstream of their targets (30, 31). Bevacizumab directly binds the soluble growth factor VEGF, preventing VEGF-dependent effects on the vasculature (32). Two monoclonal antibodies have been used to deliver cytotoxic therapy directly to tumors by conjugating them to radioactive isotypes (ibritumomab tiuxetan and 131 I tositumomab) or to toxic chemicals (gemtuzumab) (10, 12). Antibody binding alone is probably sufﬁcient to provide some antitumor activity; however, therapeutic monoclonal antibodies may also function by recruiting other elements of the immune system to malignant cells. At sufﬁcient densities, IgG1 antibodies can activate compleDougan

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ment, leading to direct cytotoxicity through the formation of complement pores in the membrane of antibody-coated cells. Some evidence indicates that complement-dependent cytotoxicity may contribute to the antitumor effects of rituximab (33). In addition to activating complement, antibodies can activate cells of the innate immune system through binding to fragment c receptors (FcRs). Ligation of activating FcRs (FcγRI, FcγRIIA, FcγRIIC, and FcγRIII) on neutrophils, monocytes, and natural killer (NK) cells can lead to antibody-dependent cellular cytotoxicity (ADCC). Antibody-sensitive tumors grown in mice that lack FcγRs become resistant to both rituximab and trastuzumab, suggesting that recognition of IgG1 by FcγRs on innate immune cells contributes signiﬁcantly to the antitumor activity of these antibodies (34). Although suggestive, the importance of ADCC in monoclonal antibody activity was not supported by clinical data until more recently. FcR polymorphisms that enhance monocyte and NK cell recognition of antibody-coated tumors are highly correlated with the clinical response to rituximab, cetuximab, and trastuzumab (35–37). One year following rituximab treatment, lymphoma patients homozygous for FCGR3A-158V, encoding the highafﬁnity FcγRIIIA, had a 90% objective response rate, compared with a 67% objective response rate for patients carrying the lowafﬁnity FCGR3A-158F polymorphism (35). Similarly, breast cancer patients homozygous for FCGR3A-158V had signiﬁcantly higher objective response rates following trastuzumab treatment compared with patients carrying FCGR3A-158F (82% versus 40%), and this higher response rate was associated with signiﬁcantly longer progression-free survival (36). A smaller study examining cetuximab treatment in colorectal cancer found that patients with either of the high-afﬁnity polymorphisms, FCGR3A-158V or FCGR3A-131R, had a median progression-free survival of 3.7 months compared with 1.1 months for patients who carried neither high-afﬁnity polymorphism (37). In addition to monoclonal antibody therapy,

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vaccination against the speciﬁc B cell receptors carried by lymphoma cells (anti-idiotype vaccines) also show increased efﬁcacy in association with high-afﬁnity FcγR polymorphisms (38). These ﬁndings implicate cells of the innate immune system in the clinical response to monoclonal antibodies. Furthermore, these ﬁndings underscore the ability of innate immune cells, when appropriately targeted, to have powerful antitumor effects. Given the capacity of immune cells to inhibit tumor growth, one of the principal goals of tumor immune therapy is to develop novel strategies for targeting immune cells toward malignant tissues.

Immune Adjuvants Cancer cells often express a variety of abnormal proteins that can serve as targets for an immune response (antigens). Although spontaneous immune responses to these antigens can occur, these reactions are rarely sufﬁcient to cause tumor regression; however, the local administration of immune-activating agents (adjuvants) can induce tumor-associated inﬂammation and protective immunity. In general, immune adjuvant–based therapies have only proven effective against early stage tumors; yet in this context they can be remarkably effective with minimal risk of serious adverse reactions. The standard of care for superﬁcial bladder cancer is surgical removal of the tumor followed by immune therapy with an intravesicular injection of live bacilli Calmette-Gu´erin (BCG). In most patients, BCG provokes a local, selflimiting inﬂammatory reaction in the bladder wall (39, 40). Therapy is associated with increased urinary frequency, hematuria, cystitis, and fever, but it is generally well tolerated and carries a very low risk of disseminated infection (40). Several clinical trials have shown that, when combined with surgery, immune therapy with BCG is more effective than conventional chemotherapy (40–42). In one trial with a tenyear follow-up, surgery and BCG combination therapy was associated with progression-free survival in 61.9% of patients, compared with 37% of patients who received surgery alone

(41). Although the precise mechanism of BCG action is not known, the degree of local immune activation correlates with disease efﬁcacy, implicating immune activation by BCG rather than direct antitumor activity (39). Microbes often elicit immune responses by activating pattern-recognition receptors such as members of the Toll-like receptor (TLR) family. Puriﬁed TLR ligands have been evaluated as immune adjuvants and have shown considerable activity in preclinical models. The TLR7 agonist imiquimod was initially approved for the treatment of external warts caused by human papilloma virus (HPV) infection; however, imiquimod has also demonstrated efﬁcacy against low-grade epithelial tumors and precancerous lesions (43, 44). Imiquimod is approved for the treatment of basal cell carcinoma, as well as actinic keratosis, the precursor lesion of cutaneous squamous cell carcinoma (43, 44). More recently, imiquimod has been evaluated in the nonsurgical treatment of vulvar intraepithelial neoplasia (VIN) (45). VIN, like cervical intraepithelial neoplasia (CIN), is typically associated with HPV 16 and 18 infection and can be managed surgically (45, 46); however, surgical treatment can be disﬁguring, making less invasive treatment strategies worthwhile. Topical treatment of grade 2 and grade 3 VIN with imiquimod led to a 25% reduction in lesion size in 21 of the 26 treated patients, with 9 complete responses. In contrast, none of the lesions regressed in the placebotreated group (45). As a result of these ﬁndings, immune adjuvant therapy with imiquimod is likely to become part of the standard treatment for VIN, and imiquimod is currently under evaluation in other neoplasias associated with HPV infection.

VIN: vulvar intraepithelial neoplasia

Cytokines Cytokines, secreted proteins with immunemodulating properties, can be delivered systemically to activate antitumor immunity. Although response rates are low, both the cytokines IL-2 and interferon (IFN)-α have been used to treat advanced melanoma and renal cell www.annualreviews.org • Immune Therapy for Cancer

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carcinoma (RCC), tumors that are generally refractory to standard chemotherapy (47–50). IFN-α is an important mediator in antiviral immunity, and IL-2 is a potent T cell growth factor, although how these functions contribute to the pharmacologic effect of these cytokines is at present unclear. Work in animal models suggests that IFN-α may play a role in antitumor immunity, and clinical responses to IFN-α are associated with therapy-induced autoimmunity, linking the effectiveness of IFN-α to an induction of an immune response (51–53). The side effects of cytokine administration are severe and often dose limiting. Typically, cytokines induce symptoms that mirror those of systemic infection, including hypotension, vomiting, diarrhea, fever, and malaise (47, 49). Despite the limitations of cytokine therapy, both IL-2 and IFN-α can induce durable responses in a subset of patients with melanoma. IFN-α is most effective prior to distant metastasis (stage III disease); in this setting, IFN-α has a 16% overall response rate and a 5% complete response rate (49). Unlike IFN-α, IL-2 can induce responses in patients with metastatic melanoma, although the overall response rate (16%) and the complete response rate (6%) are similarly low (47). In addition to treatment of melanoma, both IFN-α and IL-2 therapy have been approved for the treatment of advanced RCC (48, 50). The importance of these cytokines in advanced RCC treatment has recently been reduced owing to newer therapeutics such as inhibitors of the mammalian target of rapamycin (mTOR) pathway and anti-VEGF treatments, both of which have fewer side effects and similar efﬁcacy to IFN-α and IL-2 (54). In contrast to systemic cytokine therapy used primarily for immune modulation, local administration of the cytokine tumor necrosis factor (TNF)-α has been used to treat soft tissue sarcomas (STSs) of the limb and melanoma, making use of the toxic effects on both tumor cells and the tumor vasculature that are mediated by this cytokine (55, 56). In addition to its antitumor activity, TNF-α is the primary medi-

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RCC: renal cell carcinoma

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ator in septic shock; as a consequence, systemic release of TNF-α can be life threatening. To prevent life-threatening hypotension, TNF-α must be delivered locally using isolated limb perfusion. Isolated limb perfusion is achieved by surgically ligating the main artery and vein of the affected limb to create a bypass circuit; a cardiac bypass pump and oxygenator can then maintain limb perfusion, enabling isolated release of TNF-α without a substantial risk of escape into the systemic circulation. Although many limb STSs can be removed surgically, severe disﬁguration and amputation are often necessary in recurrent or locally advanced disease (55, 56). Using isolated limb perfusion to deliver TNF-α in combination with the alkylating agent melphalan leads to a high response rate (70–85%) with a relatively high chance of limb preservation in patients who otherwise would require amputation to prevent disease progression (55, 56).

Supportive Therapy Many forms of conventional chemotherapy have dose-limiting toxic effects on the bone marrow, including effects on cells of the immune system. Immune toxicity, in particular neutropenia, can lead to substantial morbidity and mortality. Because neutrophils are central effectors in most antibacterial responses, loss of neutrophils can predispose cancer patients to life-threatening bacterial sepsis. Supportive therapy aimed at rescuing immune cells is thus a critical component of many chemotherapy regimens for both solid and hematopoietic tumors, as well as many bone marrow transplantation protocols (57, 58). To prevent neutropenia, many high-dose chemotherapeutic regimens are followed with an infusion of recombinant granulocyte (G) or granulocyte-macrophage (GM) colony stimulating factor (CSF) (57, 58). Both G-CSF and GM-CSF act on the bone marrow to increase neutrophil production, reducing the risk of febrile neutropenia, infection-related mortality, and early mortality by more than 40% (57).

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Evidence favoring a particular CSF is minimal; however, a single head-to-head trial found that G-CSF led to more rapid neutrophil recovery, although this kinetic difference was not associated with a clinically meaningful risk reduction (59). Bone marrow rescue is also a routine component of high-dose methotrexate (MTX) regimens (60). MTX, which inhibits dihydrofolate reductase (DHFR), a critical enzyme in the folic acid pathway required for thymidine synthesis, is used to treat a variety of tumors, including acute lymphoblastic leukemia (ALL), NHL, and breast cancer (60). High-dose therapy with MTX can block the replication of normal cells, leading to substantial toxicity in both gut epithelial cells and the bone marrow. This toxic effect can be mitigated by following MTX with an activated folic acid derivative (leucovorin), which bypasses DHFR and allows thymidine synthesis to continue. For reasons that are, at present, unclear, leucovorin tends to have a greater effect on nonmalignant cells compared with malignant ones, enabling leucovorin rescue to prevent bone marrow and gut epithelial cell loss without compromising the antitumor activity of MTX (60).

Prophylactic Immune Therapy A number of cancers are caused by microbial infections, either directly or through the induction of chronic inﬂammation (61). As a result, therapies aimed at eradicating or preventing these infections act prophylactically against their associated tumors. Because microbes carry many conserved structures that can be recognized as foreign by the immune system, classic vaccination strategies to induce protective immunity can be used without having to overcome the signiﬁcant barriers inherent in targeting sporadic tumors. The hepatitis B virus (HBV) vaccine was the ﬁrst available vaccine that provided protection against an infection with known oncogenic potential. HBV infections of the liver can lead to chronic hepatitis, which can, in turn, predispose people to the de-

velopment of hepatocellular carcinoma (HCC) (61). Although the HBV vaccine induces protective antibody titers in 95% of vaccinated individuals, the large population studies necessary to demonstrate a vaccine-induced reduction in adult HCC have not been performed (62). In Taiwan, where HBV is endemic, however, vaccination has been associated with a drop in HCC incidence among children, especially boys, with HCC cases falling from 1.08 per 100,000 between 1981 and 1984 to 0.49 per 100,000 between 1990 and 1996, establishing the principle of antitumor efﬁcacy (63). More recently, a vaccine against HPV 16 and 18 has been developed speciﬁcally to prevent cervical cancer. Worldwide, cervical cancers cause more than 250,000 deaths per year, and HPV 16 and 18 are associated with 70% of these tumors, in addition to tumors of the vagina and vulva (64–66). Other strains of HPV cause genital warts, and two of these strains (HPV 6 and 11) are also included in the most widely used vaccine (64). Vaccination of girls against HPV induces potent antiviral immunity that prevents viral acquisition, leading to 98% efﬁcacy against HPV 16– and 18–associated CIN and cervical carcinoma (64). In addition to cervical and other genital epithelial tumors in women, both HPV 16 and 18 are associated with penile, perineal, and perianal cancer, as well as tumors of the head and neck; trials are currently underway to evaluate the HPV 16 and 18 vaccine as prophylactic therapy for these cancers as well (67, 68). Like viruses, bacteria, including the bacteria Helicobacter pylori, have also been associated with tumor development. H. pylori is the primary cause of stomach cancer, the second most common cause of cancer-related death in the world, and is also an important cause of mucosal-associated lymphoid tissue (MALT) lymphomas (66, 69). Although infection of the gastric mucosa with H. pylori is typically asymptomatic, these infections can lead to chronic gastritis and the development of gastric ulcers; in rare individuals, H. pylori–associated chronic inﬂammation can then precipitate tumor

www.annualreviews.org • Immune Therapy for Cancer

MTX: methotrexate ALL: acute lymphoblastic leukemia

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FAP: familial adenomatous polyposis NSAID: nonsteroidal anti-inﬂammatory drug GVL: graft-versusleukemia CML: chronic myelocytic leukemia

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formation (61, 69). H. pylori colonization can be cleared with appropriate antibiotics, and evidence suggests that treatment of H. pylori prior to alterations in the gastric mucosa can decrease the risk of stomach cancer (70). In contrast, after the development of precancerous mucosal changes, the risk of tumor formation no longer depends on the continued presence of H. pylori (70). The situation with MALT lymphomas is somewhat different. Many cases of MALT lymphoma are treatable with antibiotics even after the development of cancer, and resistance to antibiotic therapy is associated with genomic alterations in the tumor cells (71, 72). A strong correlation exists between chronic inﬂammation of the colon and colorectal cancer, even in the absence of a clear infectious cause. This inﬂammation has been directly implicated in the development of colorectal cancer owing to the efﬁcacy of several antiinﬂammatory treatments in reducing cancer risk (73–78). The efﬁcacy of anti-inﬂammatory therapy is most apparent in patients at high risk for the development of colorectal cancer. Treatment of patients with ulcerative colitis, a severe inﬂammatory disease of the colon, with the anti-inﬂammatory drug 5-aminosalicylic acid (5-ASA) is associated with a 50% decrease in the odds of colorectal cancer development (73). Similarly, anti-inﬂammatory COX-2 inhibitors are approved for reducing precancerous polyp formation in patients with the genetic colon cancer syndrome familial adenomatous polyposis (FAP). Although no anti-inﬂammatory agents have been approved for the treatment of sporadic colorectal cancer, the use of nonsteroidal anti-inﬂammatory drugs (NSAIDs) like aspirin is associated with an 18–39% decrease in the risk of sporadic colorectal tumors, although side effects mitigate the overall beneﬁt of this approach in a relatively low-risk population (76, 77).

Bone Marrow Transplantation Many hematologic malignancies are treated with bone marrow ablative therapy, which is then followed by bone marrow transplantation 4.8

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from a healthy donor. Although bone marrow transplantation has a very high response rate, in a large number of hematologic tumors relapses following transplantation are still fairly common (79, 80). Intriguingly, for many tumors the risk of relapse is substantially higher for patients who receive a syngeneic transplant (for example, from an identical twin) than for patients who receive allogeneic transplants, indicating a graft-versus-leukemia (GVL) effect associated with allogeneic transplantation (79). In ALL, the risk of relapse is 36% with a syngeneic transplant, compared with 26% with an allogeneic transplant; in AML the difference in relapse rates is 52% compared with 16%; and in chronic myelocytic leukemia (CML) the difference is 40% compared with 7% (79). Allogeneic transplant recipients also run the risk of developing graft-versus-host disease (GVHD), a potentially lethal syndrome characterized by a transplant-mediated immune assault on the recipient’s tissues. The severity of GVHD is inversely correlated with the risk of tumor relapse, suggesting that similar immune mechanisms are responsible both for GVHD and for the GVL effect (80). The GVL effect is believed to be mediated by a combination of cytotoxic T cells and NK cells, and more recent work has begun to elucidate the speciﬁc targets of the GVL response in tumor cells (81, 82). Fully mismatched major histocompatibility complex (MHC) bone marrow transplantation is now possible using nonmyeloablative treatment regimens. These nonmyeloablative conditions lead to bone marrow chimerism, with hematopoetic cells deriving from both the donor and the recipient (83). As might be expected, given the proposed GVL mechanism, nonmyeloablative transplants may reduce the risk of relapse, especially in AML. In a series of 20 patients transplanted using a nonmyeloablative protocol, only two developed relapsed disease (83). Perhaps the clearest evidence indicating an important role for donor immune cells in preventing post-transplantation relapses comes from the success of donor leukocyte infusion (DLI) (84, 85). In DLI, leukocytes harvested

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from the peripheral blood of transplant donors are infused back into transplant recipients following disease relapse. The rationale behind DLI is to provide potent antitumor immune cells to bring the expanding tumor back under immune control. This immune therapeutic approach has been remarkably successful in CML, leading to complete remission of relapsed disease in 70% of infused patients (84). DLI is also effective in relapsed ALL, AML, and multiple myeloma, although complete remissions are far less common (15–29%) (84). Because of its success, immune therapy with DLI is now considered a standard treatment for relapsed leukemia following bone marrow transplantation.

THE INTERACTION BETWEEN THE IMMUNE SYSTEM AND CANCER The immune system interacts with tumors throughout their development, and the consequences of this interaction have substantial implications for cancer therapy (Figure 1). Although some host responses may inhibit tumor growth and progression, the immune system can also promote cancer by provoking chronic inﬂammation and, in turn, elaborating factors that drive tumor growth, survival, and angiogenesis. Failure of immunity can predispose a person to tumor development, particularly in the context of oncogenic viruses.

NK cells

Microbial products?

MyD88

Endogenous adjuvant receptors

NKG2D ligands

Microbial receptors

Myeloid cells

Inﬂammatory cytokines DNA damage

TNF-α, IL-1β, IL-6

NF-κB STAT3

NF-κB

IL-23?

CD4

IL-17 Angiogenesis

Tumor

Proliferation Survival Growth

IFN-γ Tumor necrosis?

Tumor apoptosis (MFG-E8)

Tregs

CD4

CD8

T cells

Immunosuppressive cytokines (TGF-β, IL-10) Negative costimulation (PD-L1) IDO

Figure 1 Complex interactions with the immune system shape tumor development. Chronic inﬂammatory response can be initiated by microbial products or endogenous adjuvants released from necrotic cells. These signals activate nuclear factor-κB (NF-κB) in myeloid cells, leading to the production of inﬂammatory cytokines (TNF-α, IL-1β, and IL-6), which in turn activate NF-κB in the tumor. Tumor-intrinsic NF-κB activation promotes growth, survival, and proliferation. IL-23 produced by myeloid cells can promote the generation of IL-17-secreting T cells, which can further support tumor growth. Genotoxic stress in tumor cells can activate NK cell ligands, which can synergize with endogenous tumor-speciﬁc CD4+ and CD8+ T cells that produce IFN-γ and restrict tumor development. Tumors can suppress nascent immune responses through a variety of mechanisms, including immunosuppressive cytokines (TGF-β and IL-10) and metabolites [indoleamine 2,3-dioxygenase (IDO)] and the expression of negative costimulatory molecules [programmed death ligand 1 (PD-L1)]. Tumors can also promote Treg recruitment and differentiation, in part through the recognition of apoptotic cells by the MFG-E8 (milk fat globule epidermal growth factor 8) pathway. www.annualreviews.org • Immune Therapy for Cancer

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Established tumors use immune-regulatory circuits to generate an immune-suppressive environment, which can act as a substantial barrier to the induction of therapeutic antitumor immunity. Substantial progress in our understanding of the molecular mechanisms governing the interaction between tumors and the immune system has been the basis for multiple investigational immune therapies for cancer, and these fundamental advances continue to provide insights with potential applications in novel treatments.

Infections and Inflammation Several infectious microorganisms are strongly linked to tumor development yet do not appear to encode oncogenic proteins (61, 86). Both HBV and HCV are highly associated with liver cancer; similarly, schistosomal infections are associated with tumors of the bladder and colon. The bacterial species Helicobacter pylori is linked to most cancers of the stomach, as well as to MALT lymphomas. In each of these cases, the pathogen establishes a chronic infection characterized by continuous inﬂammation and failure to achieve immune clearance; the chronic inﬂammation itself appears to cause the associated cancers. In fact, even in the absence of obvious infection, chronic inﬂammation can provoke tumor formation. Several autoimmune diseases increase the risk of B cell lymphoma, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren’s syndrome (87). Similarly, both ulcerative colitis and Crohn’s disease, inﬂammatory diseases of the gastrointestinal track, increase the risk of colorectal cancer (88). As mentioned above, evidence from NSAID use suggests that the role of inﬂammation in cancer may extend beyond tumors that arise in the context of frank inﬂammatory disease. NSAID use is associated with a decreased incidence of sporadic colon cancer, and COX-2 inhibitors decrease the risk of tumor formation in patients with FAP (74–77). Inﬂammation has also been linked to lung cancer: Both cigarette smoking and asbestos inhalation lead 4.10

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to pulmonary inﬂammation, and NSAIDs can decrease the risk of lung cancer (61, 89). Over the past few years, signiﬁcant progress has been made toward elucidating the molecular mechanisms linking inﬂammation to cancer. The proinﬂammatory cytokine TNF-α plays an essential, early role in several cancer models, as well as in chronic inﬂammatory diseases of both mice and humans (61, 86). Consistent with the importance of TNF-α in human cancer, increased levels of TNF-α have been linked to multiple myeloma, HCC, breast cancer, bladder cancer, and gastric cancer (61, 86). TNF-α, largely produced by cells of the innate immune system such as macrophages and mast cells, can have myriad effects in the tumor microenvironment. TNF-α promotes cell growth and survival as well as angiogenesis and the recruitment of immune effector cells. Although the events downstream of TNF-α that are critical for tumor development are not all known, nuclear factor-κB (NF-κB) family transcription factors appear to play a signiﬁcant role in linking TNF-α to cancer (61, 86). NF-κB controls the transcription of a number of proteins involved in cell survival, division, and growth; in addition, NF-κB is important for the production of many inﬂammatory cytokines and chemokines, including TNF-α itself. NFκB can function both tumor-intrinsically and -extrinsically to promote cancer development. In a mouse model of colon cancer, speciﬁc ablation of NF-κB signaling in immune cells led to reductions in tumor growth, whereas ablation in the colonic epithelium dramatically decreased tumor incidence (90). Depending on the system examined, other acute inﬂammatory cytokines can drive cancer development, including IL-6 and IL-1. IL-6 has been implicated in many of the same processes as TNF-α, acting both as a mitogen and as an angiogenic factor, primarily through activation of the transcription factor signal transducer and activator of transcription (STAT) 3 (91). IL-6 is a central mediator in mouse models of inﬂammatory liver and colon cancer, and IL6 has been implicated in an autocrine growth pathway downstream of EGFR in a subset of

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human lung cancers (61, 86, 92). IL-1 can also activate NF-κB and promote tumor formation in carcinogen-induced skin cancer models; in humans, polymorphisms in IL-1 have been linked to gastric cancer (86, 93, 94). Given their apparent importance, strategies to target acute inﬂammatory cytokines or downstream signaling molecules are under evaluation (95). Many tumor-promoting cytokines are produced largely by innate immune cells; however, cells of the adaptive immune system can also promote tumor formation. Overexpression of the cytokine IL-23 dramatically increases the risk of cancer in mouse models, and IL-23 levels are increased in a variety of human tumors (96). IL-23 functions to support the differentiation of a subset of T cells that preferentially produce IL-17, a cytokine that is overexpressed in several human cancers, including cervical cancer and NSCLC (61). IL-17 production can drive both TNF-α and IL-6 secretion from many different cell types, linking IL-17 to innate immunity and the acute inﬂammatory cytokines and suggesting that therapies targeting IL-17 or IL-23 could limit tumor-promoting inﬂammation (61). Despite substantial progress in identifying relevant inﬂammatory mediators, many of the early steps that initiate tumor-promoting inﬂammation are, at present, unknown. In several mouse models of cancer, inﬂammation is initiated by signals downstream of microbial pattern-recognition receptors. In these models, the signaling adaptor protein myeloid differentiation factor 88 (MyD88) plays a crucial role (97–99). MyD88 is an indispensable mediator of most TLR signaling pathways as well as IL-1 receptor signaling. Loss of MyD88 reduces the number of skin tumors formed in two different carcinogen-induced skin cancer models, as well as in a carcinogen-induced model of liver cancer (97, 98). In APCmin/+ mice, a model of human FAP, MyD88 deﬁciency reduces both the size and the frequency of tumors, as well as tumor-associated cytokine production (99). Even with MyD88 positioned upstream of the initiation of inﬂammation, the factors responsible for engaging MyD88 are still ob-

scure. TLRs can recognize a wide range of microbial products, potentially implicating occult infections or recognition of endogenous ﬂora in the onset of tumor-promoting inﬂammation. Alternatively, MyD88 may function to mediate sterile inﬂammation downstream of the IL-1 receptor, which would be consistent with the importance of IL-1 in the 7,12-dimethylbenz[a]anthracene, 12O-tetradecanoylphorbol-13-acetate (DMBA/ TPA) model of skin cancer (97). Other sterile inﬂammatory signals, such as those mediated by the receptor for advanced glycation endproducts (RAGE), have also been implicated in tumor formation (100). Because acute, self-limiting inﬂammation is generally insufﬁcient to induce tumor formation, defects in the normal mechanisms of immune regulation may be common features of tumor-promoting inﬂammation. In many cases, for example in the context of infection or autoimmunity, the speciﬁc factors preventing the resolution of inﬂammation are unknown; however, in several mouse models, defects in immune-regulatory proteins increase tumor susceptibility. In the dextran sulfate sodium model of colitis, loss of TIR8, a negative regulator of TLR and IL-1 receptor signaling, exacerbates cancer development (101). Similarly, loss of the secreted IL-1 antagonist, IL-1Ra, increases the growth rate of skin tumors in DMBA/TPA-treated mice (93). A more thorough understanding of how immuneregulatory pathways control cancer development may provide novel opportunities for therapeutic intervention.

Spontaneous Immunity Whereas chronic inﬂammation generally promotes tumor development, in some contexts, adaptive immunity can suppress tumor formation. The role of adaptive immunity in controlling tumor growth is most obvious in the case of cancers of viral origin; however, immunedeﬁcient states are also associated with a small increase in the risk of tumors that are not known to have infectious etiologies. www.annualreviews.org • Immune Therapy for Cancer

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HTLV: human T-lymphotropic virus

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HHV8: human herpes virus 8

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Oncogenic viruses. Several human viruses encode proteins that can directly contribute to tumor formation, including HPV, human T-lymphotropic virus (HTLV), EpsteinBarr virus (EBV), and human herpes virus 8 (HHV8/Kaposi sarcoma virus). Control of these viruses represents perhaps the most direct mechanisms by which the immune system can suppress cancer, as well as one of the most attractive targets for tumor immune therapy. As discussed earlier, a vaccine against HPV, the primary cause of cervical cancer and several other anogenital tumors, has become the ﬁrst vaccine against an oncogene-encoding virus, serving as a model for further prophylactic anticancer immune therapy (64, 68). Like human immunodeﬁciency virus (HIV), HTLV is a retrovirus that infects CD4+ T cells; however, in contrast to HIV, HTLV promotes T cell survival and proliferation (102). Infection with HTLV provokes strong antiviral cytotoxic T cell responses, yet these responses are typically unable to clear the infection (102). In a subset of patients, over the course of 20 to 30 years, persistent HTLV leads to the development of adult T cell leukemia/lymphoma (ATLL) (103); strategies to augment antiHTLV responses thus represent an attractive avenue for treating these tumors. The herpes virus EBV primarily infects B cells and is associated with Burkett’s lymphoma and nasopharyngeal carcinoma in a minority of infected individuals (104). EBV is one of the most prevalent infectious diseases: Greater than 90% of the world’s population has been infected, with the vast majority of infected individuals living as asymptomatic carriers. EBV lymphomas are more common in immunesuppressed individuals, implicating the immune system in the control of these tumors; furthermore, EBV lymphoproliferative disease following bone marrow transplant can be controlled through DLIs that contain EBV-reactive T cells (104, 105). Like EBV, HHV8 infections typically do not cause overt disease, and most HHV8-associated skin tumors occur in patients infected with HIV (106). In the context of HIV, HHV8-associated Kaposi sarcomas occur in paDougan

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tients with low CD4+ T cell counts, and effective antiretroviral therapy can inhibit their development, strongly implicating the immune system in the control of these tumors (106). Tumors of nonviral origin. Increasing evidence suggests that beneﬁcial immune responses to tumors can occur spontaneously in humans; however, these immune responses are unlikely to control tumor development in most patients. Spontaneous immune inﬁltrates are common in many cancers, often correlating with a favorable prognosis; this correlation is particularly strong for inﬁltrates composed of activated CD8+ T cells and memory CD45RO+ T cells (107, 108). Several types of cancer are associated with the development of distinct autoimmune syndromes. These syndromes are thought to occur following the induction of spontaneous antitumor responses, and cancer patients who have autoantibodies characteristic of these paraneoplastic syndromes in the absence of frank autoimmune disease have improved prognosis (51, 107). In addition to evidence of immune activation, the microenvironment of most established human cancers is generally immune suppressive; this suggests that overcoming immune rejection may be an important feature of early tumor growth, yet whether the development of such an environment poses a legitimate barrier for early tumors is unclear (109–111). Extensive work performed in experimental systems has elucidated some of the mechanisms underlying spontaneous antitumor immunity, and has formed the basis for the cancer immunoediting hypothesis. This hypothesis divides the immune response to cancer into three phases: a tumor destructive “elimination” phase, a stable “equilibrium phase,” and “escape” phase characterized by tumor progression. Immune responses that eliminate tumors or delay their growth involve the production of IFN-γ, as well as the generation of tumorreactive cytotoxic T cells (51, 107). Mice deﬁcient in IFN-γ are more susceptible to methylcholanthrene (MCA)-induced sarcomas, as are mice lacking T cells and mice unable to produce

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perforin, a key effector protein in T cell cytotoxicity (51, 107). Several strains of immunedeﬁcient mice, including mice lacking T cells and mice deﬁcient in IFN-γ, are also more susceptible to sporadic tumor development, although a role for microbial infections in these tumors cannot yet be excluded (51, 107). In addition to preventing tumor formation, the adaptive immune system may also be able to hold established tumors in check, leading to equilibrium between the host response and tumor growth. Recent studies in animals have identiﬁed a subset of mice that develop stable tumors in response to low-dose MCA treatment; these tumors can be induced to grow by transient immune suppression, establishing a role for protective immunity in restricting tumor growth (112). Clear evidence for a similar phenomenon in human cancer patients is lacking; however, several observations suggest that such immune control may occur. Rarely, patients develop recurrent malignancies decades after the removal of their primary tumor (51, 107). Similarly, tumors have been inadvertently transmitted along with an organ transplant taken from a donor who was apparently tumor free after having undergone cancer surgery years earlier (51, 107). Plasma cells from patients with the precursor lesion of multiple myeloma, monoclonal gammopathy of undetermined signiﬁcance (MGUS), express high levels of MHC class I chain–related gene A (MICA), which can be recognized by NKG2D on NK cells and stimulate the production of anti-MICA antibodies (113). The secretion of soluble MICA, which leads to downregulation of NKG2D, is associated with progression to multiple myeloma, linking tumor development to suppression of endogenous immunity (113). Although these observations are not deﬁnitive, establishing such immune control of tumor growth is one of the principal goals of cancer immune therapy.

Immunogenic Cell Death Cell death is a ubiquitous feature of developing tumors, which are often characterized by

disorganized growth and an inadequate blood supply. The speciﬁc mechanisms underlying tumor cell death can dramatically inﬂuence interactions with the immune system, having implications for the development of tumor immune therapies. Many malignant as well as nonmalignant cells die through apoptosis, a highly regulated cell death pathway that results in loss of membrane lipid polarity and exposure of phosphatidylserine (PS) on the outer leaﬂet of the plasma membrane (114). Exposed PS is recognized by at least three distinct pathways, each of which can lead to rapid phagocytosis of apoptotic cells by macrophages and dendritic cells (DCs). Phagocytosis of apoptotic debris promotes the production of anti-inﬂammatory cytokines and immune tolerance. By antagonizing these pathways, the immune-suppressive effect of apoptosis can be circumvented, potentially enhancing strategies to generate antitumor immunity. This approach has proved successful in an experimental therapeutic vaccine for melanoma, where antagonism of one of these pathways, mediated by the binding of milk fat globule epidermal growth factor 8 (MFG-E8) to PS and the subsequent binding of MFG-E8 to αv integrins, signiﬁcantly augmented vaccine efﬁcacy (115). In many cases, tumor cells do not undergo apoptosis and instead die through necrosis. In contrast to apoptosis, necrotic cell death appears to be immune stimulatory (116). The molecular mechanisms linking necrosis to immune activation are just beginning to be elucidated; however, they appear to rely on the aberrant exposure of speciﬁc cellular components, such as uric acid and the DNA-binding protein high-mobility group box 1 (HMG-B1), which are recognized by activating receptors on innate immune cells (116). Necrotic cell death may act to prime endogenous antitumor immune responses or may augment immune therapies. In addition, some evidence indicates that immunogenic cell death may be promoted by speciﬁc chemotherapeutic agents, such as the DNA-targeting anthracyclines, indicating the potential for more conventional cancer treatments to augment antitumor immunity (117). www.annualreviews.org • Immune Therapy for Cancer

MGUS: monoclonal gammopathy of undetermined signiﬁcance

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MDSC: myeloidderived suppressor cell IDO: indoleamine 2,3-dioxygenase

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Similarly, DNA damage caused by chemotherapy or defective repair machinery may increase the expression of stress ligands such as MICA that can be targeted by NK cells (118). By increasing the expression of NK cell ligands, tumors with genetic damage can be recognized by the innate immune system. Strategies to take advantage of these immunogenic death and damage pathways may synergize with immune therapy to generate more potent antitumor responses.

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Immune Suppression in the Tumor Microenvironment Immune suppression is a common feature of the tumor microenvironment and a substantial barrier to tumor immune therapy. The microenvironment of tumors is established through the activity of both myeloid and lymphoid regulatory cells, as well as through the production of immune-suppressive factors by malignant cells themselves. Many tumor-inﬁltrating macrophages have an immune-suppressive phenotype (109). These macrophages, referred to as myeloid-derived suppressor cells (MDSCs), are characterized by the expression of both CD11b and GR1 (109). MDSCs are abundant in many tumors arising in both humans and mice and can exert powerful anti-inﬂammatory effects. The anti-inﬂammatory activity of MDSCs is mediated at least in part through the production of two enzymes involved in arginine metabolism: arginase 1 and nitrous oxide synthase (NOS) (109). Both arginase 1 and NOS exert powerful suppressive effects on T cells, although whether these effects are mediated by altered T cell metabolism, increased production of hydrogen peroxide, or other as yet undeﬁned mechanisms is not clear (109). In addition to MDSC, regulatory T cells (Tregs) also heavily inﬁltrate many tumors (110). These cells, characterized by the expression of the transcription factor FoxP3 as well as CD4 and CD25, play a key role in the regulation of adaptive immunity. Tregs can suppress immune responses through the secretion 4.14

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of suppressive cytokines like TGF-β and IL35 as well as through a poorly understood, contact-dependent mechanism (110, 119). The presence of large Treg inﬁltrates is correlated with poor prognosis in several types of cancer, including cancers of the ovary, breast, and liver (110). As is discussed below, Tregs are a potential barrier to developing productive immune therapies for cancer, and they represent an attractive target for enhancing antitumor immunity. The elaboration of immune-suppressive factors by tumor cells represents yet another powerful barrier to the generation of antitumor immunity. Tumor cells often secrete immunesuppressive cytokines such as IL-10, TGF-β, and VEGF (110). These factors not only inhibit cytotoxic immune responses but may also promote the formation or recruitment of additional regulatory cells (110). Many tumors express the immune-suppressive enzyme indoleamine 2,3dioxygenase (IDO), an enzyme that is involved in tryptophan metabolism (111). IDO appears to exert its suppressive effect through the depletion of tryptophan and the production of antiinﬂammatory tryptophan metabolites (111). In addition to immune-suppressive soluble mediators, many tumors express surface receptors that can inhibit T cell activation, such as programmed death ligand 1 (PD-L1), which may play an important role in downmodulating antitumor T cell responses (120). The possibility of blocking the PD-L1 pathway as a mechanism for enhancing tumor immunity is under active investigation, as is discussed below.

NOVEL APPROACHES TO IMMUNE THERAPY A variety of novel strategies for eliciting protective antitumor immune responses are under development. Several of these strategies attempt to target novel pathways identiﬁed through basic research. Although tumor-reactive cells are present in many experimental and therapeutic settings, a large number of regulatory pathways appear to prevent these reactive cells from generating productive antitumor responses. As

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a result, many novel immune therapies for cancer attempt to circumvent these regulatory pathways. Immune suppression can be inhibited by antagonizing regulatory molecules or by directly targeting regulatory cells. Alternatively, T cell costimulation can be augmented to reverse the effects of immune suppression. The antigenic targets and frequency of spontaneous, tumor-reactive cells may not be sufﬁcient for inhibiting tumor growth; consequently, a variety of therapeutic strategies seek to increase the number of tumor-reactive cells either through vaccination or ex vivo expansion. In addition to novel therapeutics, immune-modulating agents developed to treat nonmalignant conditions are now being tested for efﬁcacy in cancer. Similarly, immune-modulating cancer therapeutics have the potential to become important treatments for nonmalignant conditions, including autoimmunity. The frequency of such therapeutic crossovers should only increase as we expand our understanding of the molecular interactions between tumors and the immune system.

Second-Generation Monoclonal Antibodies Impressive clinical responses to monoclonal antibody therapy have already been achieved, yet many tumors remain largely refractory to approved antibody therapies. Second-generation monoclonal antibodies, addressing some of the limitations of current antibodies, are under development to enhance antitumor efﬁcacy. The high molecular weight of the four-chain antibody structure can inhibit diffusion, potentially contributing to weak responses in solid tumors. By removing the antibody Fc region to generate F(ab )2 fragments, diffusion of antibodies into tumors can be signiﬁcantly improved (121). Unfortunately, many important antibody functions are mediated by the Fc region. As a result, such F(ab )2 fragments are likely to be most useful as targeting mechanisms for antibody-conjugated cytotoxic therapy. Monoclonal antibodies have been modiﬁed to alter serum half-life, either to extend the

period over which the antibodies exert their biology effects or to accelerate the clearance of toxin-conjugated antibodies (122, 123). The neonatal FcR (FcRn) is largely responsible for determining the serum half-life of antibodies; by introducing mutations that enhance or diminish FcRn binding, antibody half-life can be extended or reduced (122, 123). Recent evidence, both from animal models and from clinical trials, supports an important role for the innate immune system in the antitumor activity of therapeutic monoclonal antibodies. In particular, the mechanism underlying monoclonal antibody–mediated killing appears to involve the activation of complement as well as the ability to direct monocyte and NK cell cytotoxicity. In addition, antibodies can assist DCs in the acquisition of tumorassociated antigen and in the presentation of these antigens to T cells, thus linking monoclonal antibody therapy to the induction of adaptive immunity (124, 125). Many strategies to improve the efﬁcacy of monoclonal antibodies seek to enhance antibody-mediated immune activation. ADCC can be augmented through modiﬁcation of the antibody Fc region to produce a more favorable binding proﬁle for the FcRs expressed on monocytes and NK cells (34, 126– 128). These modiﬁcations include mutations in the amino acid structure of the Fc region as well as alterations in the Fc glycosylation pattern (34, 126–128). A triple alanine substitution mutant trastuzumab (S298A/E333A/K334A) has signiﬁcantly improved binding to FcγRIIIA, the principal activating FcR on monocytes and NK cells; consistent with improved binding, this substituted trastuzumab has a superior ability to activate ADCC in vitro (126). Loss of fucosylation on antibody N-linked glycosyl groups has a similar effect on FcγRIIIA binding, again leading to improved activation of ADCC (127). Interestingly, loss of fucosylation appears to maximize ADCC, and the additional amino acid substitutions may be unable to enhance ADCC further (128). Antibodymediated killing can also be enhanced by decreasing binding to the inhibitory FcγR, www.annualreviews.org • Immune Therapy for Cancer

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FcγRIIB, implicating both positive and negative signals in the response to antibody therapy (34). Like ADCC, complement activation may play an important role in the antitumor activity of monoclonal antibodies. Several antibody isotypes, including IgM, IgG1, and IgG3, are capable of ﬁxing complement through the binding of complement factor C1q and the subsequent activation of the C1r/s proteases. Although IgM pentamers are the most efﬁcient complement-ﬁxing antibodies, these multimeric structures are too bulky to make useful therapeutics; as a result, strategies for enhancing therapeutic complement activation have focused on modifying IgG1 to increase its binding afﬁnity for C1q (129). Several mutations in the IgG1 Fc region enhance C1q binding and complement activation; however, some of these complement-activating mutants also negatively affect ADCC, narrowing the range of potentially useful modiﬁcations. The double alanine substitution K326A/E333A (which overlaps at E333 with the ADCC-activating triple mutant) enhances complement activation by rituximab without negatively affecting ADCC (129). In an experimental system, complement activation has also been achieved through the direct inhibition of complement regulatory proteins using a chimeric, bi-speciﬁc antibody capable of both tumor binding and complement inhibition (130).

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Novel Immune Adjuvants Immune adjuvants have already proven useful in the treatment of a range of early stage tumors. Unfortunately, neither the TLR7 agonist imiquimod nor BCG, the two immune adjuvants currently approved for cancer therapy, are suitable for systemic delivery. As a result, current research has focused on identifying systemically active immune adjuvants which could be used to treat a wider range of tumors. TLR9 agonists. Unlike imiquimod, agonists of TLR9 can activate productive immune responses when delivered into the circulation 4.16

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(131). Similar to TLR7, TLR9 is part of the recognition system for microbial nucleic acids. TLR9 is involved in transducing signals from unmethylated CpG DNA, acting to skew T cell responses toward the production of IFN-γ, a critical cytokine mediator of tumor immunity (132). In humans, TLR9 is expressed on only a subset of cells, including plasmacytoid dendritic cells (pDCs) and B cells (132, 133). A variety of synthetic TLR9 agonists have been developed, several of which are undergoing clinical testing in a wide range of tumors (131, 134–139). Intratumor injection of the TLR9 agonist PF-3512676 is being tested in phase I/II trials as monotherapy for both basal cell carcinoma and metastatic melanoma (134, 135). Systemic PF-3512676 is under evaluation in phase I trials as monotherapy for cutaneous T cell lymphoma (CTCL) and following rituximab therapy for NHL; phase II trials have also begun to examine PF-351267 combination therapy with taxanes for NSCLC (136– 138). In each of these ongoing trials, TLR9 agonist treatment has been associated with the induction of immune reactions and some evidence of antitumor activity, although complete responses have been rare (134–138). α-galactosylceramide. α-galactosylceramide (α-galcer, KRN7000), a lipid derived from marine sponges, was originally isolated more than a decade ago in a screen for biological molecules with anticancer activity (140, 141). α-galcer is a speciﬁc agonist for a subset of rapidly activated T cells known as NKT cells. Unlike most T cells, which recognize peptide bound to MHC molecules, NKT cells respond to lipid antigens displayed by the MHC class I homolog CD1d (141). Most NKT cells can recognize α-galcer bound to CD1d and respond within several hours by producing cytokines; as a result, α-galcer is now often used as a pan-NKT cell agonist (141). NKT cells may play a critical role in skewing immune responses toward the production of speciﬁc effector cytokines, including IFN-γ (141). The administration of α-galcer can have potent antitumor effects in a wide range of murine

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cancer models, suggesting that NKT cells may play a broad role in antitumor immunity (141). Additional α-galcer derivatives are also under investigation in an attempt to generate still more potent antitumor activity (142). Several phase I clinical trials have investigated the potential for using NKT cell activation through α-galcer as a cancer treatment (143–147). The intravenous administration of α-galcer is well tolerated in cancer patients and leads to measurable immune activation; however, total NKT cell numbers vary dramatically across individuals, and responses are only substantial in patients who have high baseline circulating NKT cells (143). DCs loaded with α-galcer have also been used in early clinical trials, where they have been associated with signs of immune activation and NKT cell expansion (144, 145, 147, 148). Because many malignancies are associated with an apparent reduction in NKT cells, the dependence of α-galcer therapy on baseline NKT cell numbers may pose an obstacle to in vivo activation therapies (149). In a recent attempt to circumvent this limitation, autologous NKT cells from patients with NSCLC were cultured and activated ex vivo and then reinfused into cancer patients (146). Although objective responses were not observed, this strategy did boost NKT cell numbers in most patients, despite the relatively low purity of the transfused NKT cell product (146).

Immune-Modulating Antibodies A large number of novel monoclonal antibodies with immune-modulating activity are under development for the treatment of cancer. Several of these antibodies directly antagonize negative regulatory circuits that are thought to be important in limiting antitumor responses; similarly, agonistic antibodies that activate T cell coreceptors are being developed to drive cytotoxic T cell responses. Negative regulatory receptors. The most clinically advanced immune-modulating antibodies block cytotoxic T lymphocyte antigen

(CTLA)-4, an important negative immuneregulatory receptor expressed on a variety of immune cells, including activated T cells and Tregs (150). In addition to sending its own negative signals, CTLA-4 binds to B7-1 and B72 with substantially higher afﬁnity and avidity than does CD28, enabling CTLA-4 to effectively outcompete CD28 (150). In the absence of CTLA-4, mice develop a lethal multiorgan inﬂammatory disease, underscoring the central importance of CTLA-4 in immune homeostasis (151, 152). A large number of studies in animal models have demonstrated enhanced antitumor activity following CTLA-4 blockade, particularly when used in conjunction with other tumor vaccination strategies (150, 153). Two CTLA-4-blocking antibodies (ipilimumab and tremelimumab) with afﬁnities <1 nM have entered clinical testing, predominantly in advanced melanoma (153–155). A series of phase I and phase II trials have shown that both antibodies have clinical activity, with objective response rates ranging from 5% to 15%, including some complete responses. Based on these early studies, advanced registration trials for these antibodies are currently underway (155). In addition to single-agent trials, some of these early trials have combined anti-CTLA-4 therapy with other immune-modulating therapy, and these combination regimens appear to enhance efﬁcacy (155). CTLA-4 blockade has been associated with grade 3 and grade 4 autoimmune toxicities, establishing the importance of this negative regulatory pathway in maintaining normal immune homeostasis in addition to suppressing antitumor immunity (153–155). Treatmentassociated colitis has been the most signiﬁcant autoimmune reaction following CTLA-4 antibody administration; however, many other autoimmune reactions have been observed, including dermatitis (in the case of melanoma), hypophysitis, and pulmonary alveolitis (153– 155). The precise mechanism of action of CTLA4-blocking therapy is still incompletely understood. In particular, as mentioned above, CTLA-4 is expressed on both effector and www.annualreviews.org • Immune Therapy for Cancer

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Tregs. The relative importance of activity against these two cell types is unclear, with some reports suggesting a role for CTLA-4 on Tregs in immune suppression, and others ﬁnding evidence of increased Treg numbers in lymph nodes following CTLA-4 blockade (154). The negative immune-regulatory receptor PD-1 is also under evaluation as an antibody target for cancer immune therapy. Similar to CTLA-4, PD-1 is an inhibitory receptor expressed on activated T cells, as well as on B cells and monocytes (156). PD-1 has two principal ligands, PD-L1 and PD-L2 (156). PD-L2 is expressed on antigen-presenting cells, whereas PD-L1 is expressed broadly, both within the immune system and on nonimmune tissues (156). PD-L1 expression can be upregulated by both IFN-α/β and IFN-γ, leading to high expression in a variety of inﬂamed tissues (156). Similarly, PD-L1 is often highly expressed on tumors, and expression of PD-1 is correlated with an unfavorable prognosis in cancers of lung, kidney, pancreas, and ovaries (157–161). These ﬁndings suggest a role for PD-L1 in limiting T cell responses within the tumor microenvironment. Consistent with the importance of the PD1/PD-L1 axis in tumor development, in animal models inhibition of PD-1, either through genetic ablation or with blocking antibodies, can improve antitumor immunity (156, 162). A fully human PD-1-blocking monoclonal antibody has recently been developed that is capable of augmenting T cell responses in vitro (161). A phase I trial of the PD-1-blocking antibody CT-011 was recently completed in 17 patients with various hematologic malignancies (163). In this patient population, PD-1 blockade was safe and associated with a complete response in one patient with NHL (163).

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Immune-suppressive cytokines. Tumors and tumor-associated immune cells can produce a range of cytokines with the ability to suppress tumor immunity. The antiinﬂammatory cytokines IL-10 and TGF-β are among the most important immunesuppressive cytokines produced by tumors, and 4.18

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strategies to reverse this immune suppression represent an attractive target for immune therapy (164–168). Targeting TGF-β can substantially augment antitumor immunity in animal models (165, 166), suggesting that analogous strategies in humans may show clinical efﬁcacy. Anti-TGF-β monoclonal antibodies have been safely administered in clinical trials for systemic sclerosis (CAT-192) and for corneal scarring (CAT-152/lerdelimumab) (169, 170), opening up the possibility for clinical investigation in cancer patients. Similarly, a monoclonal antibody against IL-10 has been developed for testing in SLE (171). Blocking IL-10 may be particularly useful in B cell lymphomas, where, in addition to suppressing antitumor immunity, IL-10 can induce expression of B cell growth factors (168). Inﬂammatory cytokines are also under investigation as immune therapy targets. Antibodies against TNF-α have recently been evaluated in clinical trials for RCC, where treatment was associated with partial responses in 16% of patients (95). TNF family costimulatory receptors. Monoclonal antibodies that act as agonists of stimulatory receptors can directly augment antitumor immune responses. Several such antibodies have been developed to target TNF family costimulatory receptors, including glucocorticoid-induced tumor necrosis factor receptor (GITR), CD134 (OX40), CD137 (4-1BB), and CD40. GITR, CD134, and CD137 are each costimulatory receptors expressed on T cells, and ligation of these receptors enhances cytotoxic T cell function (172). In animal models, agonistic antibodies directed against each of these proteins can signiﬁcantly increase the efﬁcacy of antitumor immune therapy (173–177). Clinical trials are planned to evaluate both GITR- and CD134-speciﬁc antibodies in cancer therapy, and an anti-CD137 antibody (BMS-663513) is now in phase I and phase II testing in a variety of solid tumors, including melanoma and NSCLC.

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CD40 plays a central role in the crosstalk between CD4+ T cells, DCs, and B cells. Antibodies against CD40 differ from other immune-stimulating antibodies because, in addition to immune-modulating activity, antiCD40 can also directly bind CD40-expressing B cell tumors, enabling the activation of conventional antibody-dependent killing mechanisms. The balance of these effects is likely dependent on whether the antibody activates or blocks CD40, and both types of antibodies are under evaluation (178, 179). Activating antibodies against CD40 have shown efﬁcacy in animal tumor models, where responses required immune activation (178). Consistent with these ﬁndings, a recent phase I trial of the anti-CD40 antibody CP-870893 showed encouraging preliminary results in patients with advanced melanoma (180). Although not an activating receptor, the receptor for the cytokine tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) is also under investigation as a target for agonistic therapy either with TRAIL itself or with monoclonal antibodies. Work in experimental systems suggests that signals through the TRAIL receptor can have potent effects on tumors cells, directly inducing apoptosis, without inducing substantial systemic toxicities (181).

Targeting Regulatory Cells The immune-suppressive tumor microenvironment is, in large part, maintained through the anti-inﬂammatory activity of both innate and adaptive regulatory cells. In particular, Tregs seem to play an important role in damping antitumor immune responses. Tregs are overrepresented in a wide range of tumors, and increased numbers of Tregs correlates with poor prognosis in a variety of tumors (182–185); in one series of 70 patients with ovarian cancer, high Treg numbers were associated with a 25-fold increase in the risk of death after controlling for disease stage and surgical debulking (182). Tregs may also pose a signiﬁcant barrier to tumor vaccination strategies, which can induce increases in antigen-speciﬁc Tregs (186). Given the poten-

tial importance of Tregs in limiting antitumor immunity, methods for directly targeting them may be of clinical use. The drug denileukin diftitox is a conjugate of Diphtheria toxin and IL-2 and was initially developed to treat T cell malignancies. In more recent work, denileukin diftitox has shown some selective toxicity against Tregs (187, 188). This selective effect may relate to the high expression of the IL-2 receptor α chain (CD25) that characterizes these cells. Inclusion of denileukin diftitox in a cancer vaccine setting has shown early promising results (188). Additional Treg-targeted therapies are under investigation in experimental systems, including interfering with Treg chemotaxis by blocking interactions between CCL22 and CCR4, blocking IL-35, and antagonizing Treg induction following the phagocytosis of apoptotic cells using a dominant negative form of MFG-E8 (115, 119, 189).

Therapeutic Cancer Vaccines Prophylactic cancer vaccines have been effective in a number of spontaneous tumor models in mice, including models of breast, prostate, and colon cancer (190–192). In cancer patients, vaccination strategies have been limited to advanced disease, owing to the investigational nature of current therapies. These therapeutic vaccines present a substantial challenge, given that they must bypass immune-regulatory mechanisms that have already led to tumor tolerance. The enormity of this challenge is underscored by the lack of effective vaccination strategies to treat chronic infectious diseases, despite the large number of foreign antigens present in infectious organisms. Many strategies for generating therapeutic immune responses to cancer have been attempted; antigen-speciﬁc vaccines, DC vaccines, and cytokine-based, whole tumor cell vaccines are among the most promising and best studied of these strategies. Antigen-specific vaccines. A wide range of vaccines based on single-tumor antigens has www.annualreviews.org • Immune Therapy for Cancer

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been tested in experimental systems, and several speciﬁc antigen vaccines are currently in clinical trials. These vaccines, which make use of recombinant proteins or antigenic peptides mixed with immune adjuvants, can elicit coordinated T and B cell immune responses and have some efﬁcacy in a variety of human tumors. The targets selected in these strategies have been diverse, yet many belong to a class of proteins referred to as cancer testes antigens. Cancer testes antigens are expressed by tumor cells as well as endogenously within the immune-privileged environment of the testes, making immune response against them highly selective for tumor tissue. Two cancer testes antigens, MAGE-A3 and NY-ESO-1, have been evaluated as vaccine targets in a series of clinical trials. MAGE-A3 is highly expressed on a wide range of tumors (193, 194), and adjuvant-mixed, recombinant MAGE-A3 protein or peptide vaccines can elicit potent antitumor B and T cell responses that can be associated with objective responses, and the tumor-speciﬁc cells persist for years following vaccination (194). Phase I and II MAGE-A3 vaccine trials have been completed in melanoma and NSCLC, and a phase III trial in NSCLC is currently underway (193, 194). Like MAGE-A3, NY-ESO1 is widely expressed in human cancers (195, 196). Multiple clinical trials have examined NYESO-1-based vaccines in a variety of cancers, including melanoma, ovarian carcinoma, and NSCLC (195, 196). Vaccines using adjuvants along with peptide or recombinant protein have elicited strong immune reactions, including coordinated antibody and cytotoxic T cell responses following vaccination with recombinant protein and CpG (196). In addition to cancer testes antigens, some tumors express proteins with a narrow distribution in nonmalignant tissues; tissue-restricted proteins have been successfully targeted in melanoma, where a variety of melanocyte-speciﬁc proteins often continue to be expressed (197). Many tumors overexpress proteins with relatively low endogenous expression, and these overexpressed proteins can be effective tar-

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gets of single-antigen vaccines. Similarly, the HER2/neu protein is highly expressed on a subset of breast cancers and, as was discussed above, has been effectively targeted by the monoclonal antibody trastuzumab. Vaccination against HER2/neu can induce coordinated antitumor immune responses that can spread beyond the initial antigenic determinants used in the vaccine (198). Targeting HER2/neu using a vaccine may be a particularly valuable approach in combination with trastuzumab, where the two therapies may synergize to augment antitumor immunity. Dendritic cell vaccines. DCs are potent inducers of adaptive immunity, driving the activation of T cells in response to invading microorganisms. The ability to culture DCs from human peripheral blood monocytes has generated signiﬁcant interest in using DCs in novel cancer vaccination strategies (199). Immature monocyte-derived DCs can be readily loaded with antigenic peptides or proteins in vitro; these antigen-loaded DCs can then be used in an autologous transplant to induce antigenspeciﬁc T cell responses (199). DCs must be activated before they are capable of inducing immunity. Activated DCs have improved antigen-presenting abilities, as well as increased expression of T cell costimulatory proteins; activation also confers migratory potential to DCs, a necessary step to bring them into contact with naive T cells in lymph nodes. DCs can be activated by a range of stimuli, including inﬂammatory cytokines and microbial products, although these stimuli do not always lead to equivalent DC activation (200, 201). Most clinical trials use an inﬂammatory cytokine cocktail to mature DCs, although other approaches, including the use of microbial pattern-recognition receptor agonists, have also been considered (202, 203). An alternative approach, which has been tested in experimental models, bypasses in vitro activation and instead injects immature, antigenloaded DCs into an inﬂamed tissue (204, 205). Once exposed to the inﬂammatory environment, antigen-loaded DCs can mature in a

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more physiological fashion and migrate to draining lymph nodes (204, 205). This vaccination approach offers at least two advantages. First, DCs acquire antigen while immature, which should ensure appropriate antigen processing. Second, this approach obviates the need for large quantities of recombinant cytokines, which can signiﬁcantly increase costs. Antigens can also be delivered directly to DCs in vivo using antibodies that bind DC surface receptors such as DEC205; targeting DCs using this strategy has proved successful in experimental models and is under development for use in patients (206–208). The delivery of antigen to cultured DCs has been accomplished through a variety of approaches, including the direct loading of antigenic peptides or long overlapping peptide mixtures, exposure to whole recombinant protein, transfection with antigen-encoding mRNA, and fusion with tumor cells (199, 209–211). Each of these approaches has its advantages, and the most effective mechanisms for generating immunity in cancer patients has not yet been established (199, 209–211). A large number of DC vaccination trials have been conducted in cancer patients using a range of protocols, some with encouraging preliminary results (212). Large-scale phase III trials of DC vaccines, including a trial in metastatic melanoma, have thus far failed to demonstrate a protective beneﬁt (212, 213). DC vaccination protocols are clearly not yet optimized, and generating more effective vaccines continues to be an area of active research. Cytokine-based tumor vaccines. Coadministered cytokines have been used as adjuvants in a variety of cancer vaccination strategies (214). When combined with vaccines, cytokines can boost immune responses through the recruitment and maturation of a wider variety of immune effector cells (214). Several cytokines, including IL-2, IL-12, IFN-α, and GM-CSF, have been evaluated as vaccine adjuvants; among these, GM-CSF has been the most widely studied (214). GM-CSF has been particularly potent when used to prime immune

responses against whole tumor cell vaccines. GM-CSF primarily acts on myeloid cells and functions to recruit and mature DCs, enhancing the presentation of tumor antigens to the immune system. In mouse models, GM-CSFtransfected tumor cell vaccines can induce complete protective immunity when used prophylactically against syngeneic tumors, and when used therapeutically these vaccines can delay tumor growth (215). Similarly, in cancer patients, injection of autologous, irradiated, whole tumor cells engineered to produce GM-CSF (GVAX) can induce coordinated B and T cell responses to a wide range of tumor antigens. In melanoma patients, these immune reactions have been associated with both partial and complete responses (216). Autologous whole-cell vaccines have the advantage of providing multiple potential tumor antigens, yet these patient-speciﬁc vaccines are also difﬁcult to manufacture on a large scale. To facilitate large-scale vaccine production, GMCSF-secreting allogeneic tumor vaccines have also been evaluated in clinical trials. These allogeneic vaccines, which are derived from tumor cell lines, have been tested in both pancreatic cancer and hormone-resistant prostate cancer, where they have shown promising early results (217–219). Based on these early studies, phase III efﬁcacy trials of GVAX in hormone-resistant prostate cancer are currently underway. Although some patients have had encouraging responses to GVAX, this strategy may be most effective when used in combination with other immune-modulating therapies (215, 220, 221). The anti-CTLA-4 monoclonal antibody ipilimumab has been tested in patients who have been previously vaccinated using DCs or GVAX (220, 221). In these patients, ipilimumab is associated with augmented antitumor immunity and objective responses in a subset of patients (220, 221). Inﬂammatory side effects were associated with therapy, yet treatment did not provoke severe autoimmunity (220, 221). Intriguingly, tumor necrosis was highly associated with an increased CD8+ -to-FoxP3+ ratio in tumor-inﬁltrating T cells (Figure 2). This suggests that clinical responses to GVAX and www.annualreviews.org • Immune Therapy for Cancer

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FoxP3

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Figure 2 The ratio of tumor-inﬁltrating CD8+ T cells to FoxP3+ Tregs after GVAX followed by ipilimumab infusion is tightly correlated with the extent of tumor necrosis. (a) Upper row: Minimal necrosis of melanoma metastasis. Lower row: Extensive necrosis of melanoma metastasis. (Magniﬁcation: H&E, × 4; CD8, × 20; FoxP3, × 40.) (b) Numbers of tumor-inﬁltrating FoxP3+ Tregs and CD8+ T cells versus tumor necrosis (reprinted from Reference 200).

CTLA-4 blockade come from a loss of immune suppression coupled with the induction of cytotoxic T cells (220, 221). These preliminary results suggest that combination immune therapies may have signiﬁcant clinical efﬁcacy, although this potential awaits testing in randomized clinical trials. 4.22

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Adoptive T Cell Therapy Adoptive T cell therapy relies on the in vitro expansion of endogenous, cancer-reactive T cells, which are harvested from cancer patients, manipulated, and then reintroduced as a mechanism for generating productive tumor immunity. Adoptive T cell therapy has had some

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promising early clinical results and has been associated with clinical responses in a minority of patients with metastatic melanoma (222–224). CD8+ cytotoxic T lymphocytes are the primary effector cells in adoptive T cell therapy (225, 226); however, CD4+ T cells may also play an important role in maintaining CD8+ cytotoxic function, and transplantation of tumorreactive CD4+ T cells has been associated with some efﬁcacy in metastatic melanoma (227). T cells used in adoptive therapy can be harvested from a variety of sites, including peripheral blood, malignant effusions, resected lymph nodes, and tumor biopsies. Although T cells harvested from the peripheral blood are technically easier to obtain, tumor-inﬁltrating lymphocytes (TILs) obtained from biopsies may contain a higher frequency of tumor-reactive cells (228). Practically, obtaining sufﬁcient cells from tumor biopsies is difﬁcult, although this approach has been used successfully in patients with melanoma (228). Two alternative approaches attempt to circumvent low levels of endogenous antitumor reactivity in the peripheral blood by directly supplying T cells with the ability to recognize tumors. T cells harvested from the peripheral blood can be engineered to express T cell receptors (TCRs) that have been selected for tumor recognition. This approach has been tested in metastatic melanoma; however, because TCR recognition of antigen is MHC restricted, each engineered TCR can only be used in patients with the required MHC allele (229). MHC restriction can be bypassed by engineering T cells to express novel chimeric fusion proteins that link the antigen-binding domain of the B cell receptor with the signaling component of the TCR complex (230–232). These “T-bodies” can directly bind tumor antigens, leading to T cell activation. Transfusion of T-body-expressing T cells have been tested in RCC, ovarian carcinoma, and pediatric neuroblastoma. Although T-body-expressing cells are generally well tolerated, severe autoimmune hepatitis was precipitated in RCC, presumably owing to expression of the target antigen, car-

bonic anhydase IX, in the biliary tract (232). This adverse event clearly demonstrates the reactive potential of T-body-expressing T cells, but it also underscores the importance of antigen selection. Once harvested, T cells can be expanded either through polyclonal stimulation with activating antibodies or through exposure to speciﬁc tumor antigens, although this second approach requires the identiﬁcation of relevant targets. Given the frequency of antigen loss variants in current clinical trials, the selection of appropriate targets may be challenging, potentially making polyclonal stimulation a more attractive approach (222–224). In addition to antigen-loss variants, adoptive T cell therapies have been limited by the replicative potential of cultured T cells. Several strategies, including the enforced expression of costimulatory proteins and telomerase, have been used to attempt to extend the life span of cultured T cells (233, 234). IL-15 has also been considered as a possible additive to cultures to enhance the production of cytotoxic cells (226). Intriguingly, engraftment of adoptively transferred T cells appears to be enhanced in lymphodepleted hosts, and strategies to combine pretreatment with lymphodepleting chemotherapy and adoptive T cell transplantation appear to increase treatment efﬁcacy signiﬁcantly (235, 236). As with other strategies to generate therapeutic antitumor immunity, adoptive T cell therapy alone may be insufﬁcient to induce clinically meaningful responses in most cancer patients. Combining adoptive T cell therapy with an autologous tumor vaccine mixed with BCG has produced promising results in RCC, where a 27% objective response rate was observed in a recent phase II trial (237). Furthermore, adoptively transferred T cells stimulated with pneumococcus can be activated in vivo using a pneumococcal vaccine (238). These results suggest that combination therapies using vaccines to increase the frequency of tumor-reactive T cells prior to, or immediately after, adoptive therapy may be another mechanism for increasing overall treatment efﬁcacy. www.annualreviews.org • Immune Therapy for Cancer

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CONCLUSION

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Single modality immune manipulations have had signiﬁcant success in some therapeutic settings; however, the complexity of the interaction between developing tumors and immune cells may often necessitate combination therapy in order to generate protective antitumor immunity. By disabling key regulatory pathways in conjunction with strategies to expand and activate tumor-reactive cells, the efﬁcacy of tumor immune therapy may improve substantially. Yet disabling regulatory pathways increases the risk of treatment-limiting autoimmunity, as has already been observed. One of the principal challenges of immune therapy for cancer will be to identify those mechanisms of immune suppres-

sion that are most valuable to speciﬁc tumors and least necessary for the maintenance of normal immune tolerance. A further challenge will be to establish meaningful criteria for the evaluation of novel immune therapies. Survival is the most important endpoint for evaluating cancer therapy; however, objective criteria based on changes in the size of tumor masses may underestimate immune therapies. Productive antitumor immune responses often lead to swelling and ﬁbrosis, which do not diminish the size of tumor masses but instead alter their composition, decreasing the overall number of viable tumor cells. As a result, using immune correlates and an assessment of tumor composition may be more a meaningful way to evaluate and optimize investigational immune therapies.

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Microglial Physiology: Unique Stimuli, Specialized Responses Richard M. Ransohoff1 and V. Hugh Perry2 1

Neuroinﬂammation Research Center, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195; email: [email protected]

2

School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK; email: [email protected]

Annu. Rev. Immunol. 2009. 27:119–45

Key Words

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

microglia, macrophage, inﬂammation, neurodegenerative disease, brain cytology, activation, regulation, central nervous system

This article’s doi: 10.1146/annurev.immunol.021908.132528 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0119$20.00

Abstract Microglia, the macrophages of the central nervous system parenchyma, have in the normal healthy brain a distinct phenotype induced by molecules expressed on or secreted by adjacent neurons and astrocytes, and this phenotype is maintained in part by virtue of the blood-brain barrier’s exclusion of serum components. Microglia are continually active, their processes palpating and surveying their local microenvironment. The microglia rapidly change their phenotype in response to any disturbance of nervous system homeostasis and are commonly referred to as activated on the basis of the changes in their morphology or expression of cell surface antigens. A wealth of data now demonstrate that the microglia have very diverse effector functions, in line with macrophage populations in other organs. The term activated microglia needs to be qualiﬁed to reﬂect the distinct and very different states of activationassociated effector functions in different disease changes. Manipulating the effector functions of microglia has the potential to modify the outcome of diverse neurological diseases.

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INTRODUCTION TO MICROGLIA

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Resident tissue macrophages are present in all tissues of the body, and the central nervous system (CNS) is no exception. An extensive literature had debated the origin of the most numerous of the brain macrophages, the microglia, speciﬁcally whether they are of myeloid lineage or of neuroectodermal origin. There is little need to rehearse these different points of view; the fact that it took so long to establish the myeloid origin of microglia tells us that these cells have a phenotype that is distinct from other tissue macrophages. Many lines of evidence have converged to establish that the microglia are of myeloid lineage, which was conclusively conﬁrmed by their absence from the CNS of PU.1-null mice (1), although the brains of PU.1-null mice are readily repopulated by microglia following bone marrow transplantation (2). Because microglia and the other populations of macrophages associated with the different structural compartments of the CNS are part of the mononuclear phagocyte system (MPS), we can identify more speciﬁc questions: When do macrophages ﬁrst invade the CNS? Are microglia derived from a speciﬁc pool of progenitors or monocytes? How are their numbers maintained in the steady state? What are the signals that lead to microglial activation? What are the phenotypes and functions of these cells in health and disease? This review relies on studies in humans, mice, and rats. It is worth mention that cells that are functionally equivalent to microglia are observed in the invertebrate CNS. Studies carried out in the leech (Hirudo medicinalis) reveal a population of neuroectodermal cells termed small glia or microglia. Leech microglial cells move rapidly to nerve crush lesions, at least partly via purinergic signaling, as described also for mammalian microglia (see below), and they phagocytose debris. Leeches show axonal sprouting and accurate reestablishment of synaptic contacts after nerve crush. Intriguingly, blockade of microglial accumulation at the lesion site impairs axon sprouting (3, 4).

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Macrophage Populations of the CNS Microglia in the adult mammalian CNS have a small cell soma, little perinuclear cytoplasm, and a number of ﬁne, branched processes that are covered in ﬁne protrusions. The cells are readily revealed in rodent and human CNS by lectin cytochemistry or immunocytochemistry for selected antigens, and they occupy a territory that does not overlap with adjacent microglia. The absence or low levels of expression of many antigens typically found on other macrophage populations indicate that the CNS microenvironment plays a critical role in deﬁning the phenotype (see the discussion below in Regulation of the Microglia Phenotype in the Normal Healthy CNS). Despite the downregulated phenotype of microglia in the normal healthy brain, in vivo imaging studies demonstrate that the ﬁne processes of microglia continually palpate and monitor their local microenvironment (5, 6). Microglia are distributed throughout the CNS and vary in density in both rodents and humans, with subtle variations in morphology in different cytoarchitectural regions (7). It is unclear what local factors determine their numerical and morphological variations or whether these variations reﬂect functional differences, although there are subtle regional diversities in expression of important immune receptors (8). In addition to the microglia, there are macrophage populations associated with the perivascular space [the perivascular macrophages (PVMs)], the circumventricular organs, the choroid plexus, and the meninges. These macrophages have different phenotypes when compared with the microglia and appear differentially constrained by their local microenvironment. PVMs express antigens not expressed on the microglia. For example, the mannose receptor is detected on PVMs of mouse and human (9) and CD163 in rat and human (10). Similarly, a signiﬁcant proportion of meningeal macrophages express these receptors. The PVMs and meningeal populations are more overtly phagocytic than are the microglia, and the expression of major

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histocompatibility complex (MHC) antigens is more widespread in these populations of cells relative to the microglia. Macrophages are also present in regions of the brain where they are exposed to the blood, including the circumventricular organs and the choroid plexus, and the phenotype of these macrophages is distinct from the microglia and PVMs, perhaps reﬂecting the role of serum proteins in the regulation of microglia phenotype (11). An important question regarding the immune privilege of the CNS is whether there exists a population of dendritic cells (DCs), the professional antigen-presenting cells (APCs) of the immune system (12). Since the ﬁrst reports demonstrating a lack of MHC class II expression in the CNS, there has been no convincing demonstration of DCs, either by expression of relevant DC surface markers or by DC function in the healthy brain parenchyma (13), although DCs are present in the meninges and the choroid plexus. This situation is radically changed in the diseased brain, however (see the section below on Multiple Sclerosis).

Origin of Microglia In postnatal rodents, immunocytochemical studies using antibodies to F4/80 (ERM1), CR3, and FcγRII/III suggested that the microglia entered the brain from the circulation and presumably were derived from circulating monocytes (14). At earlier stages of development (prior to the development of the vasculature), cells of myeloid origin can be found within the embryonic mouse CNS (embryonic day 8), where they proliferate (15). In the zebra ﬁsh, yolk sac–derived macrophages enter the developing brain and retina, where they develop into immature microglia (16). The lineage origin of these cells from the progenitor cells in the yolk sac is unclear, but evidence suggests that these primitive macrophages develop along a PU.1-independent pathway (17). Whether any of these primitive macrophages contribute to the microglial populations that appear later in development or persist into adulthood is not known. In one study (18), more

than 20% of microglia isolated from immature brain expressed the hematopoietic stem cell marker CD34, and 90% expressed the B220 antigen, an isoform of CD45 typically associated with B cells and a subset of DCs. These markers are subsequently downregulated during maturation.

Maintaining the Microglial Population Investigators widely believe that the microglia are a long-lived population of tissue macrophages, but how the resident populations of brain macrophages are maintained in homeostasis and during disease is not yet resolved. Recent studies show that resident tissue macrophage populations may arise from speciﬁc subsets of monocytes rather than simply from stochastic recruitment or entry of monocytes into tissues. At least two distinct subsets of monocytes are found in mouse, Gr-1hi /CCR2+ /Ly6Chi /CX3CR1+ and Gr-1lo /CCR2− /Ly6Clo /CX3CR1++ (19). Geissmann et al. (20) reported that Gr1lo /CCR2− cells may give rise to tissue macrophages, including the microglia, in the steady state. Those investigations did not clarify how many cells enter the brain and into which compartment; furthermore, the cells that apparently entered the parenchyma did not persist for more than a few days. It has been suggested that either CX3CR1 or homeostatic tissue macrophage turnover mediates recruitment, but Cardona et al. (21) demonstrated that CX3CR1 is not required for entry into the brain. Thymidine labeling studies in adult mice demonstrate that microglia undergo DNA synthesis, divide, and contribute to the maintenance of this population (22), akin to the situation found for other long-lived resident myeloid cell populations. To address the question of the relative contribution of circulating monocytes or intrinsic division to the steady-state population, investigators have typically used radiation chimeras or parabionts. However, the complications introduced by whole body irradiation, including irradiation of the brain, have led to some www.annualreviews.org • Microglial Physiology

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confusion. The use of radiation chimeras and parabiosis to study microglia turnover is discussed below in the section on Irradiation Bone Marrow Chimerism and Parabiosis. The extent to which other populations of brain macrophages, PVMs, or meningeal cells turn over is also poorly understood. Several bone marrow chimera studies have suggested that these cells turn over more rapidly than microglia, but again there are technical issues to consider (discussed below). By injecting India ink into the brain parenchyma, PVMs were labeled that could be identiﬁed even two years later (23), suggesting that this population may also be long-lived.

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RESEARCH METHODS—THEIR STRENGTHS AND LIMITATIONS In Vitro In vitro microglial culture. In vitro systems provide a critical tool for exploring many aspects of microglia biology. Removal of these cells from their microenvironment will release them from the normal constraints that play such a critical role in their phenotype. Thus, using in vitro systems assumes we recognize either that the cells must be studied rapidly after isolation or that the investigator must attempt to recapitulate features of the CNS microenvironment that will in turn give clues about the factors regulating their phenotype. A signiﬁcant problem is the identiﬁcation of the microglia phenotype in vitro, as there is no single microglia marker and the microglial phenotype is deﬁned in vivo by a combination of morphology and often lack of or low expression of multiple macrophage antigens. This circumstance has lead to a relatively loose use of the term “process bearing” or other terms for the characterization of microglia in vitro, and all too often no attempt is made to address whether they differ from macrophages. Despite these caveats, many studies rely on the isolation and expansion of microglia from the neonatal brain as a model for the study of resident microglia function. Another limitation of studying neonatal 122

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microglia is that they have not experienced the CNS milieu in vivo in the context of an intact, mature blood-brain barrier (BBB). Ponomarev et al. (24) recently described a protocol for isolating adult rodent microglia. The importance of astrocyte-microglia interactions as part of the environmental regulation is well illustrated in experiments by Rosenstiel and colleagues (25), who have shown that microglia and other macrophage populations grown on an astrocyte monolayer develop a highly ramiﬁed morphology that is associated with a downregulation of nuclear factor-κB (NF-κB). Whether expression of a spectrum of cell surface antigens is also downregulated has not been systematically studied. Isolation of adult microglia and neonatal microglia shows these cells to be poor or immature APCs, although treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) will lead to differentiation to a more DC-like phenotype (26). Isolation of adult microglia and subsequent culture in low levels of macrophage colony-stimulating factor-1 (M-CSF) lead to proliferation of the surviving cells that then develop a modestly ramiﬁed morphology that could be maintained for many weeks (24), consistent with the notion that M-CSF is a key component of tissue macrophage survival and phenotype. Mice lacking M-CSF do exhibit normal morphology and apparent function of microglia, however (27). Elucidation of many aspects of macrophages and microglia clearly can be readily accomplished in vitro, but the direct relevance of these observations needs to be established through in vivo studies before accepting them at face value. In most in vitro studies, there has been no attempt to replicate the CNS microenvironment, and the state of the cells grown on glass or tissue culture plastic is likely more relevant to inﬂammatory cells rather than to steady-state microglia. Brain slices. An intermediate approach to in vivo characterization or the isolation of microglia from their natural environment is to investigate their properties in accessible acute

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brain slices or organotypic cultures. Acute brain slices suffer from the obvious consequences of the slicing process, and one might imagine that the microglia would be rapidly activated, but in contrast to isolated in vitro microglia, the ion channel expression in the slice cultures is distinct from that found in cells in vitro and consistent with a downregulated or resident phenotype (28). Organotypic cultures permit the study of microglia with a resident phenotype after several days in vitro, and subsequent injury to these slices leads to somewhat predictable responses given what we know of the in vivo condition and the responses of macrophages (29). Few reported studies have used slice culture, and wider exploitation of this approach might be productive, given the evident limitations of in vitro culture of isolated microglia.

In Vivo Immunohistochemistry and in situ hybridization. Given the marked limitations imposed by studying microglia in vitro (see above), much effort has been devoted to evaluating microglial biology in the intact CNS. Immunohistochemistry (IHC) has been widely used to characterize microglia, both in human tissues and in those from experimental animals. The strengths of this approach are manifold, and no other technique can provide a comparable wealth of information. Different from RNA analysis [such as in situ hybridization (ISH), Northern blotting, or reverse transcriptasecoupled polymerase chain reaction (RT-PCR)], IHC detects protein, which is more likely to be functionally relevant than mRNA. Furthermore, unlike Western blotting or enzymelinked immunosorbent assay (ELISA), IHC enables protein both to be detected and to be localized to speciﬁc cell types with regional differentiation. With proper controls, IHC supports quantitative morphometry. In addition, using high-resolution techniques such as confocal microscopy with immunoﬂuorescence, subcellular localization can be achieved, occasionally with functional implications. For example, stimulus-dependent transcription factors,

such as the signal transducers and activators of transcription (STATs) and the NF-κB components, translocate to the nucleus upon ligand engagement of upstream receptors. Therefore, the detection of STATs in microglial nuclei implies ligation of relevant cytokine receptors (30). IHC can accordingly yield an incomparably vivid depiction, albeit static, of physiological and pathological processes. Against these strengths lies the cardinal weakness that IHC is fraught with artifact, much of which is deceptively subtle and the causes of which are legion. Artifactual immunostaining can appear crisp and speciﬁc with low or absent background. Because of IHC’s importance in clarifying neurobiological processes, guidelines for validating antibody staining in neuroscience have been proposed (31, 32). These guidelines are useful both for those performing experiments and for those evaluating experimental results. At the outset, it is evident that simple technical controls such as omission of primary antibody represent only a ﬁrst step in this crucial process. Pertinent suggestions include use of Western immunoblotting to verify presence of the antigenic target in tissue lysates and application of multiple independent antibodies directed against a single target to verify cellular localization. Where possible, antibodies can be used in negative control experiments to stain tissues of animals [such as gene-targeted mice or patients with gene mutations (33)] that deﬁnitively lack the target antigen. These guidelines also recommend that subcellular localization of target antigen should correspond to the known function of the protein; immunoreactivity of receptor antibodies should typically demonstrate plasma membrane distribution, for example. Parallel performance of ISH with IHC is extremely useful for conﬁrming that targetencoding mRNA resides in the same cell, region, and physiological context as the immunoreactivity. This analysis can be assisted in silico using the Allen Brain Atlas (http://www. brain-map.org/welcome.do), a searchable online compendium of ISH experimental results, that can be used to localize mRNAs in the www.annualreviews.org • Microglial Physiology

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mouse brain with impressive anatomical detail. Deﬁnitive experiments combine IHC and ISH. In summary, tissue IHC is a centrally important technique for microglial research because the most valid observations of these enigmatic cells need to be conducted in the intact CNS. At the same time, it has been famously difﬁcult to apply IHC to characterize microglia owing to their relatively low expression of MPS markers. Adherence to guidelines for validating IHC results is therefore critical for research into microglia.

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Two-photon imaging. Excitation ﬂuorescence imaging allows visualization of neural structures to a depth of 100–200 μm, through a thinned-skull preparation. This approach has been successfully applied to neurobiological questions related to dendritic physiology for more than a decade (34). A major advantage is that this technique affords prolonged imaging sessions, without photobleaching or photodamage. As noted above, two groups used two-photon ﬂuorescence imaging to study mice in which CX3CR1, the receptor for chemokine CX3CL1, was replaced with enhanced green ﬂuorescent protein (EGFP) (Cx3cr1+/GFP mice). Both groups made the same surprising observation: that microglia in the intact, healthy CNS continually remodel their processes, in apparent surveillance of the extracellular milieu (5, 6). This landmark ﬁnding provided an entirely new view of parenchymal cells previously stigmatized as resting microglia (35). Their dynamic surveillance of the CNS involved processes at all levels of branching, without movement of the soma, and was calculated to monitor the entire tissue every several hours. Contacts with astrocytes, vascular elements, and neurons were visualized. Blocking voltage-gated sodium channels (and thus neuronal action potentials) with tetrodotoxin did not affect process motility. Further research motivated by these ﬁndings showed that microglia walled off laser lesions extremely rapidly through process extension, again with minimal initial displacement of the soma (5). Extracellular nucleotides/nucleosides 124

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such as ATP or ADP were required for microglial process movement in response to injury (but not in surveillance of the healthy CNS) and signaled through the metabotropic purine receptor P2Y12 (5, 36). The signals that underlie physiological process movement remain to be established (35). Cx3cr1+/GFP mice were ideally suited for these studies, as the EGFP ﬂuorescence was expressed in all cortical microglia, as judged by colocalization with microglial markers such as ionized calciumbinding adapter molecule 1 (iba-1), and was restricted to microglial cells, as determined by lack of colocalization with lineage markers for neurons and glial subpopulations (21). Interpretation of data gained by the application of two-photon imaging to microglial biology will depend on which promoter-reporter is used and on detailed knowledge of promoter activity under physiological and pathological conditions. In this context, it bears repeating that no microglial-speciﬁc promoter has yet been identiﬁed. Irradiation bone marrow chimerism and parabiosis. Hematopoiesis research has long relied on introducing labeled donor cells, with or without functional alterations, into recipient animals. One widely used technique is to subject a recipient host to lethal irradiation and provide rescue by transferring bone marrow containing hematopoietic stem cells. An alternative is parabiosis, involving anastamosis of the circulations of host and recipient, allowing for mixing of the circulating elements. Irradiation bone marrow chimerism to study the immunology of the CNS was ﬁrst reported in the early 1980s and addressed questions such as the identiﬁcation of CNS cells that expressed the MHC class II antigens (37). Irradiation chimerism was suitable for studies of this type because the polymorphic MHC antigens enabled differentiation between the host and donor cells, and the experiments gave unambiguous answers. It was noted early on that parenchymal microglia did not display the markers of transferred cells, although perivascular MPS cells did (38), yielding a preliminary suggestion that perivascular

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and parenchymal MPS cells turned over with differing kinetics. The technique of irradiation chimerism was soon adapted to address key questions in CNS immunity and inﬂammation. A wide spectrum of research employed the model disease experimental autoimmune encephalomyelitis (EAE), involving sensitization of rodents with myelin protein fragments or adoptive transfer of myelin-speciﬁc T cells. In 1988, a seminal study used irradiation chimerism and adoptivetransfer EAE to demonstrate that PVMs of the CNS could stimulate myelin-reactive T lymphocytes adequately to bring about demyelination (39). These results were updated recently, using additional controls to show that PVMs of the CNS were necessary and sufﬁcient for adoptive-transfer EAE (40). Irradiation chimerism was used to address central questions regarding the turnover and proliferative capacity of microglia and perivascular cells (41–43). The response of engrafted (inﬁltrating) and resident cells to varied disease or injury models was extensively compared. Irradiation chimerism seemed well adapted for examining the comparative roles of the resident microglial cells and the inﬁltrating bloodderived elements. It now appears that data from these earlier irradiation chimerism experiments need reevaluation in light of studies that directly address potential confounds arising from the preparation (44–46). Three sources of confound are recognized: (a) Use of bone marrow cells to reconstitute the hematopoietic system might lead to nonphysiological numbers of hematopoietic stem cells or progenitors in the circulation. (b) Lethal irradiation-induced cell death in the hematopoietic system is associated with enormous ﬂuxes of cytokines through the circulation and in tissues. (c) Irradiation of the CNS causes vascular changes, permanently affecting competence of the BBB. Together, these constituents of the irradiation chimerism protocol appear to lead to nonphysiological transmigration of cells into the CNS parenchyma. Informative studies have used the facialaxotomy model, which results in brisk mi-

croglial reaction in the ipsilateral facial nerve nucleus, after facial nerve crush in the periphery (47, 48). Previous studies showed that facial axotomy of irradiation chimeric rodents leads to incorporation of donor-derived cells in the microglial reactive population (43), and parabiotic animals showed few if any donorderived microglia under physiological conditions (49). To address mechanisms for recruiting donor microglia into the axotomized facial nerve nucleus, parabiotic animals were generated, and the CNS vasculature of the recipient was conditioned by lethal irradiation, yielding recipients in which actin-GFP-labeled donor cells predominated in the circulation of the recipient but did not contain bone marrow elements beyond those found circulating physiologically (44). After facial axotomy, no GFP+ microglia were found in the reactive microglial population of the facial nerve nucleus. These results indicated that irradiation alone was not sufﬁcient to allow circulating cells to become parenchymal microglia after this type of injury (44). In complementary experiments, irradiation chimerae were generated, using a protocol that spared the cranial vasculature, and then subjected to facial axotomy (45). Again, no donor-derived microglia were found in the axotomized facial nerve nucleus. The interpretation was that irradiation was required (although not sufﬁcient) (44) to enable circulating cells into the parenchymal microglial pool. Therefore, based on direct comparisons between parabiotic and irradiation-chimeric preparations, investigators now recognize that nonphysiological processes seed the CNS with donor microglia following irradiation chimerism (46). These two complementary observations pointed to the conclusion that both cranial irradiation and transfer of bone marrow cells into the circulation were required for the nonphysiological entry of donor cells into the parenchymal microglial population (46). Corollary conclusions are that parenchymal microglia are replaced by proliferation of resident cells and that microgliosis can be exceedingly impressive in the absence of a contribution from circulating cells. www.annualreviews.org • Microglial Physiology

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PET scan. Microglia are embedded in a dense network of neural interactions and are intimately associated with neurons. Owing to its networked nature, the CNS demonstrates unique, distributed pathological patterns, by contrast to other tissues. Damage to an axon excites a brisk (retrograde) response by its remote cell body; damage to an axon also causes an (anterograde) reaction by local microglia that invests the terminus of the injured cell process; most remarkably, when a nerve cell loses input owing to elimination of a distant projecting neuron, there is a trans-synaptic response, including microglial reaction in the vicinity of the deprived neuron. Neuropathological description of these phenomena captures neither their extent nor their kinetics. Positron emission tomography (PET) scanning has been a useful adjunct in neurological practice, showing, for example, hypometabolism in affected regions of brain in neurodegenerative disease through the application of [18 F] 2-ﬂuoro 2-deoxy-dglucose (FDG) as a PET tracer. Labeled R-enantiomer of an isoquinolone [11 C] (R)PK11195 binds selectively to a mitochondrial membrane translocator protein (TP)-18, part of a protein complex previously termed the peripheral benzodiazepine-binding site (PBBS). This binding activity is expressed selectively by myeloid cells in the CNS, as shown by correlation of IHC and autoradiography. PET studies using PK11195 take advantage of the partition of this moiety into brain across the competent BBB, as well as its selectivity for activated, in contrast to surveillant, microglia. The binding activity remains relatively constant across the spectrum of morphological variations of microglial activation (activated, rodlike, amoeboid) and also binds to inﬁltrated macrophages. The technique requires signiﬁcant postprocessing and is challenged by confounds in circumstances of BBB disruption. Having accepted these limitations that, to date, preclude routine clinical application, PET with PK11195 has provided proof-of-principle for microglial activation in neurodegenerative disorders, where trans-synaptic processes must be

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operative, as well as in the lateral geniculate projection ﬁelds of optic nerves of multiple sclerosis (MS) patients suffering from severe optic neuritis (50–52). Furthermore, this technique demonstrates early microglial activation as well as remarkably prolonged activation in neurodegeneration. Documenting microglial activation in these clinical settings has been a prominent catalyst for ongoing research, but the phenotype associated with PK11195 upregulation has not been deﬁned.

Genomics, Proteomics, and Other “Omics” MPS cells originate from a clonogenic bone marrow progenitor that gives rise to macrophages and DCs (53). Despite their common origin, components of the MPS are markedly heterogeneous, beginning with the circulating elements: As noted above, inﬂammatory and resident populations of blood monocytes have been tentatively identiﬁed in both humans and mice using surface markers (20). Although the two monocyte populations are believed to originate from a bone marrow progenitor by cell-autonomous mechanisms, differential properties of tissue macrophages probably derive mainly from environmental cues (54). The properties of tissue macrophages reﬂect the demands placed on them by host tissues. Cutaneous Langerhans cells express functions associated with pathogen recognition and entrainment of host defenses. In bone, osteoclasts reﬂect the requirement for continual tissue remodeling. Pulmonary alveolar macrophages express host-defense functions associated with airborne pathogens and particulate matter, whereas thymic macrophages need high-capacity ability to engulf apoptotic cells. Growth factors related to the generation and maintenance of some of these populations have been described: Mice lacking M-CSF exhibit grossly defective osteoclast development but only subtle alterations in microglia, whereas those deﬁcient for GM-CSF show faulty alveolar macrophage function resulting in alveolar proteinosis. Deﬁciency for the ets family

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transcription factor PU.1 results in failed development of the myeloid lineage and, as noted above, provided proof of the myeloid origin of microglia (1). It is tempting to consider how concepts related to macrophage heterogeneity could be informative for microglial physiology. Macrophage functions are evidently adapted to host tissues. Even within a single organ such as spleen, regional macrophage heterogeneity has been described. The CNS MPS populations exhibit phenotypic heterogeneity, plausibly related to function. For example, parenchymal microglia may infrequently be exposed to viral pathogens but rarely to bacteria, fungi, parasites, or particulate matter. Their Tolllike receptor (TLR) complement comprises at minimum TLR1 through TLR9, but relative expression levels and signaling efﬁciency have not been deﬁned (55). Adult parenchymal microglia would, however, be confronted with lipid-rich tissue debris and, to a lesser extent, with apoptotic cells. Appropriate receptors, including scavenger receptors (SRs) and apoptosis-recognition components, appear to be expressed by parenchymal microglia. Surveillant microglia also express P2Y12, a receptor for ATP and ADP, which mediates process extension in reaction to tissue damage (36). Parenchymal microglia encounter T lymphocytes only under pathological conditions (56). Other CNS MPS cells, including PVMs, choroid plexus, epiplexus, and meningeal macrophages, may, however, be called upon to present antigen to T lymphocytes within the subarachnoid space (57). Accordingly, parenchymal microglia express MHC class II determinants at a low level compared with other CNS MPS cells; furthermore, MHC class II on parenchymal microglia appears cytoplasmic rather than associated with the plasma membrane when localized by confocal microscopy (58). Therefore, one can envision diverse MPS cells in the CNS, analogous to those found throughout the organism. Deﬁning how the varied but closely related cell populations of the MPS differ at the levels of gene expression, protein production, and

signaling has proven a tantalizing and productive ﬁeld of investigation (59). As one example, the Alliance for Cell Signaling (AfCS, http:// www.signaling-gateway.org) conducted an extensive analysis of the responses of RAW 264.7 cells (a murine macrophage-like, Abelson leukemia virus–transformed cell line on the BALB/c background). For this study, RAW cells were exposed individually to each of 22 separate ligands and, subsequently, to 231 pairwise combinations of ligands (60). Evaluation of response included cytokine secretion, second messenger generation, and signaling protein phosphorylation. Applying matrix analysis of these outputs to address how cells respond to multiple simultaneous inputs was revealing: There were remarkably selective cytokine outputs in response to combinations such as TLR- plus G protein– coupled receptor (GPCR)-mediated signaling. Evaluation following varied ligand combinations including purinergic- and prostaglandinresponsive GPCRs provided sufﬁcient information to derive a dynamic scheme for cross-regulation of cAMP and Ca2+ mobilization. Initial application of the brute-force approach was required to obtain this type of information, as existing data sets would not have allowed generation of a broad concept relating cAMP levels and Ca2+ in a dynamic interaction. Use of these multifold approaches to analyze CNS MPS cells is constrained by considerations described above for in vitro approaches to microglial biology. The most robust physiological data can likely be generated only from cells analyzed immediately ex vivo, but such cell preparations are both labor intensive and resource consumptive (61). Keeping these limitations in mind, data sets combining high-density oligonucleotide microarrays and proteomics platforms have been described for cultured microglia exposed to disease-relevant stimuli such as nitrated alpha-synuclein aggregates, a moiety present in the CNS of individuals with Parkinson’s disease (62). Comparing results from such studies with those obtained through examination of other MPS populations subjected to other stimuli will be informative (63). www.annualreviews.org • Microglial Physiology

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Systems biological studies, in which computational bioinformatics tools are applied to exploit fully the information in unbiased gene expression data sets, have provided novel insights into the MPS and will also be essential for research into microglia. One instructive report began by compiling a kinetic description of LPS-induced genes in macrophages. Cluster analysis yielded the unexpected prediction that there was an identiﬁable group of transcripts predicted to be regulated by the cAMP response element binding-protein (CREB) family member activating transcription factor (ATF)-3 (64). Network analysis suggested that ATF-3, not previously ascribed an immune or inﬂammatory function, modulated a subset of NF-κBinduced transcripts that were induced by TLR4 ligation in macrophages. Speciﬁcally, ATF-3 seemed to be a negative regulator of the late wave of NF-κB-induced genes such as IL-6. Biochemical studies validated this hypothesis both by showing closely approximated binding sites for ATF-3 and NF-κB transcription factor complexes in the promoters of the putative regulated genes and by demonstrating physical interactions between ATF-3 and NF-κB components. This type of research, beginning with unbiased descriptive compilation of regulated genes and culminating in delineation of regulatory pathways, should be applicable to dissecting how microglia respond to the distinctive stimuli with which they are confronted within their unique environment. Microarray experiments using cultured microglia have been reported but have not taken systematic advantage of the strengths of kinetic description of geneexpression changes after stimulation and have not been comprehensively exploited with bioinformatics tools (65, 66). Integrated molecular, bioinformatics, and biochemical approaches will be essential for characterizing microglial responses to stimuli and will beneﬁt from existing rich data sets. As one example, the Innate Immune Database (http://www.innateimmunitysystemsbiology.org) contains a searchable database of more than 150 microarray experiments in which TLR ligands were used to

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stimulate murine macrophages from a single inbred strain (67). The pathway to use these tools to explore microglial biology has been ably blazed by the MPS research community and now awaits utilization for CNS MPS studies.

AFFERENT SIGNALS FOR MICROGLIAL ACTIVATION— NEURODEGENERATION AND INFLAMMATION Regulation of the Microglia Phenotype in the Normal Healthy CNS One of the striking features of microglia beyond their morphology is their distinct downregulated phenotype when compared with other tissue macrophage populations. Surprisingly, only recently have factors emerged that might be responsible for this phenotype.

Soluble Factors A ﬁrst place to seek factors that regulate microglia phenotype is in the interstitial ﬂuid, the solution that bathes them. In contrast to most other macrophages, these cells are shielded from serum proteins that might lead to their activation, and there is evidence that serum constituents can selectively and potently activate macrophages (68). Furthermore, the cytokine mediator proﬁle in the healthy adult CNS is consistent with a downregulated phenotype with relatively readily detectable levels of cytokines such as transforming growth factorβ (TGF-β) and prostaglandin E2 (PGE2 ). A cytokine both soluble and bound that plays a key role in tissue macrophage survival, proliferation, and differentiation is M-CSF. Mice deﬁcient in M-CSF (op/op) show reduced numbers of microglia with minor alterations in morphology (27), but no difference in the PVM population.

Cellular Interactions The microglia are in intimate contact with cells in their immediate environment, and the

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expression of receptor-ligand pairs on the microglia and their neighbors will have a potent effect on their phenotype. The state of activation of myeloid cells is determined in part by the relative levels of expression of receptors expressing immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibition motifs (ITIMs) (69). Following receptor ligation, receptors bearing an intracellular ITAM consensus sequence are phosphorylated by Src family protein kinases, which leads to docking of SH2 domains of Syk protein kinases and an intracellular activation cascade. In contrast, ITIM-bearing receptors are phosphorylated by Src with recruitment of SHP-1 and SHP-2 phosphatases, with consequential decreased intracellular activation. The receptor CD200R is expressed on microglia and has an ITIM motif. In mice lacking CD200, normally expressed on neurons, the microglia show constitutive upregulation with changes in morphology and expression of MHC class II, and these changes also lead to more severe disease in EAE (70). There are a number of other receptor-ligand pairs that might be expected to play a similar role. CD172a/SIRPa expressed on macrophages binds the relatively ubiquitous ligand CD47, leading to downregulation of phagocytosis via an ITIM motif (71). Neumann and colleagues (72) have shown that microglia express TREM2 (triggering receptor expressed on myeloid cells-2) and that this receptor is involved in the phagocytosis of debris and downregulation of proinﬂammatory cytokine expression. Loss or ablation of this receptor leads to deﬁcient removal of cellular debris from apoptotic cells but enhanced expression of inﬂammatory mediators. Precisely how signaling via TREM2 leads to inactivation of the macrophage is as yet unclear because this receptor signals via the adaptor molecule DAP12 normally associated with cell activation. Recent studies show that deletion of TREM2 or of DAP12 from macrophages leads to identical phenotypes (namely, enhanced inﬂammatory cytokine production) when challenged with TLR agonists (73). Importantly, homozygous deﬁciency of either TREM2 or

DAP12 in patients leads to adult-onset dementing leukoencephalopathy, providing proof-ofprinciple that microglial functions are required for CNS homeostasis. The expression of these receptors and others such as the Siglecs (74) will likely contribute to maintaining the state of inactivation of the microglia. Accordingly, disturbances or loss of their ligands during pathological processes will contribute to microglial activation.

Defining Microglial Activation 2008 marked the 150th anniversary of a seminal description of microglia by Virchow, and morphological accounts of resting and activated microglia were embedded in that ﬁrst report. Subsequent studies of microglial activation began with a premise that activated microglia emerged from a resting state and underwent morphological transformation from ramiﬁed to varied activated forms, including amoeboid, rodlike, phagocytic, and so on. The notion of alternative forms of microglial activation seemed implicit in these studies but remain incompletely deﬁned. At present, one can begin to integrate contemporary macrophage biology with further understanding of microglia to attempt to reframe concepts of microglial activation (Table 1). Studies of the MPS have led to varied formulations of macrophage activation. Early during a pathological process, tissue macrophages may be stimulated either by nonself pathogens (stranger signals) or by injured-self components (danger signals). Both stimuli can activate pattern-recognition receptors such as the TLRs, SRs, and the NOD system. Effector outputs of these stimuli focus on clearance of tissue debris, generation of cues for tissue restoration, and resistance to pathogens. Together, these reactions comprise innate immune responses. Subsequent events may require establishment of responses, including lymphocyte effector functions, antibodies, and immunological memory, collectively termed adaptive immunity. MPS cells contribute to this process by antigen presentation, including the instruction www.annualreviews.org • Microglial Physiology

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CD11b/CD18 (Mac-1) mGluR2 P2Y12 P2Y6 Unknown CX3CR1 CD200L

Glutamate ATP, ADP (brief exposure) UDP (prolonged exposure) Peripheral nerve injury CX3CL1 CD200

Receptor

Serum proteins such as ﬁbrinogen

Stimulus

TGF-βR, IL-10R, GC-R, CD200L

TGF-β, IL-10, glucocorticoids, CD200

ITIM

GPCR

Src-family kinases

GPCR

GPCR

GPCR/NF-κB

Rho-family GTPases

Signaling

Smads, Jak/STAT, nuclear hormone receptor, unknown

Jak/STAT

Jak/STAT + NF-κB/IRFs

NF-κB/IRFs

Activation pattern

Contact-dependent inhibition

Inhibition

Axotomy and loss of input

Danger

Danger

Neurotoxic

Danger

Activation pattern

Alternativedeactivating (M2c)

Alternative (M2a)

Classical (M1)

Innate danger

Innate stranger

Output

Reduced inﬂammatory cytokines, less MHC class II

Reduced IL-1, ROS, iNOS/RNS

Via P2X4, mediate neuropathic pain

Phagocytosis

Process extension

TNF-α

Cytoskeletal rearrangement, enhanced phagocytosis

Output

Reduced MHC class II, reduced inﬂammatory cytokines, anti-inﬂammatory prostaglandins

Endocytic activity, mannose receptor, β-glucan receptor ﬁbrogenic cytokines, parasitocidal activity, arginase

Inﬂammatory cytokines, ROS, iNOS/RNS, increased antigen presentation via upregulated MHC II and costimulatory molecules, bacteriocidal activity, iNOS

Inﬂammatory cytokines, ROS, iNOS/RNS

Inﬂammatory cytokines, ROS, RNS

Abbreviations: GPCR, G protein–coupled receptors; iNOS, inducible nitric oxide synthase; IRF, interferon regulatory factor, ITIM, immunoreceptor tyrosine-based inhibition motif; LPS, lipopolysaccharide; RNS, reactive nitrogen species; ROS, reactive oxygen species; TLR, Toll-like receptor.

a

Microglia

IL-4/IL-13R

IFNGR plus TLR4

TLR

Signaling NF-κB/IRFs

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IFN-γ + LPS

Receptor TLR

ARI

Endogenous TLR ligands (dsDNA, HSPs)

LPS, peptidoglycan, dsRNA

Stimulus

Alternate activation of macrophages and microgliaa

Macrophage

Table 1

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of T cells to adopt varied effector programs (Th1, Th2, Th17) and in some cases directing them to the tissue from which pathogenic material originated. As part of the immunological effector program, MPS cells respond to the products of activated T cells, including IFN-γ, TNF, and IL-1. One useful exercise has been to organize MPS effector responses along varied pathways by which macrophages can be polarized by exposure to pathogen or to T cell–derived products (Table 1). Classically (or M1) activated macrophages respond to LPS plus IFN-γ, whereas alternatively activated macrophages “see” either IL-4/IL-13 (M2a); immune complexes along with IL-1 (M2b); or an aggregation of modulatory stimuli, such as glucocorticoids, TGF-β, or IL-10 (M2c) (75). Microglia differ decisively from peripheral macrophages, and their activation likely does not follow these precise pathways; nevertheless, it is instructive to consider how the underlying concept of macrophage heterogeneity might apply to microglial responses. Given the results discussed above from twophoton imaging (35), microglia may now be regarded as surveying the healthy CNS and engaging varied modes of progression from the surveillant state to effector microglia. This concept should replace the notion that microglia proceed from resting to activated (76). Next, stimuli for microglia are analogous to, but clearly distinct from, those confronted by peripheral macrophages. Three (at least) clear distinctions between microglia and macrophages apply here: 1. Microglia reside behind the BBB so that serum products represent danger signals indicating BBB breach. 2. Microglia are MPS family members but need also to be equally regarded as brain glial cells, so that altered synaptic activity with perturbation of neurotransmitter availability will affect microglial activation states and effector properties. Regulation by neurotransmitter signal-

ing may intermittently affect peripheral macrophages, as described for the cholinergic anti-inﬂammatory response (77), but neurotransmitter effects are more diverse and pervasive for microglia. 3. Microglial cells, perhaps as a consequence of living among fragile, nonrenewing neurons, exhibit an actively repressed phenotype, so that removal of (mainly neuronally derived) inhibitory constraints constitutes a type of danger signal, indicating that neuronal function is impaired. Here, we cite examples of each of these special considerations for microglial activation. BBB disruption as a danger signal. Serum constituents activate microglia (which is a key limitation of using in vitro cultures to examine their physiology). One molecular interaction that underlies microglial activation during demyelinating disease may be exposure to ﬁbrinogen, which engages CD11b/CD18 integrin heterodimers (68). Suppressing this stimulatory pathway reduces the severity of EAE. Microglial responses to neurotransmitter alterations. Glutamate is an excitatory neurotransmitter, and excess glutamate stimulation is neurotoxic. Glutamate excess can result from increased release (by neurons) or decreased clearance (which is mainly the responsibility of astrocytes). Glutamate receptors can be coupled to calcium entry (ionotropic) or to GPCR signaling (metabotropic or mGluRs). Microglia express mGluRs and respond to ﬂuxes in extracellular glutamate. Microglial responses to acute and chronic injury are regulated in part by selective activation of mGluRs, culminating either in neurotoxic or neuroprotective outcomes (78, 79). Adding complexity, extrinsic inﬂammatory stimuli regulate mGluR expression. Danger signals from damaged cells and from altered neurotransmitter levels. As a composite example of specialized danger responses by microglia, ATP is a purinergic

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neurotransmitter, signaling both to P2X ionotropic and P2Y metabotropic receptors. Microglia are endowed with both P2X and P2Y purinergic receptors. As noted above, extracellular ATP, released in part from damaged neurons, acts through microglial P2Y12 to mediate process extension after a cortical laser lesion (5, 36). Therefore, P2Y12 can be regarded as a specialized PRR for danger signaling within the CNS. Neuronal cell death or injury confronts microglia with a requirement to function as phagocytes, and another purinergic receptor, P2Y6, acting in a longer time frame than P2Y12 (which transduces signals within seconds), appears to play a signiﬁcant role in microglial phagocytosis (80). Intriguingly, the endogenous danger ligand for P2Y6 is extracellular UDP, again providing an instance of selectivity in the microglial reaction to injury.

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Microglial activation by loss of neural input. P2X4 receptors are induced on spinal microglia via Src family kinases after peripheral nerve injury and are critically involved in the CNS mediation of neuropathic pain (tactile allodynia) in pain models, although the speciﬁc mechanism by which this effect occurs is uncertain (81–83). This phenomenon, given its speciﬁcity, can be viewed as an alternative mode of microglial activation, evidenced by selective upregulation of a purinergic receptor. Microglia are actively repressed. CX3CL1 is a membrane-tethered chemokine that is tonically released from CNS neurons through the action of ADAM proteases. Microglia receive tonic inhibitory inputs through the CX3CL1 receptor, CX3CR1 (21). If this tonic inhibition is removed, microglial neurotoxicity is unleashed in response either to systemic inﬂammatory stimuli or to damage to resident neurons (21). Release from constitutive inhibition is also believed to underlie the devastating neurological consequences of deﬁciency either for microglial TREM2 or its intracellular adaptor DAP12 (84, 85). Although TREM2 is a transmembrane receptor–like molecule with clear-cut inhibitory function for varied stimuli, 132

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its endogenous ligands remain to be fully characterized.

EFFECTOR FUNCTIONS OF MICROGLIA: DEVELOPMENT AND REPAIR The early entry of macrophages into the embryonic brain was described above (Origin of Microglia section). It is uncertain whether these cells persist and give rise to adult microglia, but their potential roles in development have attracted interest. Microglial developmental functions may be informative for roles in disease, and conversely.

Synaptic Remodeling: Pruning, Stripping, and Plasticity During CNS development, axonal connections with synaptic targets exceed those required and are reduced in a process termed pruning. For some years, it has been clear that synaptic pruning is aberrant in mice lacking speciﬁc MHC class I determinants, although mechanisms continue to unfold (86). Recently, Stevens et al. (87) found that mice lacking complement components C1q or C3 exhibit defects in visual system synaptic reﬁnement that are similar to those found in MHC class I–deﬁcient animals. They and others (86, 87) proposed assigning microglia an effector role in this aspect of CNS development, as microglial cells are the CNS elements that express the appropriate complement receptors. Stevens et al. (87) also provided evidence that synaptic loss might be associated with aberrant engagement of complement pathways in a mouse glaucoma model. The opposite possibility has also been proposed: speciﬁcally, that microglia assist in synaptic repair after brain lesions. Synaptic repair is incorporated along with learning as different aspects of synaptic plasticity. By extrapolation from developmental synaptic pruning, Cullhein & Thams (88) suggested that microglia contribute to plasticity after lesions, by pathways similar to those used for pruning excess synapses during development and facial axotomy, but direct evidence is lacking.

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Inducing Apoptosis in Supernumerary Purkinje Cell Neurons During Development CNS development is characterized by waste: excess production of synaptic connections and generation of a surplus of cells. Supernumerary cells undergo apoptotic cell death and are engulfed without generating inﬂammation by microglia (14, 89, 90). It was long speculated that microglia might also provide inputs that promote developmental neuronal apoptosis (89), and data have been forthcoming to support this contention. Cerebellar slice cultures from neonatal mice demonstrate apoptosis of Purkinje cell neurons, which are elaborately invested with microglial processes. Furthermore, elimination of microglia with toxic liposomes reduces the efﬁciency of Purkinje cell commitment to apoptosis (91). Can microglia also provide growth factors for neurons during development? Here the data are indirect and rely solely on observations in adult mice. PU.1-deﬁcient mice lacking microglia exhibit grossly intact CNS structure and function. However, studies of adult neural stem cell niches provide tantalizing hints of supportive microglial-neural progenitor relationships. In adult mice, neural stem/progenitor cells (NSPCs) are localized in the hippocampal subgranular zone (SGZ) and in the rostral subventricular zone (SVZ). In culture, NSPCs give rise to both neurons and glia, thereby fulﬁlling criteria of multipotency. Conditioned medium of microglial cultures contained factors capable of supporting prolonged neurogenesis, implying an instructive function for microglia toward NSPCs of the adult SVZ (92). Microglia secrete factors that direct NSPC migration in vitro (93). Microglia also promoted neuronal differentiation in these studies (93). Taken together, these data suggest a role for microglia in promoting CNS lesion repair by NSPCs. This concept was given relevance for neurodegenerative disease, through studies of microglia from transgenic mice expressing mutant forms of human presenilin-1 (PS1) associated with familial Alzheimer’s disease (AD). NSPCs in these

mice fail to exhibit environmental enrichmentmediated proliferation and neurogenesis. In vitro, NSPCs from transgenic mice expressing either mutant or wild-type PS1 proliferated and differentiated equally. By contrast, co-culture of microglia from mice expressing mutant PS1 secreted with wild-type NSPCs recapitulated the phenotype of impaired proliferation and neuronal differentiation. This effect was mediated by microglial-derived secreted factors. The data supported an important role for microglia in regulating NSPCs during physiology and pathology (94, 95).

Vascular Development It is not clear whether microglia exert functions that promote CNS vascularization. The notion that they might exert such functions comes by inference from two observations: ﬁrst, the provocative ﬁnding that microglia are present in the CNS before vasculogenesis and that invading vessels are closely invested with microglia; second, that experimental CNS tissue implants are colonized by microglia before they are vascularized (89).

PATHOLOGY Introduction Microglia are exquisitely sensitive to disturbances of their microenvironment, and they have been dubbed the sensors of pathology (96). The early and rapid response of microglia is entirely consistent with the role of tissue macrophages as the ﬁrst line of defense against infection or injury. The response of the microglia is not a linear process varying simply by degree, but rather, as has been documented in other macrophage populations, their response is dictated by the nature of the stimulus, the receptor repertoire that is engaged, and the prior state of the macrophage (97) (see section on Deﬁning Microglial Activation). In the context of neurodegeneration, a number of important variables must also be considered in the temporal and spatial domains. The two most

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common neurodegenerative conditions of the CNS are stroke and AD. In the former, the death of neurons is extremely rapid, a matter of minutes; in the latter, the pathology driven by the presence of a misfolded protein may lead to death of a slowly increasing number of neurons over years or even decades. The former stimulus is a step function, the latter a slow ramp function. The slow degeneration of neurons—their synapses, soma, axons, and myelin sheath—may continue for many years, providing a stimulus that must lead to adaptive changes in the surrounding microglia. The adaptive changes may then be inﬂuenced by other comorbidities and systemic inﬂuences that communicate with the brain (98).

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Neurodegeneration versus Neuroprotection A major theme in studies of the role of microglia in neuropathology is the dichotomy between their contributions to neurodegeneration versus neuroprotection. The role of macrophages in wound repair is well documented and part of the innate immune response (99). The thinking behind this dichotomization is that if we can understand the two components we can minimize the harmful and capitalize on the beneﬁcial (100, 101). This is a hugely ambitious aim when we recognize that this has yet to be achieved for signiﬁcant clinical advantage in any organ, let alone within the complex context of the CNS. Furthermore, the CNS is an immune-privileged organ in which evolutionary pressures have ensured that the innate and acquired immune responses are tightly controlled. Overcoming the immune privilege per se is unlikely to beneﬁt the host except perhaps in combating infections. Finally, microglia themselves might be targeted by a pathological process, which also affects adjacent tissues. Recent data from studying the genetically determined inﬂammatory demyelinating metabolic disorder X-linked adrenoleukodystrophy suggest that such a mechanism might play a part in the cerebral form of this disease (102). 134

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SPECIFIC NEUROLOGICAL DISORDERS Alzheimer’s Disease It is now nearly 20 years ago that an innate inﬂammatory response in AD, the presence of morphologically activated microglia, was described, rapidly followed by studies indicating that people taking nonsteroidal antiinﬂammatory drugs were in some way protected from the onset or progression of AD. The role of inﬂammation in AD has been extensively researched and exhaustively reviewed (103, 104), but the contributions to the disease of the microglia and the associated innate inﬂammatory response are by no means clear. A wide variety of inﬂammatory mediators are expressed by microglia when they are challenged in vitro with the Aβ peptides derived from the amyloid precursor protein (APP). However, there is a lack of consensus on which cytokines or other mediators are indeed present in the postmortem brain tissues of patients that died with AD. In APP transgenic mice (which mimic the deposition of Aβ amyloid in the brain, a hallmark of AD pathology), the microglia do indeed appear morphologically activated, particularly in the immediate vicinity of the plaques. But again, there is little agreement on the cytokine proﬁle associated with these microglia (98), which is consistent with the difﬁculty in performing assays of low abundance and highly soluble proteins such as the cytokines (see above). A more direct route to assessing precisely which molecules are synthesized by microglia that might either promote or delay disease is to study their impact on the plaque load. The plaques in various mouse models, and in human AD patients, inexorably increase with age, and the conclusion must be that the microglia are rather inefﬁcient phagocytes, which is perhaps not surprising given their very slow removal of neuronal and myelin debris after a stroke (105). However, indirect activation of the microglia by intracranial challenge with LPS leads to a decrease in plaque load (106). Others suggest that bone marrow–derived macrophages selectively invade the region of the plaques and play a role

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in removal of the amyloid material (107). How such ﬁndings translate to the clinical situation is unclear because these models are not progressive neurodegenerative disease models. The use of bone marrow chimeras in these studies will also have to be reassessed. An exciting development in the attempts to manipulate the immune system for beneﬁt in AD comes from studies in APP transgenics, which show that systemic immunization against Aβ leads to removal of the amyloid plaques (108). Several hypotheses have been suggested as to how systemic antibodies might achieve this, including microglia phagocytosis via FcRs. Alternatively, antibody-mediated clearance of systemic/circulating Aβ would promote increased drainage of Aβ from the brain and inhibit Aβ ﬁbril aggregation (109). These mechanisms are not mutually exclusive, and it is by no means clear that the microglia are essential for effective removal of the amyloid. This intriguing experimental approach has been rapidly translated into human: Despite the fact that it is unclear how the amyloid is removed, this approach does permit a direct test of the hypothesis that removal of the amyloid plaques might slow disease progression. A recent neuropathlogical analysis of the brains from a small number of patients who were vaccinated in the AN1792 Elan Pharmaceuticals trial shows a positive correlation between the degree of plaque removal and the antibody response. However, clinical analysis of the immunized cohort revealed evidence neither of improved survival nor of an improvement in the time to severe dementia (109a). It is too early to conclude that a vaccination strategy will not work, and it is possible that earlier or different vaccination strategies will be required to achieve a beneﬁt.

Multiple Sclerosis MS is an acquired inﬂammatory demyelinating disorder of the CNS and is the leading cause of nontraumatic disability among young adults in the United States and Europe. The cause of MS is unknown but includes both en-

vironmental and genetic factors, some of which have been identiﬁed (110). MS research has depended largely on an autoimmune animal model, EAE, that exhibits features of CNS inﬂammatory demyelination and paralysis that resemble aspects of MS. The model disease begins with peripheral immunization, usually with peptide epitopes of myelin proteins. EAE is a T cell–dependent disease, most commonly mediated by CD4+ T cells, including those that express both Th1 and Th17 cytokine proﬁles. EAE proceeds either by monitoring animals until disease onset around two weeks postimmunization (active EAE) or by isolation and transfer of primed cells from draining lymph nodes or spleen (adoptive-transfer EAE). Roles of microglia in EAE illustrate their ability to contribute to adaptive (auto)immune reactions that target CNS antigens. We have long known that, to elicit EAE after adoptive transfer, T cells require restimulation with antigen in the CNS compartment. Classic experiments used radiation bone marrow chimeras to generate animals in which bone marrow– derived cells expressed MHC determinants distinct from those present on parenchymal microglia. Adoptive transfer of myelin-speciﬁc T cells that were restricted by the parenchymal cells did not cause disease, whereas those that recognized antigen in the context of PVMs derived from the bone marrow inoculum led to EAE (39). In follow-up studies, eliminating all potential APCs except for CD11c+ PVMs produced mice that remained susceptible to adoptive-transfer EAE (40). A variant form of adoptive transfer, in which EAE was primed by one myelin epitope and mice with ongoing disease received unprimed TCR transgenic T cells speciﬁc for a different epitope, was used to show that myelin-speciﬁc T cell proliferation occurred mainly in the CNS after disease onset (111). This experiment extended concepts of the capabilities of CNS DCs, which arise in the context of active CNS inﬂammation (112). Nevertheless, it has yet to be shown that the healthy CNS parenchyma contains DCs competent to initiate immune responses by presenting antigen to naive T cells in vivo (113). www.annualreviews.org • Microglial Physiology

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Following priming, do parenchymal microglia contribute to neurobehavioral impairment in EAE? This issue was addressed by generating CD11b-HSV-TK mice that expressed a lineage-restricted Herpes virus thymidine kinase (HSV-TK) suicide gene in myeloid cells, so that administration of gancyclovir would eliminate proliferating CD11b+ cells. Radiation chimerism with wild-type bone marrow produced animals in which only the radiationresistant parenchymal microglia remained susceptible to gancyclovir-mediated death. Combining EAE immunization with gancyclovir treatment led to mice exhibiting EAE with microglial paralysis. These mice exhibited mild EAE signs, indicating a role for microglia in the severity of EAE (114). Are these studies informative for MS? The journey from EAE mechanism to MS pathogenesis is notoriously tortuous, the route bafﬂing, and the destination uncertain. Proof-ofprinciple that MS is autoimmune has not been forthcoming. For that reason, immunopathologic mechanisms of EAE seem more directly pertinent for MS than do applications of the principles of autoimmunity. From EAE, we could predict that MPS cells would exhibit DClike characteristics in MS tissues, a hypothesis with experimental support (115, 116). In white matter, actively demyelinating MS lesions are identiﬁed by the presence of myelin debris in macrophages. Early-active zones in which demyelination began within a few days can be distinguished from late-active zones in which demyelination has been present for 2–4 weeks, by deﬁning whether labile or relatively stable myelin breakdown products are found within phagocytes (117). Early-active MS lesions contain a mixture of inﬁltrating hematogenous monocytes and microglia, which are differentiated by morphology, localization, and surface markers; late-active MS lesions are typiﬁed by a monomorphic population of phagocytic macrophages (117). Numbers of MPS cells in individual early-active and late-active MS lesions are identical, suggesting that phagocytic macrophages in late-active regions arose both from monocytes and microglia. Enumeration of

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monocytic and microglial cells in early-active zones suggests that between one-half and twothirds of phagocytic macrophages in late-active MS white matter lesions arise from microglia (117, 118). Recently, it has become clear that MS is not exclusively a white matter disease: Remarkably, cortical (or gray matter) demyelination is more extensive, as a fraction of total myelin, than is white matter demyelination. The largest and most mysterious demyelinating foci of MS gray matter affect the most superﬁcial three layers of the cortical ribbon and are termed subpial lesions (119). Among inﬂammatory demyelinating diseases, subpial lesions are relatively speciﬁc for MS (120). Although the mechanisms underlying subpial lesions remain unclear, the inﬂammatory characteristics of such lesions in chronic-progressive disease are noteworthy: There is a paucity of lymphocyte or monocyte inﬁltration, as evidenced by sparseness of perivascular cuffs, whereas microglial activation is remarkably robust (119). The data suggest the possibility that microglia are key effectors in the process of subpial demyelination, possibly activated by diffusible factors from the cerebrospinal ﬂuid. Subpial demyelination may be a critical determinant of disability in MS patients (121), so its mechanisms are crucially important for therapeutic development. Equally vital, data to date comprise only chronic material; imaging studies indicate that cortical damage occurs very early during the course of MS, and the mechanisms underlying cortical demyelination in the initial phases of disease have yet to be characterized. Roles of microglia, which seem prominent in chronic cortical demyelination, may be different in the earlier stages of MS. Can microglia promote repair of MS lesions? Remyelination is clearly a prevalent and poorly understood attribute of MS lesions (122). Many factors elaborated by activated microglia might support oligodendroglial survival or function (123). However, present MS therapy aims at complete elimination of MSassociated neuroinﬂammation. Until this goal is shown by evidence to be impossible, it is most

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logical and straightforward to strive to abrogate CNS inﬂammation, rather than allow damage while trying simultaneously to bias the process toward enhanced repair.

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Prion Disease Prion disease offers a tractable laboratory model to study many aspects of fatal neurodegeneration associated with protein misfolding and has the additional attraction that it is the same disease in both humans and animals. In murine prion disease, the microglia become activated early in the disease process, even in the absence of widespread histologically detectable amyloid protease–resistant PrPsc deposits (124). The cytokine proﬁle associated with this activated phenotype was not dissimilar from that observed in APP transgenics, featuring low levels of inﬂammatory cytokines but, in addition, readily detectable levels of TGF-β and PGE2 , a phenotype previously associated with macrophages that had phagocytosed apoptotic cells (125). This phenotype appears prior to the onset of neuronal apoptosis but in association with synaptic loss (126), indicating ﬁrst that prion disease is a member of diseases with an early synaptic degeneration (a synaptopathy), and second that this may lead to an atypical microglial activation that has been referred to as anti-inﬂammatory or benign. In prion disease induced on a wild-type background, there is no evidence that the enhanced levels of PGE2 are detrimental, nor that TGF-β is detrimental. However, when TGF-β is neutralized by decorin, delivered by an otherwise harmless adenoviral vector, this leads to acute and marked neuronal degeneration, highlighting the importance of anti-inﬂammatory regulation in chronic neurodegenerative disease (127). To investigate how the activated microglia phenotype might be affected by systemic inﬂammation, a common occurrence in clinical neurodegenerative disease, mice were challenged with endotoxin to mimic a systemic infection. This maneuver led to a dramatic switch in the microglial phenotype with an ag-

gressive inﬂammatory cytokine proﬁle, exacerbation of the sickness behavior associated with endotoxin challenge, and increased neuronal apoptosis (128). Investigators have proposed that the microglia in the prion diseased brain are primed by the ongoing pathology and that a secondary stimulus, the signaling of systemic inﬂammation across the BBB, switches these cells to an aggressive phenotype (98). Whether microglia switched to an inﬂammatory phenotype phagocytose or degrade the prion protein by secretion of relevant proteases remains unclear. The concept of the rapid switching of the microglia phenotype is of course entirely in keeping with what we know of the considerable degree of plasticity of the cells of the macrophage lineage. Systemic inﬂammation has a profound impact on a number of other animal models of neurological disease (98) and accelerates cognitive decline in Alzheimer’s patients (129).

Amyotrophic Lateral Sclerosis (ALS) ALS is a fatal neurodegenerative disease that also involves a misfolded protein. In contrast to prion diseases, in which the protein accumulates predominantly extracellularly, the toxic protein in ALS is an intracellular protein and exerts its action intracellularly. Approximately 5–10% of ALS is an autosomal-dominant familial form of the disease, of which a signiﬁcant proportion has mutations in the enzyme superoxide dismutase-1 (SOD-1). Microglial activation is present in the vicinity of the degenerating motorneurons (130). Transgenic mice expressing the mutant SOD-1 have been a valuable model for understanding disease pathogenesis, revealing that the demise of the motorneurons is not a cell-autonomous event, but rather depends on the surrounding nonneuronal cells (131). Dissection of the role of microglia has come from several different approaches. Mice in which the microglia are no longer able to respond to the downregulatory inﬂuence of the chemokine CX3CL1 have been www.annualreviews.org • Microglial Physiology

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crossed with SOD-1-expressing mice, and these mice show a modest shortening of survival time (21). More dramatic effects have been uncovered in studies in which mice expressing Crerecombinase driven by the CD11b promoter are crossed with a LoxSOD1G37R mouse, leading to levels of mutant SOD-1 that are signiﬁcantly reduced in microglia (132). These mice lived longer than LoxSOD1G37R mice, and, in particular, the late stage of the disease was prolonged. The role for microglia expressing SOD-1 in accelerating disease progression was also demonstrated by another group using a different approach in which SOD-1 mice crossed with PU.1 mice were transplanted with either wild-type or SOD-1-expressing bone marrow that would then populate the tissues, including the brain, with either wild-type or SOD1-expressing macrophages (2). The mice transplanted with wild-type cells lived longer than those transplanted with SOD-1 bone marrow, and again it was the later stage of the disease that appeared to be prolonged. SOD-1-expressing microglia generated more superoxide and nitric oxide when challenged with LPS in vitro than did wild-type microglia. The general principle, namely that action of a mutant gene in microglia can promote selective loss of neuron subpopulations, has since been validated in other diseases (133). In both these studies, the impact of the Cre-recombinase or bone marrow transplantation will affect not only the microglia, but also the systemic macrophage populations and any monocytes that might be recruited to the vicinity of the degenerating motor neurons. The differential contributions of the local microglia and of the systemic macrophage have yet to be separately assessed and may be consequential for studies on prion disease, as described above.

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Tumor Primary brain tumors are primarily derived from the astroglial lineage. These lesions are inﬁltrative and typiﬁed by vigorous angiogenesis; their metastatic potential is relatively low. Considerations of the roles of microglia in 138

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brain tumor follow from a robust literature about tumor-associated macrophages (TAMs) (75). TAMs are considered to provide a variety of proneoplastic functions, while residing in an immunosuppressive environment, which restrains antitumor properties such as capacity to generate reactive oxygen and nitrogen species. Investigators have proposed that TAMs represent variant M2 macrophages that have been exposed to immunosuppressive prostaglandins, TGF-β, and IL-10. Beyond inhibiting their antitumor potential, these environmental cues could stimulate TAMs to produce growth and angiogenic factors for tumor cells. Such principles translate readily to an evaluation of the roles of microglia in primary brain tumors. Microglia were detected in brain tumors more than 80 years ago (134). Subsequent research focused on three issues: (a) Why do tumor-associated microglia appear quiescent rather than tumoricidal? (b) What microglial factors promote tumor growth or survival? (c) How can microglia be stimulated to express tumoricidal functions? As a generalization, immunosuppressive factors present in other tumor beds are also detected in brain tumors, prominently including TGF-β, prostaglandins, and IL-10. The origin of tumor-associated microglia is uncertain. Glioma cells exuberantly express CCL2, so much so that the human protein was ﬁrst isolated from glioma culture ﬂuid (135). CCL2 acts toward hematogenous monocytes, but CCR2, the relevant receptor, is not expressed on microglia. Furthermore, ﬂow cytometric analysis of human tumor samples revealed inﬁltrating CD45hi cells, unlike the mixed population of CD45hi and CD45dim cells found in rodent glioma models (134). It is plausible therefore that tumor-inﬁltrating MPS cells are mainly monocytic in origin, attracted through an impaired BBB by chemoattractants such as CCL2. Tumor-inﬁltrating microglia, stimulated by local IL-6, produce IL-10, which suppresses macrophage antitumor effector functions and also promotes Type 2–related angiogenic factors. Type 2 macrophage/microglia may also support invasiveness by expression of matrix metalloproteases. The presence of

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MPS cells within brain tumors has elicited therapeutic endeavors, mainly aimed at stimulating antitumor immunity through peripheral immunization with DCs. To present tumor antigens, DCs are pulsed with antigenic peptides or programmed to express glioma cell cDNAs (136). One particularly interesting approach involves use of cDNA from glioma stem cells (136). These strategies rely in part on the APC properties of resident MPS cells to restimulate tumor-speciﬁc CD4+ T cells within the CNS. Microglia may also be called upon to execute tumoricidal functions following stimulation by activated CD4+ T cells.

Human Immunodeficiency Virus (HIV) Infection with HIV-1 imposes a vast burden of neurological disease, exclusive of that caused by opportunistic infection and metabolic compromise (137, 138). During early years of the pandemic, HIV-associated dementia (HAD) was the most feared and most prominent of these complications. HAD is a clinical syndrome comprising cognitive, affective, and motor symptoms. With highly active antiretroviral therapy (HAART) and prolonged survival, the incidences of HAD have lessened and those of simpairment along with that of painful sensory neuropathy have increased. Two considerations placed microglia and inﬁltrating MPS cells squarely in the center

of HAD pathogenesis: First, mononuclear phagocytes are the major infected population in the CNS, and second, neurons, whose function is most evidently impaired, are very poorly infectable and are not grossly reduced in number. Given their prevalence and severity, HAD and related conditions represent the most signiﬁcant disease for which primary pathogenic pathways involve microglia. HIV-1, like other lentiviruses (139), enters the CNS in Trojan horse mode within trafﬁcking MPS cells. In HAD tissue sections, most cells containing viral antigens are macrophages and microglia. In vitro studies suggest that microglial physiology is disrupted by infection, so that potentially neurotoxic functions are engaged and neuroprotective responses are blunted. Microglia can elevate levels of excitotoxins such as glutamate and quinolinic acid by diverse mechanisms, many of which seem active in HAD; combined with oxidative stress, these components may lead to neuronal structural pathology and functional compromise (62, 140). Microglial activation may also operate through production of inﬂammatory cytokines to recruit astrocytes and BBB elements into the HAD process. There is evidence for unapparent BBB dysfunction and for the participation of astrocytes in mediating neuronal dysfunction. Understanding mechanisms underlying HAD is regarded as signiﬁcant for therapeutics, for extension to other dementing illnesses, and for addressing neuron-glial interactions during disease.

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS Research in the Ransohoff lab has been supported by the U.S. National Institutes of Health, the National Multiple Sclerosis Society, the Charles A. Dana Foundation, the Nancy Davis Center Without Walls, and the Williams Family Fund for Multiple Sclerosis Research. Research in the Perry lab has been supported by the Medical Research Council (UK), the Wellcome Trust, the Multiple Sclerosis Society (UK), and the European Union. We thank Dr. Natalia M. Moll and Anna Rietsch for generating images shown in the Table. www.annualreviews.org • Microglial Physiology

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The Liver as a Lymphoid Organ Ian Nicholas Crispe David H. Smith Center for Vaccine Biology and Immunology, Aab Institute for Biomedical Research, University of Rochester Medical Center, Rochester, New York 14642; email: nick [email protected]

Annu. Rev. Immunol. 2009. 27:147–63

Key Words

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

antigen presentation, hepatitis, Kupffer cells, innate immunity, sinusoid, stellate cells

This article’s doi: 10.1146/annurev.immunol.021908.132629 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0147$20.00

Abstract The liver receives blood from both the systemic circulation and the intestine, and in distinctive, thin-walled sinusoids this mixture passes over a large macrophage population, termed Kupffer cells. The exposure of liver cells to antigens, and to microbial products derived from the intestinal bacteria, has resulted in a distinctive local immune environment. Innate lymphocytes, including both natural killer cells and natural killer T cells, are unusually abundant in the liver. Multiple populations of nonhematopoietic liver cells, including sinusoidal endothelial cells, stellate cells located in the subendothelial space, and liver parenchymal cells, take on the roles of antigen-presenting cells. These cells present antigen in the context of immunosuppressive cytokines and inhibitory cell surface ligands, and immune responses to liver antigens often result in tolerance. Important human pathogens, including hepatitis C virus and the malaria parasite, exploit the liver’s environment, subvert immunity, and establish persistent infection.

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INTRODUCTION sinusoid: thin-walled blood space through which blood passes in the liver Kupffer cell: intravascular macrophage lining the liver sinusoids

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DC: dendritic cell TLR: Toll-like receptor APC: antigenpresenting cell

The liver stands at a hemodynamic conﬂuence. In distinctive, thin-walled vessels termed sinusoids, oxygenated blood from the arterial system mixes with portal venous blood returning from the intestine. The sinusoids contain a diversity of immunologically active cell types, including both lymphocytes and myeloid cells (Figure 1). The Kupffer cells form a large intravascular macrophage bed and, with liver dendritic cells (DCs), come in both immunogenic and tolerogenic forms. The liver also contains diverse lymphocytes, including T cells, natural killer T (NKT) cells, and natural killer (NK) cells. Portal venous blood contains the products of digestion, along with antigens and microbial products that originate from the bacteria in the small and large intestine. Among

Hepatocytes HSC

mDC/ pDC

KC LSEC

these bacterial products is lipopolysaccharide endotoxin (LPS), derived from the cell walls of Gram-negative bacteria. Under normal conditions, LPS is undetectable in the systemic circulation, but it is present at up to 1.0 ng/ml in portal venous blood (1). The cells of the hepatic sinusoids express the LPS receptor and effectively remove this molecule so that the systemic circulation is protected from endotoxemia (2). Many cells of the innate immune system express the LPS receptor, which consists of Toll-like receptor-4 (TLR4) together with the molecules CD14 and MD2; engagement of this receptor on most cell types delivers a strong activating signal. However, in the liver these receptors are continuously exposed to low levels of LPS, resulting in altered responsiveness to an LPS challenge. At the same time, the adaptive immune cells of the liver are exposed to food-derived antigens, the majority of which are harmless. The continuous presence, under normal conditions, of both TLR ligands and antigens has resulted in a distinctive set of mechanisms to maintain self-tolerance yet deliver immunity to infection. Liver immunity features a local concentration of overlapping innate immune mechanisms, together with the capacity of unusual cell types to act as antigen-presenting cells (APCs). The liver’s resident immune cells are not passive in the face of continuous exposure to antigens and LPS; instead they exist in a state of active tolerance, which results in liver allograft tolerance (3) but also creates a window of vulnerability for welladapted pathogens. This state of tolerance is metastable; the right combination of signals can reverse tolerance and activate immunity locally.

Figure 1

INNATE IMMUNITY IN THE LIVER

Antigen-presenting cells (APCs) in liver sinusoids. The liver contains multiple subsets of dendritic cells, including myeloid and plasmacytoid dendritic cells (mDCs and pDCs). There are abundant mononuclear phagocytes in the form of Kupffer cells (KCs), and these can express costimulatory molecules. In addition, there are two additional populations of APCs in the form of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs). The evidence for APC function in each of these cell types is summarized in the text.

The LPS from intestinal bacteria is not the only immune stimulus to which the liver is exposed. Pattern-recognition receptors in the liver sense the presence of enteric pathogens. These include cell surface and endosomal TLRs, cytoplasmic nucleotide-binding oligomerization

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domain (NOD)-like receptors, and RNA helicases, including retinoic acid inducible protein-I (RIG-I). The TLRs recognize a diverse array of bacterial and viral molecules, including bacterial lipopeptides (heterodimers of TLR1 and TLR2, and of TLR2 with TLR6); LPS and ﬂagellin (TLR4 and TLR5, respectively); and exogenous dsRNA, viral ssRNA, and bacterial unmethylated DNA (TLR3, TLR7, TLR8, and TLR9). The NOD receptors recognize bacterial peptidoglycans, whereas the RIG-I molecules recognize structural features of viral ssRNA. These receptors, their speciﬁcity, and their roles in host defense were recently reviewed (4, 5). From the perspective of a global discussion of liver immunity, the key point is that signals from these diverse receptors converge on two signaling pathways. Thus, all of the TLRs except TLR3 transmit signals via the adaptor protein MyD88 (myeloid differentiation factor-88), which results in the activation of the kinases p38, JNK, and IκB kinase, leading to NF-κB activation. NOD receptors also activate NF-κB. The TLR4 receptor complex, in addition to activation via MyD88, recruits the adaptor protein TRIF (TIR-domain containing adaptor recruiting interferon-β), which acts via TBK1 (TRAF family member–associated NFκB activator–binding kinase 1) to cause phosphorylation and nuclear localization of IRF-3 (IFN regulatory factor 3), the transcription factor that drives synthesis of type 1 interferon (IFN). Ligation of TLR3 selectively activates this signaling pathway. Similarly, RIG-I and its homolog MDA-5 promote the activation of mitochondrial IPS-1 (IFN-β promoter stimulator 1), resulting in IRF-3 activation and type 1 IFN secretion. These pathways are optimized such that pattern-recognition receptors engaged by bacterial products generally promote NF-κB activation, whereas those pathways activated by viral infection strongly induce IFN-β. The continuous, low-level stimulation of the former pathway is one of the distinctive features of the liver environment (Figure 2).

NK cells are present at higher frequency in the liver than in most tissues. Thus, in human liver leukocytes obtained by elution from donor livers (6), in cell suspensions obtained from human liver tissue (7), and in the cells isolated from the mouse liver by enzymatic digestion (8), NK cells make up as many as 50% of liver lymphocytes. Similarly to NK cells elsewhere, these cells respond both to cytokine activation and to engagement of an excess of activating receptors over inhibitory receptors (9). Once activated, they manifest their function through cytokine synthesis and cytotoxicity. NK cells express two key adaptor molecules: DAP10 and DAP12. DAP10 associates with the activating NKG2D receptor, and the ITAM (immunoreceptor tyrosine-based activation motif)-bearing DAP12 adaptor protein associates with several receptors, including the CD94-NKG2C heterodimer and the Ly49H receptor. Ligands for NKG2D are expressed in the liver under diverse circumstances. Low amounts of NKG2D ligands are expressed constitutively in the liver (20), and these ligands can be upregulated after viral infection or transformation of hepatocytes. The NKG2D ligands include MHC class I–related proteins A and B (MICA and MICB), which are expressed on human hepatocellular cancer cells (10), and mouse retinoic acid early inducible-1 (RAE-1), which is transcriptionally upregulated by cytomegalovirus infection (11). Liver NK cells are induced to synthesize IFN-γ in response to IL-12 (12) and to manifest perforin-dependent cytotoxicity in response to the Kupffer cell–produced cytokine IL-18 (13). They are also cytotoxic owing to the expression of TRAIL (TNF-receptor apoptosis-inducing ligand), which can engage death receptors that are induced on hepatocytes by hepatitis B virus (HBV) (14). The liver lymphocytes also contain an unusually high frequency of NKT cells. These include both canonical NKT cells expressing an invariant T cell receptor (TCR) that binds to CD1d complexed with α-galactosylceramide and the noncanonical NKT cells that recognize other ligands. Liver NKT cells are abundant

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HBV: hepatitis B virus

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Constitutively engaged by gastrointestinal ligands Bacterial peptidoglycans

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Figure 2 Effects of microbial products. The model explains the effects of the liver environment on patternrecognition receptors. The presence of detectable LPS in portal blood suggests that other gut-derived microbial molecules may be present also. We therefore propose the model that NOD proteins and the subsets of TLRs that recognize microbial products are constitutively engaged in the liver. In contrast, receptors for viral elements (TLR3, RIG-I, MDA-1) are not engaged under normal conditions. We propose that this changes the balance between NF-κB and IRF-3-dependent signaling pathways. In this diagram, heterodimeric TLRs are indicated as TLR1/2 and TLR2/6.

both in mouse (15) and in human (6). These cells were recently ﬁlmed patrolling the hepatic sinusoids, based on their expression of a GFP reporter molecule driven by the endogenous CXCR6 promoter (16). Despite their thymic origin (17) and expression of a TCRαβ generated by V(D)J recombination, these CD1d-reactive T cells show evolutionary convergence with leukocytes expressing innate pattern-recognition receptors; their TCRs recognize glycolipid antigens that are conserved features of bacterial cell walls. These include glycosphingolipids from the soil bacterium, Sphingomonas sp. (18), and a diacylglycerol derived from the pathogenic spirochaete Borrelia burgdorferi (19). The NKT cells may also have the potential to respond to the bacterial cell wall components derived from the intestinal bacteria, and this could account not only

HCV: hepatitis C virus

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for their abundance in the liver, but also for their expression of markers of activation (6). Like many other T cells, the NKT cells express DAP10- and DAP12-associated receptors, and the NKG2D receptors on these cells are implicated in immunopathology in a mouse model of hepatitis B in which RAE-1 is induced to engage these receptors (20). The signiﬁcance of innate immunity in the defense of the liver is evident from the multiple adaptations through which pathogens subvert it. Thus, HCV RNA interacts with RIG-I to activate NF-κB and IFN-β secretion; however, HCV also subverts this pathway because its NS3/4 protease cleaves the IPS-1 adaptor protein of RIG-I signaling (21). IRF-3 may also be activated through the TLR3 pathway, but HCV NS3/4 also targets this pathway through the cleavage of TRIF (22). Strikingly, this

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mechanism of innate immune escape is also seen in hepatitis A virus infection, where the protease 3ABC cleaves IPS-1 (also known as MAVS), again resulting in subversion of NF-κB activation (23). This convergence supports the argument that the IRF-3 pathway plays a critical role in antiviral immunity in liver. Apart from inhibiting innate immune signaling pathways within the infected cells, HCV targets NK cells though a completely independent mechanism. The HCV envelope protein, E2, binds to CD81 on human NK cells, and this results in suppression of their cytotoxic function and of IFN-γ synthesis (24). An important feature of HCV is therefore both a concerted attack on IFN-β synthesis and a major cellular source of IFN-γ. Because the pathogen so actively subverts IFN delivery and function, it is reasonable to suppose that IFN-responsive genes play a key role in anti-HCV immunity. It is not, therefore, surprising that the mainstay of treatment is high-dose exogenous IFN-α. Complex protozoan pathogens also manipulate the phagocytic function of Kupffer cells. Thus, the malaria parasites Plasmodium sp. enter Kupffer cells as part of the process by which they cross liver endothelium and gain access to hepatocytes. Evidence in favor of the mechanism comes from direct visualization of malaria sporozoite behavior in vivo (25) and from the observation that parasitization of the liver is reduced in osteopetrotic mice, which lack mature macrophages (26). As part of their interaction, the malaria sporozoites disable the Kupffer cells’ respiratory burst by increasing intracellular cyclin AMP (27). This effect is mediated by an abundant malaria protein, the circumsporite protein (CSP), which binds to a Kupffer cell’s surface receptor, LRP-1 (the low-density lipoprotein receptor-related protein). Thus, the most likely model is that the malaria sporozoite’s CSP engages LRP-1, inducing cyclic AMP and suppressing the Kupffer cell’s normal response to phagocytosed pathogens. This converts the Kupffer cells from effective elements in innate host defense into portals through which the parasite traverses the endothelium (28).

WORLD PREVALENCE OF LIVER DISEASE Malaria causes severe disease in 500 million people each year; many more are infected, and 40% of the world population is at risk. There is no effective vaccine. More than 350 million people have chronic infection with HBV, which results in one million deaths per year from cirrhosis and liver cancer. A recombinant subunit vaccine is effective, so this total will probably decline. Around 180 million people are infected with HCV, and, of these, 130 million are chronically infected and at risk for cirrhosis and liver cancer. There is no effective vaccine.

THE DIVERSITY OF POTENTIAL ANTIGEN-PRESENTING CELLS The liver contains plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), and pDCs are more abundant than they are in lymphoid tissue. These pDCs are a major source of IFN-α, consistent with the importance of innate immune mechanisms in the liver. But in addition, the mouse liver contains two other identiﬁable subsets of DCs, the CD8α+ DCs (29) and the less well-deﬁned natural killer DCs (NKDCs) (30), neither of which has yet been identiﬁed in humans. In addition to synthesizing IFN-α, pDCs synthesize both IL-10 and IL-12. In LPS-treated mice, liver pDCs synthesized less IL-12 than did splenic pDCs (31) and were poor APCs compared with splenic DCs (32). Conversely, liver mDCs synthesized IL-10, and this was increased in HCV patients (33). As discussed below, diverse other potential APCs in the liver respond to TLR ligation by secreting IL-10. The liver contains a large macrophage population; these are the Kupffer cells. In addition to their role as phagocytes, these cells express MHC and costimulatory molecules, rendering them potential APCs (Figure 3). However, relatively little work has addressed the APC function of Kupffer cells. Early experiments suggested that Kupffer cells were primarily immunosuppressive. Thus, addition of Kupffer cells to a mixed leukocyte reaction performed in the presence of low arginine resulted in www.annualreviews.org • The Liver as a Lymphoid Organ

sporozoite: the developmental stage of the malaria parasite that infects liver cells

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LPS Proteins Particulates

TLR4

IL-10 IL-12/18

IFN-γ T cell NK cell

LSEC

Figure 3 Immunology of Kupffer cells. These cells can take up LPS, proteins, and particulates from the blood and secrete a number of cytokines, including TNF-α, IL-12, and IL-18, but also IL-10. The balance between IL-12/-18 and IL-10 production regulates NK cell activity. Kupffer cells express PD-L1 and have the capacity to inactivate T cells, a function shared with other liver APCs. Both T cells and NK cells secrete IFN-γ, which powerfully activates Kupffer cells. (Abbreviations: KC, Kupffer cell; HSC, hepatic stellate cell.)

LSEC: liver sinusoidal endothelial cell stellate cell: a distinctive liver cell type, located between the liver endothelial cells and the hepatocytes

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immunosuppression, mediated in part by PGE2 (34). Kupffer cells may also mediate suppression through their synthesis of nitric oxide (35) and respond to TLR4 ligation by secreting IL-10 (36). In a liver transplant model, Kupffer cells expressed FasL, leading to alloreactive CD4+ T cell apoptosis (37). On the basis of such experiments, investigators have invoked Kupffer cells to explain such diverse phenomena as oral tolerance, portal vein tolerance, and liver allograft tolerance. However, Kupffer cells may also act as effective APCs. In HCV infection, human Kupffer cells became MHC I and II high, expressed CD40 and CD80, and formed clusters with CD4+ T cells, consistent with their acting as APCs (38). Perhaps the capacity of Kupffer cells to stimulate or inhibit T cell activation depends on the signals to which these cells have been exposed. In a study of liver NK cell activation by human Kupffer cells, the selective Crispe

activation of either the TRIF pathway or the MyD88 pathway of TLR signaling resulted in the predominant expression of either IL-18 or IL-10, leading to higher or lower levels of NK cell activation (39). The liver sinusoidal endothelial cells (LSECs) have been implicated in antigen presentation (Figure 4). These endothelial cells are unusual in several respects: They do not secrete an organized basement membrane, and they are perforated by numerous fenestrations, clustered into sieve plates. These cells express the scavenger receptor, which renders them competent to take up circulating proteins. They also express MHC class I and class II and costimulatory molecules including CD40, CD80, and CD86, giving them the surface characteristics of highly active stimulatory APCs, such as DCs (40). However, they respond to TLR4 ligation with the secretion of IL-10, to which they also respond by downregulating their APC functions (41), and their main effect seems to be the induction of T cell tolerance. Thus, when LSECs were isolated from mice that received ovalbumin parenterally or orally, they engaged T cells, resulting in immune deviation to a CD4+ T regulatory phenotype, or in CD8+ T cell tolerance (42, 43). Hepatic stellate cells reside in the subendothelial space of Disse and constitute the primary site for the storage of vitamin A. They regulate hepatic sinusoidal blood ﬂow and can also transdifferentiate into myoﬁbroblasts during the process of liver ﬁbrosis (44). Recent studies suggest that these cells also belong among the liver APCs (Figure 5). Thus, they express MHC class I, MHC class II, and CD1d, and they have the potential to respond to innate immune signals though their expression of TLR4, CD14, and MD2, which renders them LPS responsive (45, 46). Ex vivo, stellate cells can activate NKT cells and classical T cells (47), although their coexpression of the inhibitory molecule PD-L1 also renders them capable of T cell inactivation, leading to tolerance (48). With so many potential liver cell subsets manifesting APC activity ex vivo, the issue of cell puriﬁcation becomes particularly

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acute. Classical experiments by Steinman and colleagues (49) demonstrated that, among spleen cells, the most powerful APC activity was concentrated in a very rare subset of cells, the DCs; indeed, the alleged APC activity of other subsets of spleen cells, including splenic macrophages, could have been attributed to rare, contaminating DCs. This same concern applies to studies that document the APC action of ex vivo liver cell subsets, but now we have more complexity. Which of the APC functions of a culture of stellate cells may be attributed to rare, contaminating DCs or LSECs? And in the case of the LSECs, how many contaminating DCs or Kupffer cells is enough to account for their ability to activate or to silence a T cell response? The optimum reagents for the analysis of the signiﬁcance of APC function would be a series of transgenic mice in which molecules of interest, such as MHC class I or MHC class II molecules, are expressed or selectively inactivated under the control of promoters of absolute cell-type speciﬁcity. These ideal tools are not yet at hand; nevertheless, we can draw some solid conclusions from the extant in vivo experiments.

LOCAL PRIMING OF T CELLS IN THE LIVER The presence in the liver of so many distinct subsets of cells with APC function raises the question of whether T cells are in fact activated locally in vivo. The distinctive architecture of the hepatic sinusoids permits circulating T cells to make direct contact with underlying hepatocytes and also with stellate cells, as well as with LSECs and intravascular Kupffer cells. Such interactions have in fact been revealed by electron microscopy (50), providing a structural basis for primary T cell activation by hepatocellular antigens. This idea was ﬁrst supported by experiments in which CD8+ T cells made a rapid, antigen-driven, local, intrahepatic immune response in transgenic mice expressing the HBV genome in both liver and other tissues (51) and subsequently in transgenic mice expressing nonself MHC class I molecules (52). In addi-

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MHC I/II CD40/80/86

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Transcytosis?

Figure 4 Immunology of liver sinusoidal endothelial cells (LSECs). These cells respond to LPS via TLR4 and can acquire circulating proteins via the scavenger receptor and the mannose receptor. LSECs may transport proteins across themselves, and into hepatocytes, a process termed transcytosis. They process and present antigens in association with costimulatory ligands (CD40, CD80, and CD86) but respond to ambient LPS by secreting IL-10, biasing T cells toward tolerance. These cells also express multiple adhesion molecules, including ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), and VAP-1 (vascular adhesion protein-1), all of which are implicated in T cell retention in the liver sinusoids. Thus, these cells can promote immune tolerance, both through the local trapping of activated T cells and the induction of regulatory T cells. This concept is summarized in the diagram as “trapping, Tregs, tolerance.”

tion to transgenic antigens, CD8+ T cells made a local response to antigen delivered speciﬁcally to hepatocytes using an AAV-2 vector, and this resulted in the subsequent seeding of activated cells to the lymph nodes and the spleen (53). Furthermore, the transplanted mouse liver, depleted of bone marrow–derived APCs, was fully competent to activate naive CD8+ T cells in response to a peptide antigen (54). Abundant evidence therefore supports the concept that the liver is a secondary lymphoid organ, acting as a site of primary T cell activation. In such experiments, it is not always clear which cell population is the main APC. The most compelling evidence that hepatocytes www.annualreviews.org • The Liver as a Lymphoid Organ

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Figure 5 Immunology of hepatic stellate cells. Stellate cells perceive LPS via TLR4 and also respond to multiple cytokines, including IL-10 and TGF-β1. They express a high surface density of the nonclassical MHC class I–like molecule CD1d and thereby activate NKT cells.

parenchymal cell: the most abundant cell type in the liver, also known as hepatocytes cross-presentation: transfer of an antigen from the antigenexpressing cell to a distinct APC, resulting in T cell engagement

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(also termed liver parenchymal cells) themselves act as primary APCs comes from parallel studies in vivo and in vitro, using allogeneic MHC antigens. In liver allograft experiments, cytotoxic T cells underwent spontaneous apoptosis in the transplanted liver (55), and both hepatocytes and nonparenchymal cells activated, then caused apoptosis of, the activated T cells in vitro (56). Similarly, both activation and apoptosis of T cells were observed in vivo following the adoptive transfer of alloreactive T cells to transgenic mice expressing a nonself MHC class I molecule on hepatocytes (52, 57). Furthermore, the T cell apoptosis was also manifest in vitro when T cells were cultured with hepatocytes, leading to the hypothesis that a feature of liver tolerance was “death by neglect,” the engagement of T cells by APCs deﬁcient in costimulatory activity (58). In these experiments, the lack of susceptibility to cross-presentation of intact MHC molecules was key in the interpretation of the data and strongly suggested that hepatocytes Crispe

themselves were engaging the CD8+ T cells. However, it is also noteworthy that in all these studies the ﬁnal outcome was T cell inactivation or apoptosis. In a distinct transgenic model, the deliberate priming of antigen-speciﬁc T cells at an extrahepatic site was not enough to break tolerance to a transgenic liver antigen expressed in hepatocytes, supporting the argument for an active state of antigen-driven local tolerance (59). The presentation of cell-intrinsic MHC antigens by hepatocytes constitutes direct presentation. Similarly, the in vitro analysis of the APC functions of stellate cells dealt primarily with direct presentation of cellular antigens, or the presentation of exogenous peptides (47). In vivo experiments revealed the capacity of stellate cells to act as immunosuppressive APCs, protecting pancreatic islet allografts against rejection (60). In this capacity, also, stellate cells were presenting their intrinsic antigens. In contrast, we know that LSECs are capable of presenting exogenous antigens encountered in vivo. This is the case for soluble antigens given parenterally or orally (42, 43). The LSECs also appear to be capable of true cross-presentation, in which tumor cell– derived antigen was acquired by LSECs and resulted in CD8+ T cell tolerance (61). The mechanism of this kind of tolerance is likely to involve PD-L1, based on antibody blocking experiments (48); in addition, LSECs deﬁcient in this molecule failed to induce CD8+ T cell tolerance (62). In addition to all these nonclassical APCs, DCs also trafﬁc through the liver. Thus, classical mDC precursors expressing CD11c, but not B220, are recruited to the liver during granulomatous inﬂammation initiated by Propionibacterium acnes and subsequently detected ﬁrst in the Disse space, then in granulomas, and subsequently in lymphoid aggregates in the portal tracts (63). These movements were orchestrated by chemokines, with CCL3 driving the initial localization to the granulomas, and CCL21 causing the subsequent relocalization to portal-associated lymphoid tissue (PALT) (64). This certainly suggests, though it does not

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prove, that DCs localize to areas of lymphoneogenesis in the inﬂamed liver and engage T cells there. In humans, the same chemokine, CCL21, is expressed by vascular endothelium in portal areas of the liver, but not in lymph node vessels (65). On this basis, investigators argue that PALT is a distinct immunological compartment; sadly, the acronym lacks distinctiveness because it has been applied to lymphoid tissue in the human prostate (66) and in the chicken pineal gland (67). But the existence of PALT, in the hepatic context, adds to the complexity of liver immunology. In addition to examining diverse APCs resident in the sinusoids, we also need to consider more conventional lymphoid tissue threaded through the liver in the network of portal tracts.

THE RISE AND FALL OF LIVER-PRIMED T CELLS Whereas the immune system is competent to eliminate infection with hepatitis A virus, a state of persistent infection is a common outcome in HBV infection and the usual outcome in HCV infection. We may therefore ask why liver immune responses frequently fail. Does this simply indicate the sophistication of several well-adapted pathogens, or, in some sense, is the liver to blame? In the case of chronic HCV infection, antigen-speciﬁc CD8+ T cells frequently assume a stunned or exhausted phenotype, in which they express a low level of the IL-7 receptor-α (CD127) and a high level of the inhibitory receptor PD-1 (68). In several infections, including HIV and HCV in humans and lymphocytic choriomeningitis virus in mice, CD8+ T cells with this phenotype are able neither to secrete IFN-γ nor to make IL-2 (69–71). In the case of HCV, another striking feature of chronic infection is selective IL-10 production by CD4+ T cells (72). Furthermore, chronic HCV in humans may be associated with very weak or absent CD4+ T cell responses (73), whereas in chimpanzees, viral escape mutants that prevent CD4+ T cell recognition fa-

vor chronicity (74), suggesting that the CD8+ T cells may be incapacitated owing to the lack of CD4+ T cell help, a state termed “helpless” (75). To what extent, then, are stunning, exhaustion, PD-1 expression, IL-10 intoxication, and helplessness all effects of the liver environment? As far as PD-1/PD-L1 interactions are concerned, the liver seems to be a preferential site of action. PD-L1 is expressed on several liver cell types (76). Mice deﬁcient in PD-L1 developed an immunoinﬂammatory hepatitis caused by CD8+ T cells, suggesting a key role for PD-L1 in regulating both CD8+ T cell abundance and immunopathology in the liver (77). The immunosuppressive effects of IL-10 secreted by LSECs, Kupffer cells, and liver pDCs have already been emphasized, along with the interpretation that this is an effect of low-level TLR4 ligation. Why should this bias exist? One possibility is that the LPS-responsive cells of the liver are manifesting a mechanism that has evolved to suppress chronic immune inﬂammation. Thus, in an acute immune response, IL-10 synthesis occurs after the peak of proinﬂammatory cytokines and may help to restore the system to a resting state. Under conditions of chronic activation, this mechanism predominates, limiting tissue injury. In the liver, the continuous presence of low levels of LPS may emulate chronic inﬂammation, calling forth IL-10 as a regulatory response. Does the liver promote CD8+ T cell helplessness? The liver’s unique vasculature permits circulating T cells to engage with hepatocytes (50), which act as APCs (56, 78). Therefore, we can consider the liver as a tissue that favors CD8+ T cell priming on cells that can neither prime nor be engaged by CD4+ T cells. The helpless phenotype would naturally follow from direct priming of HCV-speciﬁc and possibly also HBV-speciﬁc CD8+ T cells on hepatocytes. In fact, there is one report of apparently directly primed CD8+ T cells in the context of liver transplantation. A liver expressing HLA-A2 was transplanted into an HLA-A2-negative recipient, who subsequently developed HLA-A2-restricted anti-HCV

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CD8+ T cells (79). This would be consistent with direct priming on newly infected donor hepatocytes. A further test of the inadequacy of liver-primed CD8+ T cells comes from a comparison of the response of TCR-transgenic T cells to a neo-self-MHC antigen expressed either exclusively in the liver or also in lymph nodes. Extrahepatic priming resulted in fully differentiated CD8+ T cells that could localize to the liver and cause autoimmune immunopathology. In contrast, the exclusively liver-primed CD8+ T cells were relatively innocuous (80). In summary, multiple mechanisms account for the rise and fall of T cells speciﬁc for liver pathogens, but many of these effects are linked to the liver’s unique immunobiology. This is a clear case of contributory negligence; HBV and HCV are subtle and devious pathogens, to be sure, but the liver offers them opportunities for immune subversion.

TGF-β1

IL-10 Trapping, FasL, TRAIL, phagocytosis

T

T

HSC

PD-L1

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Figure 6 Mechanisms of T cell tolerance in liver. The expression of adhesion molecules facilitates the trapping of activated T cells in liver sinusoids, where they may undergo apoptosis owing to FasL and TRAIL expressed on Kupffer cells and may also be phagocytosed. In addition, T cells that recognize antigen in the liver are exposed to immunosuppressive cytokines, including IL-10 and TGF-β1, and to inhibitory ligands, including PD-L1 (also known as B7-H1). 156

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HOW DOES THE LIVER INDUCE SYSTEMIC TOLERANCE? In view of the bias toward tolerance when T cells encounter antigens in the liver (summarized in Figure 6), it is not surprising that such a large organ can impose systemic immune tolerance. This phenomenon was ﬁrst recognized in the context of allogeneic liver transplantation. In the classic experiments conducted at the University of Oxford, renal transplants between unrelated pigs were promptly rejected, whereas liver transplants between equally unrelated pigs were generally accepted. Strikingly, the transplantation of a kidney and a liver from the same donor enhanced the survival of the kidney. In the half-century since the description of these “Strange English Pigs”1 (3, 81), this phenomenon has not been fully explained. Many explanations have been considered. For example, serial transplantation experiments implicated passenger leukocytes as playing a role in the induction of kidney allograft rejection and suggested that their loss plays a role in the induction of allograft tolerance (82). However, the loss of passenger leukocytes cannot be implicated in the tolerance associated with liver transplantation because of the abundance of long-lived donor hematopoietic cells within the liver graft (83). It was therefore reasonable to propose that, unlike other passenger leukocytes, those originating in the liver were tolerogenic. Investigators (84, 85) suggested that the detection of a low frequency of graft-derived leukocytes in multiple tissues of a liver transplant recipient (microchimerism) explains liver transplantation tolerance, but the survival of these cells would be equally well explained if the liver were imposing allospeciﬁc tolerance by some other mechanism. If recirculating passenger leukocytes were an effect rather than a cause of liver allograft tolerance, what other mechanisms would be candidates? The sessile Kupffer cells, LSECs, and

1

“Strange English Pigs” was the title of an anonymous editorial in The Lancet, November 1, 1969, on the topic of liver allograft tolerance.

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stellate cells all have the capacity to present antigens to T cells, along with cosignals including IL-10 and PD-L1 that induce tolerance. Therefore, we may conjecture that liver allograft tolerance is induced by this mechanism, with persistent microchimerism as a by-product. In this model, allospeciﬁc precursor T cells would enter the transplanted liver, undergo activation by liver-speciﬁc APCs, and then either undergo paralysis or deletion owing to liver-speciﬁc local signals. We might consider how this mechanism could account for the effect of the liver in oral tolerance. The delivery of a protein antigen, usually ovalbumin, into the stomach results in systemic tolerance, particularly of Th1 CD4+ T cells and CD8+ T cells. If the venous drainage of the gut is surgically diverted, this oral tolerance is lost (86). In the context of an oral tolerance model, isolated and ex vivo cultured LSECs interacted with ova-speciﬁc T cells, activating them but then causing them to deviate their immune function toward an antiinﬂammatory pattern of cytokine synthesis (43). While the caveats already raised in relation to the absolute purity of cultured liver APCs certainly apply to these experiments, they make a prima facie case for the induction of systemic tolerance by liver APCs. Similarly, systemic immune tolerance can be induced by the injection of APCs into the portal vein (87), although it is unclear whether these APCs take up residence in the liver and become tolerogenic or whether cross-presentation by resident liver APCs accounts for this effect. The induction of systemic tolerance by liver APCs has been attributed both to peripheral deletion and to the induction of antigen-speciﬁc Tregs. Supporting the deletion model is the abundant evidence that activated, circulating CD8+ T cells are sequestered in the liver, even in the absence of antigen. This is most strikingly illustrated in the context of inﬂuenza infection, where both virus RNA and protein are limited to the respiratory system, but inﬂuenza-speciﬁc CD8+ T cells are found in the liver, associated with Kupffer cell–rich inﬂammatory foci and with subclinical hepatocyte damage (88). This sequestration depends on integrin lig-

ands, including ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) (89), and, like other aspects of liver tolerance, it may be driven by LPS acting via TLR4 (90). The evidence that this form of antigen-independent CD8+ T cell sequestration results in systemic tolerance is thin; however, in the context of a CD8+ T cell response to antigen-pulsed DCs, the interruption of the TLR4-dependent element of T cell trapping in the liver resulted in exaggerated systemic immune responses, followed eight weeks later by an enhanced secondary response. On the basis of this experiment, John et al. (91) proposed the model that the liver regulates the magnitude of CD8+ T cells responses. However, it is abundantly clear that deletion is not the fate of every T cell that enters the liver because liver-derived T cells can repopulate systemic memory (92). It has been argued that this observation invalidates the model that the liver acts as a sink for activated CD8+ T cells (93), but this argument fails to take into account the distinction between activated blast T cells and CD8+ memory T cells. The data are best reconciled by a model in which recently activated lymphoblasts preferentially localize to hepatic sinusoids owing to adhesion molecules expressed on LSECs, and then recruit and are killed by Kupffer cells, creating a “sink” for excess T cell blasts. Resting memory cells, in contrast, may be overrepresented in the liver owing to their adhesion receptors, but they do not activate Kupffer cells nor are they phagocytosed. These cells ﬁnd the liver quite hospitable, as we have argued previously (94). The alternative to a deletion model is the idea of active regulation by suppressor T cells, also known as Tregs. These cells exist in a variety of subsets, some of which arise as Tregs in the thymus, whereas others differentiate from apparently uncommitted peripheral CD4+ and CD8+ T cell precursors. The impact of liver transplantation on these cells is controversial. A perfusate of human liver was enriched in CD4+ 25+ FoxP3+ CD127low cells, and such donor-derived Tregs were found in the circulation of liver transplant www.annualreviews.org • The Liver as a Lymphoid Organ

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recipients, an aspect of the phenomenon of microchimerism (95). Investigators agree that, after liver transplantation in humans, the overall frequency of Tregs in the circulation falls (96, 97). Among subsets of CD4+ 25+ T cells, the number of cells that expressed high amounts of CD127 increased in human liver transplant recipients with stable allografts, whereas the number of CD4+ 25+ 127low cells with regulatory function decreased (98); this is hard to reconcile with the idea that the CD127low regulatory subset was maintaining tolerance. However, in mouse liver transplantation, CD4+ 25+ FoxP3+ CTLA4+ Tregs increased in abundance after liver grafting, and depletion of these cells using an anti-CD25 antibody caused acute rejection of the graft, consistent with an important role for these kinds of Tregs in maintaining liver allograft tolerance and also, by extrapolation, systemic tolerance (99). It does not follow that mouse and human are fundamentally different. The most straightforward resolution of the apparent conﬂict between mouse and human data is that spontaneous liver allograft acceptance involves CD4+ 25+ FoxP3+ Tregs but that, in human, liver allografts are tolerated because of immunosuppression and despite the depletion of Tregs by immunosuppressive drugs.

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THE LIVER AS A LYMPHOID ORGAN: CONCLUSIONS The liver is an important site for primary T cell activation, but this takes place in an environment biased toward tolerance. Several mech-

anisms contribute to this suppressive milieu. First, the constitutive exposure of liver cells to traces of endotoxin and other microbial products results in the down-modulation of costimulatory molecules and the synthesis of IL-10 by Kupffer cells and LSECs. Second, the open architecture of the liver endothelium results in ready access of naive T cells to diverse subsets of APCs, including hepatocytes. This may result in selective CD8+ T cell priming without concomitant CD4+ T cell activation, resulting in a “helpless” phenotype that leads to longterm CD8+ T cell dysfunction and a lack of immune memory. Third, the liver endothelium expresses adhesion molecules that facilitate the sequestration of circulating activated T cells, particularly CD8+ T cells. This gives the liver a role in systemic immunoregulation. An important epiphenomenon resulting from these mechanisms is liver allograft tolerance. Because of the high threshold for the initiation of an adaptive T cell response in the liver, innate immune mechanisms assume greater signiﬁcance. Abundant NK cells and NKT cells may be activated by pathogen-associated structures via TLRs, invariant TCRs, or alternative sensing systems such as RIG-I, and by cytokines. Kupffer cells respond to inﬂammatory cytokines such as IFN-γ by the synthesis of their own inﬂammatory mediators: TNF-α, IL-12, and IL-18. These in turn deliver positive signals to both adaptive and innate immune cells. Awash with potential activating signals, the liver’s immune system is held in a baseline state of active tolerance, which can be reversed by sufﬁciently strong pathogen-speciﬁc signals.

SUMMARY POINTS 1. The liver receives blood from the intestine, which is rich in microbial products. These engage TLRs, which modify innate immunity in the hepatic environment. 2. The liver lymphocytes are enriched in CD8+ T cells, activated T cells, memory T cells, NKT cells, and NK cells. 3. Multiple cell populations can act as APCs in the liver. These include hepatocytes, endothelial cells, and subendothelial stellate cells, as well as several subsets of dendritic cells.

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4. The liver is rich in immunosuppressive cytokines including IL-10, and several liver cell subsets express the inhibitory ligand PD-L1. The consequence of this is that many encounters between T cells and liver APCs end in immune tolerance.

FUTURE ISSUES

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1. The mechanisms of antigen presentation and T cell activation by the many subsets of liver APCs must be clariﬁed. In particular, do these cell types form distinctive synapses with T cells? Which of them engage in cross-presentation of hepatocellular antigens? What mechanisms promote such cross-presentation? 2. In hepatitis B, hepatitis C, hepatocellular cancer, and the liver stage of malaria, how are hepatocellular antigens presented to the immune system? 3. Which immune mechanisms favor the elimination of hepatocellular antigens? Which mechanisms cause liver immunopathology? How can we promote the former, while limiting the effects of the latter?

DISCLOSURE STATEMENT The author is not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review. LITERATURE CITED 1. Lumsden AB, Henderson JM, Kutner MH. 1988. Endotoxin levels measured by a chromogenic assay in portal, hepatic and peripheral venous blood in patients with cirrhosis. Hepatology 8:232–36 2. Catala M, Anton A, Portoles MT. 1999. Characterization of the simultaneous binding of Escherichia coli endotoxin to Kupffer and endothelial liver cells by ﬂow cytometry. Cytometry 36:123–30 3. Calne RY, Sells RA, Pena JR, Davis DR, Millard PR, et al. 1969. Induction of immunological tolerance by porcine liver allografts. Nature 223:472–76 4. Thompson AJ, Locarnini SA. 2007. Toll-like receptors, RIG-I-like RNA helicases and the antiviral innate immune response. Immunol. Cell Biol. 85:435–45 5. Abreu MT, Fukata M, Arditi M. 2005. TLR signaling in the gut in health and disease. J. Immunol. 174:4453–60 6. Tu Z, Bozorgzadeh A, Crispe IN, Orloff MS. 2007. The activation state of human intrahepatic lymphocytes. Clin. Exp. Immunol. 149:186–93 7. Norris S, Collins C, Doherty DG, Smith F, McEntee G, et al. 1998. Resident human hepatic lymphocytes are phenotypically different from circulating lymphocytes. J. Hepatol. 28:84–90 8. Crispe IN. 1996. Isolation of mouse intrahepatic lymphocytes. Curr. Protoc. Immunol. 3:22–28 9. Lanier LL. 2008. Up on the tightrope: natural killer cell activation and inhibition. Nat. Immunol. 9:495–502 10. Jinushi M, Takehara T, Tatsumi T, Kanto T, Groh V, et al. 2003. Expression and role of MICA and MICB in human hepatocellular carcinomas and their regulation by retinoic acid. Int. J. Cancer 104:354–61 11. Lodoen M, Ogasawara K, Hamerman JA, Arase H, Houchins JP, et al. 2003. NKG2D-mediated natural killer cell protection against cytomegalovirus is impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules. J. Exp. Med. 197:1245–53 12. Nguyen KB, Salazar-Mather TP, Dalod MY, Van Deusen JB, Wei XQ, et al. 2002. Coordinated and distinct roles for IFN-αβ, IL-12, and IL-15 regulation of NK cell responses to viral infection. J. Immunol. 169:4279–87 www.annualreviews.org • The Liver as a Lymphoid Organ

3. The discovery of liver tolerance, leading to the recognition of the liver as a distinct immunological site.

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22. A nice example of how HCV-encoded proteins attack immune signaling pathways.

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36. Knolle P, Schlaak J, Uhrig A, Kempf P, Meyer zum Buschenfelde KH, Gerken G. 1995. Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge. J. Hepatol. 22:226– 29 37. Miyagawa-Hayashino A, Tsuruyama T, Egawa H, Haga H, Sakashita H, et al. 2007. FasL expression in hepatic antigen-presenting cells and phagocytosis of apoptotic T cells by FasL+ Kupffer cells are indicators of rejection activity in human liver allografts. Am. J. Pathol. 171:1499–508 38. Burgio VL, Ballardini G, Artini M, Caratozzolo M, Bianchi FB, Levrero M. 1998. Expression of costimulatory molecules by Kupffer cells in chronic hepatitis of hepatitis C virus etiology. Hepatology 27:1600–6 39. Tu Z, Bozorgzadeh A, Pierce RH, Kurtis J, Crispe IN, Orloff MS. 2008. TLR-dependent cross talk between human Kupffer cells and NK cells. J. Exp. Med. 205:233–44 40. Knolle PA, Limmer A. 2001. Neighborhood politics: the immunoregulatory function of organ-resident liver endothelial cells. Trends Immunol. 22:432–37 41. Knolle PA, Uhrig A, Hegenbarth S, Loser E, Schmitt E, et al. 1998. IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules. Clin. Exp. Immunol. 114:427–33 42. Limmer A, Ohl J, Kurts C, Ljunggren HG, Reiss Y, et al. 2000. Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance. Nat. Med. 6:1348–54 43. Limmer A, Ohl J, Wingender G, Berg M, Jungerkes F, et al. 2005. Cross-presentation of oral antigens by liver sinusoidal endothelial cells leads to CD8 T cell tolerance. Eur. J. Immunol. 35:2970–81 44. Friedman SL. 2008. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol. Rev. 88:125–72 45. Paik YH, Schwabe RF, Bataller R, Russo MP, Jobin C, Brenner DA. 2003. Toll-like receptor 4 mediates inﬂammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells. Hepatology 37:1043– 55 46. Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, et al. 2007. TLR4 enhances TGF-β signaling and hepatic ﬁbrosis. Nat. Med. 13:1324–32 47. Winau F, Hegasy G, Weiskirchen R, Weber S, Cassan C, et al. 2007. Ito cells are liver-resident antigen-presenting cells for activating T cell responses. Immunity 26:117–29 48. Yu MC, Chen CH, Liang X, Wang L, Gandhi CR, et al. 2004. Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice. Hepatology 40:1312–21 49. Nussenzweig M, Steinman R. 1980. Contribution of dendritic cells to stimulation of the murine syngeneic MLR. J. Exp. Med. 151:1196–212 50. Warren A, Le Couteur DG, Fraser R, Bowen DG, McCaughan GW, Bertolino P. 2006. T lymphocytes interact with hepatocytes through fenestrations in murine liver sinusoidal endothelial cells. Hepatology 44:1182–90 51. Ando K, Guidotti LG, Cerny A, Ishikawa T, Chisari FV. 1994. CTL access to tissue antigen is restricted in vivo. J. Immunol. 153:482–88 52. Bertolino P, Bowen DG, McCaughan GW, Fazekas de St Groth B. 2001. Antigen-speciﬁc primary activation of CD8+ T cells within the liver. J. Immunol. 166:5430–38 53. Wuensch SA, Pierce RH, Crispe IN. 2006. Local intrahepatic CD8+ T cell activation by a nonself-antigen results in full functional differentiation. J. Immunol. 177:1689–97 54. Klein I, Crispe IN. 2006. Complete differentiation of CD8+ T cells activated locally within the transplanted liver. J. Exp. Med. 203:437–48 55. Qian S, Lu L, Fu F, Li Y, Li W, et al. 1997. Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T cells and implications for tolerance induction. J. Immunol. 158:4654–61 56. Lee YC, Lu L, Fu F, Li W, Thomson AW, et al. 1999. Hepatocytes and liver nonparenchymal cells induce apoptosis in activated T cells. Transplant. Proc. 31:784 57. Bertolino P, Trescol-Biemont MC, Rabourdin-Combe C. 1998. Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival. Eur. J. Immunol. 28:221–36 58. Bertolino P, Trescol-Biemont MC, Thomas J, de St Groth BF, Pihlgren M, et al. 1999. Death by neglect as a deletional mechanism of peripheral tolerance. Int. Immunol. 11:1225–38 www.annualreviews.org • The Liver as a Lymphoid Organ

36. This paper links LPS, Kupffer cells, and IL-10, setting up a major paradigm for liver tolerance.

42. A classic paper documenting the APC activity of LSECs.

47. This paper adds stellate cells to the diverse inventory of liver APC.

55. This paper reveals the apoptosis of T cells infiltrating a liver allograft, arguing for peripheral deletion as a key aspect of liver tolerance.

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68. The lack of effective T cell immunity to HCV is linked to the “exhausted” phenotype.

80. This paper suggests that intrahepatic T cell priming is incomplete, resulting in poor function, in contrast to lymph node priming.

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59. Limmer A, Sacher T, Alferink J, Kretschmar M, Schonrich G, et al. 1998. Failure to induce organspeciﬁc autoimmunity by breaking of tolerance: importance of the microenvironment. Eur. J. Immunol. 28:2395–406 60. Chen CH, Kuo LM, Chang Y, Wu W, Goldbach C, et al. 2006. In vivo immune modulatory activity of hepatic stellate cells in mice. Hepatology 44:1171–81 61. Berg M, Wingender G, Djandji D, Hegenbarth S, Momburg F, et al. 2006. Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-speciﬁc CD8+ T cell tolerance. Eur. J. Immunol. 36:2960–70 62. Diehl L, Schurich A, Grochtmann R, Hegenbarth S, Chen L, Knolle PA. 2008. Tolerogenic maturation of liver sinusoidal endothelial cells promotes B7-homolog 1-dependent CD8+ T cell tolerance. Hepatology 47:296–305 63. Yoneyama H, Matsuno K, Zhang Y, Murai M, Itakura M, et al. 2001. Regulation by chemokines of circulating dendritic cell precursors, and the formation of portal tract-associated lymphoid tissue, in a granulomatous liver disease. J. Exp. Med. 193:35–49 64. Yoneyama H, Ichida T. 2005. Recruitment of dendritic cells to pathological niches in inﬂamed liver. Med. Mol. Morphol. 38:136–41 65. Grant AJ, Goddard S, Ahmed-Choudhury J, Reynolds G, Jackson DG, et al. 2002. Hepatic expression of secondary lymphoid chemokine (CCL21) promotes the development of portal-associated lymphoid tissue in chronic inﬂammatory liver disease. Am. J. Pathol. 160:1445–55 66. Di Carlo E, Magnasco S, D’Antuono T, Tenaglia R, Sorrentino C. 2007. The prostate-associated lymphoid tissue (PALT) is linked to the expression of homing chemokines CXCL13 and CCL21. Prostate 67:1070–80 67. Mosenson JA, McNulty JA. 2006. Characterization of lymphocyte subsets over a 24-hour period in PinealAssociated Lymphoid Tissue (PALT) in the chicken. BMC Immunol. 7:1 68. Radziewicz H, Ibegbu CC, Fernandez ML, Workowski KA, Obideen K, et al. 2007. Liverinfiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression. J. Virol. 81:2545–53 69. Gruener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, et al. 2001. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. J. Virol. 75:5550–58 70. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, et al. 2006. PD-1 expression on HIV-speciﬁc T cells is associated with T-cell exhaustion and disease progression. Nature 443:350–54 71. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, et al. 2006. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439:682–87 72. Godkin A, Jeanguet N, Thursz M, Openshaw P, Thomas H. 2001. Characterization of novel HLA-DR11restricted HCV epitopes reveals both qualitative and quantitative differences in HCV-speciﬁc CD4+ T cell responses in chronically infected and nonviremic patients. Eur. J. Immunol. 31:1438–46 73. Wedemeyer H, He XS, Nascimbeni M, Davis AR, Greenberg HB, et al. 2002. Impaired effector function of hepatitis C virus-speciﬁc CD8+ T cells in chronic hepatitis C virus infection. J. Immunol. 169:3447–58 74. Puig M, Mihalik K, Tilton JC, Williams O, Merchlinsky M, et al. 2006. CD4+ immune escape and subsequent T-cell failure following chimpanzee immunization against hepatitis C virus. Hepatology 44:736– 45 75. Janssen EM, Droin NM, Lemmens EE, Pinkoski MJ, Bensinger SJ, et al. 2005. CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death. Nature 434:88–93 76. Iwai Y, Terawaki S, Ikegawa M, Okazaki T, Honjo T. 2003. PD-1 inhibits antiviral immunity at the effector phase in the liver. J. Exp. Med. 198:39–50 77. Dong H, Zhu G, Tamada K, Flies DB, van Deursen JM, Chen L. 2004. B7-H1 determines accumulation and deletion of intrahepatic CD8+ T lymphocytes. Immunity 20:327–36 78. Bertolino P, Trescol-Biemont MC, Rabourdin-Combe C. 1998. Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival. Eur. J. Immunol. 28:221–36 79. Lauer GM. 2005. Hepatitis C virus-speciﬁc CD8+ T cells restricted by donor HLA alleles following liver transplantation. Liver Transpl. 11:848–50 80. Bowen DG, Zen M, Holz L, Davis T, McCaughan GW, Bertolino P. 2004. The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity. J. Clin. Invest. 114:701–12 Crispe

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81. Calne RY, White HJ, Binns RM, Herbertson BM, Millard PR, et al. 1969. Immunosuppressive effects of the orthotopically transplanted porcine liver. Transplant. Proc. 1:321–24 82. Welsh KI, Batchelor JR, Maynard A, Burgos H. 1979. Failure of long surviving, passively enhanced kidney allografts to provoke T-dependent alloimmunity. II. Retransplantation of (AS X AUG)F1 kidneys from AS primary recipients into (AS X WF)F1 secondary hosts. J. Exp. Med. 150:465–70 83. Ng IO, Chan KL, Shek WH, Lee JM, Fong DY, et al. 2003. High frequency of chimerism in transplanted livers. Hepatology 38:989–98 84. Qian S, Demetris AJ, Murase N, Rao AS, Fung JJ, Starzl TE. 1994. Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology 19:916–24 85. Starzl TE, Demetris AJ, Trucco M, Ramos H, Zeevi A, et al. 1992. Systemic chimerism in human female recipients of male livers. Lancet 340:876–77 86. Yang R, Liu Q, Grosfeld JL, Pescovitz MD. 1994. Intestinal venous drainage through the liver is a prerequisite for oral tolerance induction. J. Pediatr. Surg. 29:1145–48 87. Gorczynski RM. 1992. Immunosuppression induced by hepatic portal venous immunization. Immunol. Lett. 33:67–77 88. Polakos NK, Cornejo JC, Murray DA, Wright KO, Treanor JJ, et al. 2006. Kupffer cell-dependent hepatitis occurs during inﬂuenza infection. Am. J. Pathol. 168:1169–78 89. John B, Crispe IN. 2004. Passive and active mechanisms trap activated CD8+ T cells in the liver. J. Immunol. 172:5222–29 90. John B, Crispe IN. 2005. TLR-4 regulates CD8+ T cell trapping in the liver. J. Immunol. 175:1643–50 91. John B, Klein I, Crispe IN. 2007. Immune role of hepatic TLR-4 revealed by orthotopic mouse liver transplantation. Hepatology 45:178–86 92. Polakos NK, Klein I, Richter MV, Zaiss DM, Giannandrea M, et al. 2007. Early intrahepatic accumulation of CD8+ T cells provides a source of effectors for nonhepatic immune responses. J. Immunol. 179:201–10 93. Bertolino P, Bowen DG, Benseler V. 2007. T cells in the liver: there is life beyond the graveyard. Hepatology 45:1580–82 94. Crispe IN. 2003. Hepatic T cells and liver tolerance. Nat. Rev. Immunol. 3:51–62 95. Demirkiran A, Bosma BM, Kok A, Baan CC, Metselaar HJ, et al. 2007. Allosuppressive donor CD4+ CD25+ regulatory T cells detach from the graft and circulate in recipients after liver transplantation. J. Immunol. 178:6066–72 96. San Segundo D, Fabrega E, Lopez-Hoyos M, Pons F. 2007. Reduced numbers of blood natural regulatory T cells in stable liver transplant recipients with high levels of calcineurin inhibitors. Transplant. Proc. 39:2290–92 97. Demirkiran A, Kok A, Kwekkeboom J, Kusters JG, Metselaar HJ, et al. 2006. Low circulating regulatory T-cell levels after acute rejection in liver transplantation. Liver Transpl. 12:277–84 98. Codarri L, Vallotton L, Ciuffreda D, Venetz JP, Garcia M, et al. 2007. Expansion and tissue inﬁltration of an allospeciﬁc CD4+ CD25+ CD45RO+ IL-7Rαhigh cell population in solid organ transplant recipients. J. Exp. Med. 204:1533–41 99. Li W, Carper K, Liang Y, Zheng XX, Kuhr CS, et al. 2006. Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance. Transplant. Proc. 38:3207–8

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Contents

Volume 27, 2009

Frontispiece Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Translating Molecular Insights in Autoimmunity into Effective Therapy Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Structural Biology of Shared Cytokine Receptors Xinquan Wang, Patrick Lupardus, Sherry L. LaPorte, and K. Christopher Garcia p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Immunity to Respiratory Viruses Jacob E. Kohlmeier and David L. Woodland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 61 Immune Therapy for Cancer Michael Dougan and Glenn Dranoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 83 Microglial Physiology: Unique Stimuli, Specialized Responses Richard M. Ransohoff and V. Hugh Perry p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p119 The Liver as a Lymphoid Organ Ian Nicholas Crispe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Immune and Inﬂammatory Mechanisms of Atherosclerosis Elena Galkina and Klaus Ley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p165 Primary B Cell Immunodeﬁciencies: Comparisons and Contrasts Mary Ellen Conley, A. Kerry Dobbs, Dana M. Farmer, Sebnem Kilic, Kenneth Paris, Soﬁa Grigoriadou, Elaine Coustan-Smith, Vanessa Howard, and Dario Campana p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 The Inﬂammasomes: Guardians of the Body Fabio Martinon, Annick Mayor, and Jürg Tschopp p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p229 Human Marginal Zone B Cells Jean-Claude Weill, Sandra Weller, and Claude-Agn`es Reynaud p p p p p p p p p p p p p p p p p p p p p p267

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Aire Diane Mathis and Christophe Benoist p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p287 Regulatory Lymphocytes and Intestinal Inﬂammation Ana Izcue, Janine L. Coombes, and Fiona Powrie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p313 The Ins and Outs of Leukocyte Integrin Signaling Clare L. Abram and Clifford A. Lowell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p339

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Recent Advances in the Genetics of Autoimmune Disease Peter K. Gregersen and Lina M. Olsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p363 Cell-Mediated Immune Responses in Tuberculosis Andrea M. Cooper p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p393 Enhancing Immunity Through Autophagy Christian Munz ¨ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p423 Alternative Activation of Macrophages: An Immunologic Functional Perspective Fernando O. Martinez, Laura Helming, and Siamon Gordon p p p p p p p p p p p p p p p p p p p p p p p p451 IL-17 and Th17 Cells Thomas Korn, Estelle Bettelli, Mohamed Oukka, and Vijay K. Kuchroo p p p p p p p p p p p p p p485 Immunological and Inﬂammatory Functions of the Interleukin-1 Family Charles A. Dinarello p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p519 Regulatory T Cells in the Control of Host-Microorganism Interactions Yasmine Belkaid and Kristin Tarbell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p551 T Cell Activation Jennifer E. Smith-Garvin, Gary A. Koretzky, and Martha S. Jordan p p p p p p p p p p p p p p p591 Horror Autoinﬂammaticus: The Molecular Pathophysiology of Autoinﬂammatory Disease Seth L. Masters, Anna Simon, Ivona Aksentijevich, and Daniel L. Kastner p p p p p p p p p621 Blood Monocytes: Development, Heterogeneity, and Relationship with Dendritic Cells Cedric Auffray, Michael H. Sieweke, and Frederic Geissmann p p p p p p p p p p p p p p p p p p p p p p p p669 Regulation and Function of NF-κB Transcription Factors in the Immune System Sivakumar Vallabhapurapu and Michael Karin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p693

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Immune and Inﬂammatory Mechanisms of Atherosclerosis∗ Elena Galkina1 and Klaus Ley2 1

Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507-1696; email: [email protected]

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Division of Inﬂammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037; email: [email protected]

Annu. Rev. Immunol. 2009. 27:165–97

Key Words

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

inﬂammation, immune cells, pathophysiology

This article’s doi: 10.1146/annurev.immunol.021908.132620

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0165$20.00 ∗ The authors dedicate this review to the memory of Dr. Ross Gerrity (1945–2008), who discovered monocyte recruitment to atherosclerotic lesions.

Atherosclerosis is an inﬂammatory disease of the wall of large- and medium-sized arteries that is precipitated by elevated levels of lowdensity lipoprotein (LDL) cholesterol in the blood. Although dendritic cells (DCs) and lymphocytes are found in the adventitia of normal arteries, their number is greatly expanded and their distribution changed in human and mouse atherosclerotic arteries. Macrophages, DCs, foam cells, lymphocytes, and other inﬂammatory cells are found in the intimal atherosclerotic lesions. Beneath these lesions, adventitial leukocytes organize in clusters that resemble tertiary lymphoid tissues. Experimental interventions can reduce the number of available blood monocytes, from which macrophages and most DCs and foam cells are derived, and reduce atherosclerotic lesion burden without altering blood lipids. Under proatherogenic conditions, nitric oxide production from endothelial cells is reduced and the burden of reactive oxygen species (ROS) and advanced glycation end products (AGE) is increased. Incapacitating ROS-generating NADPH oxidase or the receptor for AGE (RAGE) has beneﬁcial effects. Targeting inﬂammatory adhesion molecules also reduces atherosclerosis. Conversely, removing or blocking IL-10 or TGF-β accelerates atherosclerosis. Regulatory T cells and B1 cells secreting natural antibodies are atheroprotective. This review summarizes our current understanding of inﬂammatory and immune mechanisms in atherosclerosis.

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INTRODUCTION

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Atherosclerosis: a chronic inﬂammatory process characterized by plaque formation within the vessel wall of arteries and extensive necrosis and ﬁbrosis of surrounding tissues Atherosclerotic plaques: plaques consisting of foam cells, immune cells, vascular ECs, SMCs, extracellular matrix, and a lipid-rich core SMC: smooth muscle cell EC: endothelial cell Foam cells: cells derived from macrophages or SMCs that took up modiﬁed LDL through scavenger receptors LDL: low-density lipoproteins

Atherosclerosis is the most common pathological process that leads to cardiovascular diseases (CVD), a disease of large- and medium-sized arteries that is characterized by a formation of atherosclerotic plaques consisting of necrotic cores, calciﬁed regions, accumulated modiﬁed lipids, inﬂamed smooth muscle cells (SMCs), endothelial cells (ECs), leukocytes, and foam cells (1). These features of atherosclerotic plaques illustrate that atherosclerosis is a complex disease, and many components of the vascular, metabolic, and immune systems are involved in this process. Although low-density lipoprotein (LDL) remains the most important risk factor for atherosclerosis, immune and inﬂammatory mechanisms of atherosclerosis have gained tremendous interest in the past 20 years (1–3). This review focuses on the role of inﬂammatory cells in atherosclerosis; the molecular mechanisms of their recruitment and retention in atherosclerotic plaque; differentiation, activation, and production of cytokines; as well as other pro- and anti-inﬂammatory mediators that regulate atherosclerosis and chronic inﬂammation that accompanies this process. We discuss the association of atherosclerosis with other inﬂammatory diseases, as well as existing and potential anti-inﬂammatory treatments for the prevention of atherosclerosis. In 1829, the term arteriosclerosis was ﬁrst introduced by Jean Lobstein (4). Within a few years, the associated cellular immune alterations within the arteries were described by

two different schools of pathology, resulting in two theories of atherosclerosis. Rudolf Virchow postulated an initial role for aortic cellular conglomerates, emphasizing that cellular pathology is critical in atherosclerosis. In contrast, Carl von Rokitansky suggested that initial injury of the vessel wall owing to mechanical injury and toxins led to endothelial dysfunction and further inﬂammation (5). Two centuries later, Mayerl et al. (4) analyzed human samples from von Rokitansky’s collection and showed T cell accumulation already in early lesions, suggesting that lymphocytes play an essential role in atherosclerosis. In the 1970s, the responseto-injury model was described (1). However, a large number of recent papers have emphasized that the chronic inﬂammatory response also has an immune component (3). How and why endothelial functions, lipid metabolism, and lipid retention become unbalanced and disturbed is still unclear. Atherosclerosis entails reactivity to self-antigens, but we do not yet know why this occurs so late in life or what role this response might play in atherosclerosis.

INFLAMMATORY CELL INVOLVEMENT The presence of leukocytes within atherosclerotic arteries was reported in the early 1980s (6). Initially, investigators thought that only macrophages are predominantly present within atherosclerotic vessels. However, several studies reported the presence of most known leukocytes in both mouse and human aortas

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 Immune and inﬂammatory cells in atherosclerosis. Atherosclerotic lesion ( foreground, bottom) and relatively unaffected areas. The endothelial cells above the lesion are polygonal in shape (cobblestone), whereas normal endothelial cells are aligned with the direction of ﬂow. The normal intima is so thin as to be invisible at this level of resolution, but it is greatly expanded in the lesion area, where it contains vascular dendritic cells, macrophages, and foam cells (blue) as well as occasional T lymphocytes ( gray). The foam cells surround the necrotic core (brown), which is thought to be composed of foam cells that have undergone secondary necrosis. The normal media is populated by smooth muscle cells that are organized by several elastic laminae (magenta lines). These laminae move apart as the smooth muscle cells assume a secretory phenotype and may form foam cells. Myeloid cells (blue) invade the media in the lesion area. The normal adventitia is populated by sparse T cells ( gray), B cells ( green), and other lymphocytes (brown) as well as vascular DCs (blue). In the lesion area (bottom), the lymphocytes organize into tertiary lymphoid structures, containing high endothelial venules and other vessels. The angiogenic process eventually leads to neovessels invading the intima, a process that is thought to destabilize plaque and precipitate rupture events. The normal adventitia contains some microvessels (vasa vasorum, in background) that do not penetrate the elastic lamina separating the media from the adventitia. 166

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proliferation in the vessel wall, and almost none on apoptosis. The reasons for this imbalance are both technical and philosophical. Conducting lymphocyte homing studies is possible, yet ﬁnding apoptotic cells in vivo is almost impossible because they are immediately taken up by surrounding phagocytes. Egress studies are technically demanding, and one of the

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(3, 7). The occurrence of inﬂammatory cells in atherosclerotic lesions (Figure 1) depends on the rate of their recruitment and egress and the balance of proliferation, survival, and apoptosis within the arterial wall. So far, most published studies have investigated short-term leukocyte recruitment, with a small minority studying egress, very few reporting on local

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well-established models of egress involves the transplantation of atherosclerotic aortic arches into nonatherosclerotic mice (8). Proliferation can be assessed by dyes such as BrdU (bromodeoxy-uridine) or CFSE (carboxyﬂuorescein succinimidyl ester), but ﬁnding proliferating cells in the vessel wall does not prove that the proliferation occurred locally. Development of more advanced techniques is needed to examine these questions in more detail.

IN AND OUT: MONOCYTE RECRUITMENT TO, RETENTION IN, AND EGRESS FROM THE ARTERY WALL DCs and macrophages already reside within the aortas of C57BL/6 mice before atherosclerosis development (9, 10). DCs are observed in the intima of atherosclerosis-predisposed regions of C57BL/6 aorta, and abundant macrophages are found throughout the aortic adventitia (10). Elegant experiments using bone marrow transfer between mice carrying allelic variants of the CD45 common leukocyte antigen demonstrated that monocyte-derived cells in atherosclerotic plaques are of bone marrow origin (11). The mechanisms of monocyte recruitment into noninﬂamed aortas are not well deﬁned. More is known about monocyte homing to aortas during atherogenesis (12). Monocyte rolling on inﬂamed endothelium occurs in a P-selectin-dependent manner, and absence of P-selectin results in decreased fatty streaks with concomitant reduction of

MACROPHAGE POLARIZATION Both M1 and M2 macrophages are found in atherosclerotic lesions. M1 macrophages are the result of classical activation by lipopolysaccharide in the presence of IFN-γ, which leads to production of high levels of IL-12, IL-23, IL-6, IL-1, and TNFα. Alternatively activated M2 macrophages differentiate in the presence of IL-4, IL-13, IL-1, or vitamin D3 and tend to produce large amounts of IL-10 and express scavenger receptors, mannose receptors, and arginase (20).

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emigrated macrophages within plaques (13). E-selectin overlaps with P-selectin in supporting rolling. Followed by rolling on inﬂamed aortic endothelium, monocytes use vascular cell adhesion molecule (VCAM)-1 for slow rolling and tight adhesion (13). Vcam1D4/D4 Ldlr−/− mice expressing only 8% of normal VCAM-1 showed decreased early lesion formation (14). There is some evidence that β2 integrins and intercellular adhesion molecule (ICAM)-1 might be involved in the support of monocyte recruitment into aortas, but this was not found in all studies (13). The chemokine/chemokine receptor network is essential for direction of leukocyte migration in homeostatic and inﬂammatory conditions (Figure 2). Numerous reports described the important role of the chemokines CXCL1, CCL2, MIF (macrophage migration inhibitory factor), CXCL16, and CX3CL1 and of their receptors CXCR2, CCR2, CXCR2 and CXCR4, CXCR6, and CX3CR1 in the regulation of leukocyte recruitment during atherosclerosis (15).

MONOCYTE SUBSETS In the circulation of mice, monocytes can be distinguished by differential expression of the Ly6C antigen, CX3CR1, and CCR2 (16). Circulating through lymphoid and nonlymphoid organs under homeostatic conditions, Ly6Chigh CCR2+ CX3CR1low monocytes are involved in the inﬂammation caused by microbial infections (17) and have been named inﬂammatory monocytes. Ly6Clow CCR2low CX3CR1high resident monocytes may patrol the inside of blood vessels under homeostatic conditions (18) and extravasate during infection with Listeria monocytogenes with differentiation to M2-like, alternatively activated macrophages (see side bar, Macrophage Polarization) (17). Similar subsets of inﬂammatory and resident monocytes have also been described in human blood (19), but it is not clear whether the mouse and human subsets are truly corresponding. Recent studies suggest that Ly6Chigh monocytes preferentially migrate into

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CXCL1, 2, 8 CCL5 CX3CL1 CCL2 P-selectin VCAM-1

High endothelial venule Lesion

Figure 2 Monocyte recruitment to the atherosclerosis-prone vessel wall. Monocytes use an overlapping network of adhesion molecules and chemokine receptors to enter the artery wall. P-selectin supports rolling and monocyte-platelet interactions. Monocyte α4 β1 integrin interacting with endothelial VCAM-1 reduces rolling velocity and leads to ﬁrm adhesion. Surface-immobilized chemokines including CXCL1, CXCL2, CXCL4, CCL5, and others can activate monocytes as they roll by, leading to increased adhesiveness of α4 β1 integrin through inside-out signaling and receptor clustering. Ly6Chigh monocytes use CCR2, CX3CR1, and CCR5 to migrate to aortas, whereas Ly6Clow monocytes use CCR5. L-selectin, interacting with an unknown ligand, and CXCR6, likely interacting with CXCL16, are partially responsible for lymphocyte recruitment, likely from vasa vasorum and under lesions from high endothelial venules.

atherosclerosis-prone arteries and predominantly differentiate into aortic macrophages (21), utilizing CX3CR1, CCR2, and CCR5 chemokine receptors (22). These conclusions are based on monocytes loaded with latex beads (22) or cultured in vitro for 24 h (21), both procedures that likely alter the monocyte phenotype (23). The functions of CX3CL1, the only known ligand for CX3CR1, and CCR2 are not completely overlapping because atherosclerosis in Cx3cl1−/− Ccr2−/− Apoe−/− mice is reduced compared with Ccr2−/− Apoe−/− and Cx3cl1−/− Apoe−/− mice (24). Combined inhibition of CCL2, CX3CR1, and CCR5 abrogated monocytosis and almost abolished atherosclerosis (up to 90%) in Apoe−/− mice (25). LyC6low monocytes require CCR5 for migration to aortas (22), but unknown additional chemokines are likely involved in monocyte recruitment and differentiation in the arterial wall. Monocyte recruitment is also determined by monocyte release from the bone marrow. It is not clear how hyperlipidemia affects monocyte maturation and functions. It is possible

and indeed likely that Ly6Chigh and Ly6Clow monocyte-derived macrophages and DCs alter their phenotype under conditions of hypercholesterolemia.

MACROPHAGES Macrophages were the ﬁrst inﬂammatory cells to be associated with atherosclerosis. In a groundbreaking paper, Gerrity and coworkers (6) identiﬁed these cells as the main component of the atherosclerotic plaque in porcine specimens. Macrophages produce proinﬂammatory cytokines, participate in lipid retention and vascular cell remodeling, and express pattern-recognition receptors (PRRs), including scavenger receptors (SRs) and Toll-like receptors (TLRs) that connect the innate and adaptive immune response during atherosclerosis. Macrophages use PRRs to phagocytose different microbes and microbial components. They can also take up modiﬁed LDL and form foam cells. There is some controversy about the function of SRs in atherogenesis. Early studies showed that SRs play a strictly

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ApoE: apolipoprotein E Apoe−/− mice: these mice develop severe hypercholesterolemia and atherosclerosis plaques that have some similarities to human atherosclerotic plaques SR: scavenger receptor TLR: Toll-like receptor

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proatherogenic role in atherosclerosis, given that Cd36−/− Apoe−/− mice showed protection from atherosclerosis (26). In a separate study, Apoe−/− mice lacking SR-A or CD36 showed increased aortic sinus lesion areas with abundant foam cells, suggesting alternative lipid uptake mechanisms and a possible atheroprotective role for SR-A and CD36 (27). Apoe−/− mice with a combined deﬁciency of CD36 and SR-A I/II showed no further reduction of atherosclerosis compared with Cd36−/− Apoe−/− mice (28). Macrophages are extremely plastic cells, taking on many different phenotypes. M1 and M2 macrophages play opposite roles during inﬂammation, but both are present in atherosclerotic lesions. Using an elegant model of CD11b diphtheria toxin (DT) receptor transgenic mice, Stoneman et al. (29) showed that CD11b+ cells are critical to atherogenesis, but once plaques are established, killing CD11b+ cells does not reduce plaque burden. Because in this model DT administration would eliminate all CD11b+ cells in mice, it is unclear whether the effect from the deletion of CD11bexpressing cells was due to an exclusive role played by macrophages or to a combined role of macrophages, myeloid DCs, and perhaps neutrophils in this model. The ability to form foam cells can also be stimulated by the exposure to lipopolysaccharide (LPS) of Chlamydia

Macrophage SR

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VEGF IL-10, TGF-β Figure 3 Macrophage functions. Macrophages express scavenger receptors (SRs), TLRs, and other receptors for pathogen-associated molecular patterns (PAMPs). Engagement of these receptors results in release of proinﬂammatory cytokines IL-1, IL-6, IL-12, IL-15, IL-18, TNF-α, and MIF, as well as anti-inﬂammatory IL-10 and TGF-β. Vascular endothelial growth factor (VEGF) promotes angiogenesis. 170

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pneumoniae through TLR-dependent activation of macrophages resulting in the production of IL-18, IL-12, and IL-15 and promoting a Th1 response with further inﬂammation within the wall (30). The production of some inﬂammatory mediators by macrophages is summarized in Figure 3. An increase in macrophage apoptosis in early lesions appears to cause the attenuation of atherogenesis, whereas impairment in macrophage apoptosis in the late stage may contribute to secondary necrosis, leading to increased proinﬂammatory responses and further apoptotic signals for SMCs, ECs, and leukocytes within the plaques (31). Deﬁciency of phospholipase C β3 resulted in enhanced sensitivity of newly recruited macrophages to 25-OHC- or oxLDL-induced apoptosis in early lesions with concomitant decrease of atherosclerosis (32). Because elimination of phospholipase C β3 leads to no visible effect on the mouse phenotype (32), this may be an attractive target for the modulation of macrophage apoptosis. The adipocyte fattyacid-binding protein aP2 has an important role in regulating systemic insulin resistance and lipid metabolism and plays a protective role in atherosclerosis (33). Dysregulation in the balance between the inﬂux and efﬂux of modiﬁed LDLs leads to the formation of lipid-laden foam cells. ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) initiate macrophage reverse cholesterol transport in vivo. Combined deﬁciency of ABCA1 and ABCG1 results in foam cell formation and further acceleration of atherosclerosis (34). Investigators (35) also found that macrophagespeciﬁc overexpression of cholesteryl ester hydrolase, which participates in cholesterol efﬂux, resulted in atherosclerosis reduction in Ldlr−/− mice, demonstrating that enhanced cholesterol efﬂux and reverse cholesterol transport play important roles in atherosclerosis prevention.

VASCULAR DENDRITIC CELLS About a decade after the discovery of vascular macrophages and foam cells, vascular

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DCs were described in atherosclerotic lesions (36), forming a network of DCs within the intima of arteries but not veins of healthy humans and rabbits. These arterial DCs are CD1a+ S-100+ lag+ CD31− CD83− CD86− and phenotypically similar to Langerhans cells of the skin. Further studies revealed that aortic CD68+ CD11c+ DCs have extended long, dendritic-like processes and are mostly located in the intima layers of the lesion-susceptible lesser curvature of the aortic arch (10). Interestingly, the number of intimal DCs, but not the number of CD68+ adventitial macrophages, was signiﬁcantly reduced in Vcam1D4D/D4D mice, suggesting a role for VCAM-1 in intimal DC recruitment (10). The function of vascular DCs within healthy and atherosclerosis-prone arteries is poorly understood. DCs are seen in contact with T cells in the atherosclerotic plaques within the zones of neovascularization and near the zones of vasa vasorum with the adventitia. In the immune system, DCs are deﬁned as cells that can present antigen to naive T cells. They constitutively migrate through nonlymphoid organs to secondary lymphoid tissues (17). Whether vascular DCs can present antigen has not been formally demonstrated, but two efforts support

Dendritic cell CD11c +

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this concept: (a) experiments using TCR transgenic mice and antigenic peptide-treated DCs (9) and (b) a study showing that isolated vascular DCs are capable of presenting antigen to transgenic T cells as effectively as bone marrow– derived myeloid DCs (T. Deem & K. Ley, unpublished data) (Figure 4). In shoulder regions of human unstable plaques, CD83+ DCs are in close proximity to CD40L+ T cells. These DCs produce CCL19 and CCL21 chemokines that might accelerate naive lymphocyte recruitment into atherosclerotic vessels (37). Some DCs within atherosclerosis-prone vessels are IFN-α+ plasmacytoid DCs that responded to pathogenderived motifs (38) and might lead to accelerated apoptosis of CD4+ T cells. Blood monocytes enter different tissues and differentiate into tissue-resident macrophages or DCs that likely leave nonlymphoid tissue within a few days and migrate back to lymphoid organs through the lymphatic vessels. The number of DCs is signiﬁcantly elevated in atherosclerosis-prone arteries. Reasons may include accelerated migration into the aorta, reduced emigration out of the vessel wall, increased local proliferation, or decreased apoptosis. In a model of atherosclerosis

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Antigen presentation Costimulation IFN-γ CD40 — CD40L OX40L — OX40 CD80, 86 — CD28

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Figure 4 Interactions between DCs and T cells. DCs may present possible atherosclerosis antigens (possibly derived from HSP-60 and oxLDL) to T cells in the context of costimulatory molecules like CD40, OX40L, CD80, and CD86, eliciting T cell differentiation and proliferation. Although a Th1-biased response is documented in atherosclerosis, there is also a signiﬁcant body of evidence suggesting a possible role for Th2 cells in mature lesions. Whether newly discovered Th17 cells also play a role remains to be investigated. Treg cells have antiatherogenic effects and play a protective role against atherosclerosis, mainly by secreting IL-10 and TGF-β. www.annualreviews.org • Immune and Inflammatory Mechanisms of Atherosclerosis

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Figure 5 Egress of macrophages and DCs from the arterial wall. The factors that control retention of macrophages and DCs in atherosclerotic vessels are not well deﬁned, but sphingosine-1-phosphate (S1P) may have a role in this process. CCR7 expression is necessary for the exit of DCs and macrophages from atherosclerotic plaques.

regression, CCR7-dependent trafﬁcking of monocyte-derived cells out of atherosclerotic plaques was detected during lesion regression (Figure 5), but little emigration was detected from progressive plaques (8, 39). Mechanisms that might regulate DC responses to modiﬁed LDL have not been deﬁned, but some studies suggest that oxLDL plays a role in DC maturation and activation in vivo. Oxidized phospholipids (ox-PLs) alter DC activation; prevent their maturation by blocking TLR3- and TLR4-dependent induction of CD40, CD80, CD83, and CD86; and block IL-12, TNF, and lymphocyte stimulatory capacity (40). OxLDL during the late stage of monocyte differentiation gives rise to phenotypically mature DCs that secrete IL-12 but not IL-10, and it supports both syngeneic and allogeneic T cell stimulation (41).

T LYMPHOCYTES T lymphocytes mainly reside within the adventitia of normal, noninﬂamed arteries (7). Short-term adoptive-transfer experiments suggest that this T cell localization is a consequence

HSP: heat shock protein

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of constitutive homing of T cells into the aortic wall, which is partially L-selectin dependent (9), and multiphoton microscopy imaging conﬁrms this observation (42). Atherosclerosisprone conditions accelerate T cell recruitment into the aortas in early and advanced atherosclerosis (13). Most of the T cells are TCRαβ+ CD4+ cells with an activated phenotype, and a few express CD8 or TCRγδ (43, 44). Oxidized lipoprotein- and heat shock protein (HSP)speciﬁc T cells are found in atherosclerotic plaques, suggesting local activation and clonal expansion during atherogenesis (45, 46). The set of Vβ and Vα segments are limited within the atherosclerotic lesions with preferential expression of Vβ6 TCR (45). Several studies have also demonstrated CD80-, CD86-, and CD40CD154-dependent T cell responses to oxLDL (47). Atherosclerosis is a dynamic process, and the inﬂammation that accompanies atherosclerosis goes through different stages. There is evidence that early atherosclerosis shows a Th1 response with prevalent production of IFN-γ, IL-6, and IgG2a antibodies against modiﬁed LDL (48). T-bet deﬁciency results in the reduction of lesional SMCs, a switch in the response to HSP-60 toward Th2, an alteration in the T cell–dependent isotype of oxLDL-speciﬁc antibodies, an increase in atheroprotective B1-derived antibodies, and reduced atherosclerosis (49). These results suggest that the Th1 response is proatherogenic and affects atherosclerosis not only through the production of proinﬂammatory cytokines, but also through the regulation of B cell function and antibody production. Severe hypercholesterolemia in Apoe−/− mice induces a switch to a Th2 response and IL-4 production in atherosclerotic lesions, but this Th2 response does not prevent further atherosclerosis development (48). Adoptive transfer of CD4+ T cells into scid/scid Apoe−/− mice clearly demonstrates the proatherogenic role of CD4+ T cells in these immunodeﬁcient conditions (47). Further studies have demonstrated a more complex

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role for CD4+ T cells in atherosclerosis. Immunization with MDA-LDL results in CD4+ T cell–independent atheroprotection; however, the atheroprotective immunization with adjuvant injections is CD4+ T cell– dependent (47). It is remarkable and intriguing how CD4+ T cells can play such opposite roles under different conditions. There are at least two sites of action during atherosclerosis: systemic through the secondary lymphoid organs and local within the arteries. Administration of the sphingosine-1-phosphate (S1P) analog FTY720 results in reduced lymphocyte proliferation, IFN-γ production, decreased plasma levels of IL-6, IL-12, and CCL5, and a switch of macrophages to the M2 phenotype, resulting in reduced atherosclerosis in Ldlr−/− (50) and Apoe−/− mice (51). Because FTY720 affects monocyte, T lymphocyte, and B lymphocyte trafﬁcking, it is not clear which population is most responsible for the atheroprotective effect of FTY720.

Regulatory T Cells The balance between Th1 and Th2 responses is tightly controlled by Tregs, which are critical in maintaining immunological tolerance (52). Atherosclerosis can be considered an autoimmune disease, as signiﬁcant evidence shows a response to self-antigens such as HSP-60 and LDL during atherosclerosis. Therefore, impaired function or absence of Tregs is likely among the reasons for the local inﬂammation and proinﬂammatory response in atherosclerosis. Adoptive transfer of cognate peptidespeciﬁc Tregs (Tr1) into Apoe−/− mice diminishes the production of the Th1 cytokine IFN-γ and of IgG2a. Together with elevated IL-10 levels, these phenomena may explain reduced atherosclerosis in recipient mice (53). Adoptive transfer of bone marrow cells deﬁcient in CD4+ CD25+ Tregs into irradiated Ldlr−/− donors results in increased lesion size (54). In the same study, transfer of splenocytes deﬁcient in CD4+ CD25+ Tregs into Rag2−/− Apoe−/− mice doubled the lesion size

compared with mice transferred with wild-type splenocytes. Thus, naturally occurring Tregs that maintain immunological tolerance may play an important role in atherosclerosis prevention (Figure 4). Which factors might affect the generation and maintenance of Tregs? Recent studies suggest that inducible costimulatory molecule (ICOS) is involved in the Treg response during atherosclerosis. Tregs from Icos−/− mice have an impaired TGF-β-dependent suppressor function compared with wild-type cells (55), suggesting that ICOS is an important molecule controlling Treg functions. Interestingly, the number and activity of Tregs can also be modulated by CD31 (56), but the underlying mechanisms are not known. Unexpectedly, obesity increases Treg numbers and improves Treg function in atherosclerosis-prone mice. Leptin deﬁciency in Ldlr−/− mice reduces Th1 polarization and improves Treg cell functions, with a signiﬁcant reduction in lesion development (57). These results identify a critical role for the leptin/leptin receptor pathway in the modulation of the regulatory immune response in atherosclerosis. Another study shows that the oral administration of HSP-60 can induce an increase in CD4+ CD25+ Foxp3+ Treg cells in HSP-60-treated mice in parallel with decreased atherosclerosis (58). Increased production of IL-10 and TGF-β by lymph node cells in response to HSP-60 was observed after tolerance induction, suggesting key roles for IL-10 and TGF-β in the balancing of the immune response against possible antigens in atherosclerosis.

γδ T Cells γδ T cells account for 5% of all T cells but are enriched at sites of exposure to antigens such as skin and gastrointestinal mucosa as well as at sites of chronic inﬂammation. γδ T cells are also observed within the intima of human atherosclerotic vessels at the early stages of atherosclerosis. Interestingly, γδ T cell–deﬁcient Apoe−/− mice show no difference

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ATHEROSCLEROSIS AND OBESITY A growing body of evidence suggests a close association between the immune system, obesity, diabetes, and atherosclerosis (189). Obesity is characterized by an excess of adipose tissue mass and is associated with low-grade inﬂammation in white adipose tissue, resulting in chronic activation of the immune system. Adipose tissues include adipocytes, preadipocytes, ﬁbroblasts, and macrophages and other types of leukocytes that secrete various cytokine-like hormones, including adiponectin, leptin, resistin, and visfatin, as well as TNF-α, IL-6, IL-1, and CCL2 (190). Adiponectin and leptin are the most abundant adipocyte products and are considered key components in regulating inﬂammation within the adipose tissues. Serum levels of adiponectin are markedly decreased in obesity, insulin resistance, type 2 diabetes, and atherosclerosis (191). Several reports suggest that adiponectin has antiatherogenic and antithrombotic effects by reducing lipid accumulation in macrophage-derived foam cells (192) and suppressing the production of CXCL10, CXCL11, and CXCL9, leading to reduced T cell homing into atherosclerosisprone aortas (193). Adiponectin can dampen the inﬂammatory phenotype of ECs and SMCs (190) and curb platelet aggregation (194). In contrast, leptin is considered a proinﬂammatory and proatherogenic cytokine. Leptin increases the secretion of CCL2 and endothelin-1 by endothelial cells, initiates proliferation and oxidative stress in ECs, and promotes migration and proliferation of SMCs (195). Leptin also facilitates thrombosis by increasing platelet aggregation (196). Treatment with recombinant leptin accelerates atherosclerosis and thrombosis in Apoe−/− mice (197). Paradoxically, leptin resistance is also proatherogenic. Apoe−/− mice lacking the long form of the leptin receptor (db/db) show elevated atherosclerosis compared with control Apoe−/− mice (182). The discovery of adipokines has spawned the concept of an association between atherosclerosis and low-degree inﬂammation not only within the aortic wall, but also within the surrounding adipose tissues.

in atherosclerosis development throughout the aortic root (59).

NATURAL KILLER CELLS Natural killer (NK) cells were found in early and advanced human atherosclerotic lesions, mainly in the shoulder regions. There is no mouse model that can provide complete NK 174

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cell deﬁciency, but some models, including Il15−/− and Il15ra−/− mice, lack fully functional NK cells (60). Defective cytolysis by NK cells has no effect on atherosclerosis in the model of Ldlr−/− perforin-deﬁcient mice (61). However, Ldrl−/− Lystbeige mutant mice that have defective protein release from cytoplasmic granules show reduced atherosclerosis. Ldrl−/− Lystbeige Rag1−/− mutant mice demonstrate increased atherogenesis and lipid levels, which complicate interpretation (61). Because in those models NK cells are still present, other NK cell functions might be involved in atherosclerosis. To further address a possible role of NK cells in atherosclerosis (Figure 6), bone marrow cells from transgenic mice expressing Ly49A under the control of the granzyme A promoter were transferred into lethally irradiated Ldlr−/− mice (62). Absence of fully functional NK cells in this model resulted in 70% reduction of atherosclerotic lesion formation, although it cannot be excluded that some natural killer T cells (NKT cells) and a subset of CD8+ T cells expressing Ly49A might also be affected.

NATURAL KILLER T CELLS Glycolipid antigens can be presented by CD1 (Figure 7), a major histocompatibility complex (MHC)-like glycoprotein, to CD1-restricted T cells (63). Cellular lipid homeostasis and metabolism play a critical role in atherosclerosis; however, modiﬁed lipids may also regulate inﬂammation through CD1-mediated antigen presentation. The proatherogenic role of NKT cells has been convincingly demonstrated in different mouse models (60). Cd1d−/− /Apoe−/− mice have a decrease in lesion size up to 25% (64). To show that this effect is dependent on CD1-restricted NKT cell activation, α-galactosylceramide (α-GalCer), a glycolipid that activates NKT cells, was injected into Apoe−/− mice, and it induced a 50% increase in atherosclerosis. In parallel, inﬂammatory Th1 as well as Th2 cytokines have been observed in Apoe−/− mice that received α-GalCer (64). Interestingly, administration of α-GalCer to

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Apoe−/− mice with established lesions had no signiﬁcant effect on size, but it did decrease their collagen content (65). Cd1d−/− Ldlr−/− mice express both IL-12 and IL-10, with unaltered levels of IFN-γ, suggesting that the absence of NKT cells does not signiﬁcantly alter Th1/Th2 balance (66).

Granzymes

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Perforin IFN-γ Figure 6 NK cells. Activated NK cells produce IFN-γ, which promotes a Th1 response, and release perforin and granzymes, which causes apoptosis in target cells.

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Mast cells are major effector cells in allergy and host defense responses. Upon activation, mast cells release a broad spectrum of proinﬂammatory cytokines, growth factors, vasoactive substances, and proteolytic enzymes (Figure 8). Although vascular mast cells are rare, they are found within the adventitia and lesions of atherosclerotic plaques, especially in the location of rupture-prone shoulder regions (67). Because mast cells are loaded with proteases such as tryptase and chymase, they might destabilize atherosclerotic plaques. Indeed, mast cells are colocalized within the regions of plaque rupture, and the activation of perivascular mast cells correlates with intraplaque hemorrhage, macrophage and EC apoptosis, vascular leakage, and CXCR2- and very late antigen (VLA)-4-mediated recruitment of leukocytes to the plaque (68). Mast cells alter lipid metabolism by interfering with ApoEand ApoA-II-dependent cholesterol efﬂux (69). Mast cell–deﬁcient KitW-s h/W-s h Ldlr−/− mice show increased collagen content, ﬁbrous cap development, and reduced local inﬂammation with diminished numbers of T cells and macrophages and reduced atherosclerosis (70). Adoptive transfer of wild-type, but not IL-6- or IFN-γ-deﬁcient, mast cells restores atherosclerosis progression, suggesting that mast cells provide IL-6 and IFN-γ in atherosclerosis.

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ciate the impact of B cells on atherosclerosis, recent studies have evaluated the role of B cells in directing the immune response during atherosclerosis (Figure 9). Adoptive transfer of bone marrow from B cell–deﬁcient mice into lethally irradiated Ldlr−/− mice resulted in up to 40% increased lesion size in parallel with decreased production of anti-oxLDL antibodies. This B cell deﬁciency did not seem

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?

B CELLS B cells in atherosclerosis were initially discovered within the adventitia, and immunoglobulin-positive cells were detected within atherosclerotic plaques (7). Although investigators did not initially appre-

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Figure 8 Mast cells. Interactions of mast cells with DCs may promote release of proatherogenic TNF-α, INF-γ, and IL-6, a broad spectrum of proteases, the 5-lipoxygenase product leukotriene (LT) B4, and GM-CSF. Mast cells may direct the development of Th1 or Th2 responses.

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B cells. The B1 subset of B cells is independent of T cell help and produces IgM natural antibodies that appear to have atheroprotective functions. They may be triggered by foreign or self-antigens through their B cell receptor (BCR). Th1-dependent B2 cells produce IgG2a and IgG1. B cells also produce IL-10.

Natural antibodies: these antibodies are found in the absence of exogenous antigens early after birth; they ﬁght bacterial and viral infections, but also recognize self-antigens and carry out a protective function in atherosclerosis

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to affect Th1 or Treg responses speciﬁcally because a simultaneous decrease in IFN-γ, IL-10, and TGF-β was observed (71). Absence of splenocytes upon splenectomy aggravated atherosclerosis in Apoe−/− mice with an associated reduction of anti-oxLDL antibodies. Adoptive transfer of splenic B cells, but not of T cells, from atherosclerosis-prone Apoe−/− mice into young Apoe−/− recipients reduced atherosclerosis (72). These studies indicate that atheroprotective immunity develops during the progression of atherosclerosis and that B cells or their immunoglobulin products may perform protective functions. Extensive atherosclerosis in Apoe−/− mice is associated with increased natural antibody titers to oxLDL (73). These IgM autoantibodies to oxLDL recognize ox-PLs containing the phosphorylcholine (PC) head group, and they block binding and degradation of oxLDL by macrophages in vitro (73). The IgM antibodies found in atherosclerosis are structurally and functionally identical to classic natural T15 anti-PC antibodies that are produced by B1 and marginal zone B cells (74, 75). Immunization with malondialdehyde (MDA) leads to the expansion of antigen-speciﬁc Th2 cells, elevated production of IL-5, noncognate stimulation of B1 cells, and thus increased production of these antibodies (76). Currently, little information is available about the presence of B1 cells and plasma cells in secondary lymphoid organs and in the aorta under atherosclerosis-prone conditions. In atherosclerotic mice, the spleen is a major source of oxLDL-speciﬁc IgM antibodies Galkina

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(73). Much less is known about the local production of natural antibodies within the aorta or their role in preventing modiﬁed LDL uptake by macrophages and other cell types.

NEUTROPHILS Neutrophils are short-lived phagocytic cells with a broad spectrum of biologically active molecules such as myeloperoxidase (MPO) and proteinases (Figure 10). Leukocytosis and especially neutrophilia are independent risk factors for coronary heart disease. CXCR4 and its ligand CXCL12 are involved in the egress of neutrophils from bone marrow and also regulate recruitment of neutrophils to atherosclerotic lesions (77). Chronic blockade of CXCR4 causes neutrophilia and increases neutrophil content in plaques, associated with apoptosis and a proinﬂammatory phenotype, suggesting

Neutrophil

ROS MPO Proteases CXCL1 CXCL8

Figure 10 Neutrophils. Neutrophils, although rare in mature atherosclerotic lesions, interact with the endothelium covering atherosclerotic lesions and may release reactive oxygen species (ROS), myeloperoxidase (MPO), proteases, and the chemokines CXCL1 and CXCL8.

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a proinﬂammatory role for neutrophils in atherosclerosis (77). Further studies are necessary to dissect how neutrophils might affect functions of vascular ECs, SMCs, and aortic leukocytes through the production of ROS, enzymes, metalloproteinases, and proinﬂammatory cytokines. It is unclear whether neutrophils are equally important in human atherosclerosis.

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PLATELETS Platelets play a major role in the hemostatic process and in thrombus formation upon injury. They can regulate the inﬂammatory and immune responses through the secretion of inﬂammatory mediators that modulate leukocyte recruitment into inﬂamed tissues (78). Activated platelets expressing P-selectin are detected at different stages of atherosclerosis (Figure 11). Bone marrow transfer experiments with P-selectin-deﬁcient mice show that platelet P-selectin contributes to lesion development and assists in calciﬁcation of atherosclerotic plaques (79). Activated

Monocyte

PSGL-1

platelets transiently interact with the endothelium of atherosclerotic carotid arteries of Apoe−/− mice in vivo. This transient interaction results in immobilization of plateletderived CCL5 and CXCR4 on atherosclerotic endothelium (80, 81). Platelet glycoproteins GPIIb/IIIa, GPIb, and endothelial von Willebrand factor are at least partially responsible for the platelet-endothelial interactions. Immobilized platelets also interact with leukocytes through P-selectin/P-selectin glycoprotein ligand (PSGL)-1 interactions that activate Mac-1 and VLA-4 integrins and may facilitate ﬁrm monocyte adhesion (80). Platelets also initiate rolling of DCs through PSGL-1-dependent interactions between Mac-1 and junctional adhesion molecule ( JAM)-C in injured carotid arteries (82). Surprisingly, platelets are also required for CX3CL1-induced leukocyte adhesion at high shear rates. Both soluble and membrane-bound CX3CL1 trigger P-selectin expression on adherent platelets, which facilitates the local accumulation of leukocytes under arterial shear (83). Platelets also help recruit and differentiate progenitor cells into ECs through the interactions of P-selectin and PSGL-1 and by using the β1 and β2 integrins. Platelets bridge inﬂamed endothelium and circulating blood cells and support recruitment of leukocytes to inﬂamed atherosclerotic endothelium.

ROS: reactive oxygen species

P-selectin

THE CASE FOR A VASCULAR IMMUNE RESPONSE

Platelets

Anatomical Proximity CD40L

TGF-β

CXCL4 CCL5 CXCL12

IL-1β

Figure 11 Platelets. Activated platelets release proinﬂammatory IL-1β, CD40L, CXCL12, CXCL4, and CCL5 as well as anti-inﬂammatory TGF-β. Through P-selectin binding to PSGL-1, platelets interact with monocytes.

Although much evidence supports the involvement of the immune system in a systemic response to hyperlipidemia, a signiﬁcant body of data also suggests a local immune response within the aortic wall. Atherosclerotic plaques are formed in very speciﬁc regions of the aortic tree where ﬂow is disturbed (84). In contrast, very little inﬂammation or atherosclerosis is found in laminar ﬂow regions (10). Activated by disturbed ﬂow, ECs elevate expression of adhesion molecules and chemokines,

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VALT: vascularassociated lymphoid tissue

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which accelerate leukocyte recruitment. Rolling and ﬁrm adhesion of monocytes to aortic endothelium was documented in an ex vivo model of the carotid artery and in vivo (13). Eriksson et al. (85) demonstrated a role for L-selectin-dependent secondary capture in leukocyte accumulation in inﬂammation and atherosclerosis in vivo. However, there is no direct intravital microscopic evidence for lymphocyte recruitment from the arterial lumen into the vessel wall. Several reports demonstrate T and B lymphocyte accumulation in the aortic adventitia in normal (9) and atherosclerotic vessels (9, 85, 86). Adoptive transfer experiments suggest that lymphocytes accumulate in the adventitia through the migration from the adventitial vasa vasorum rather than from the intimal lumen site (9). Local revascularization correlates with an increase in cellular composition within vulnerable regions of human atherosclerotic plaques (Figure 1). In contrast, the inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis (87). Recently, investigators have shown that vasa vasorum can penetrate the media, enter atherosclerotic plaques, and come close to the arterial lumen (88). This is an important direct demonstration of the existence of a vascular network connecting the adventitia with the plaque tissue. Thus, we now better understand the role of neovascularization in atherosclerosis (87), but further studies are necessary to elucidate the role of small adventitial vessels in the immune response during this disease. The presence of antigen-presenting cells and T cells within atherosclerosis-prone artery walls is well documented, but there is little information about local antigen-dependent activation of T cells. It remains to be determined whether elevated numbers of lymphocytes, which have been seen in atherosclerotic vessels, are a consequence of the accelerated recruitment of activated cells from draining lymph nodes or of local antigen-induced proliferation that leads to the increased aortic lymphocyte numbers. Galkina

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One of the possible sites of T cell activation in aorta may be vascular-associated tertiary lymphoid structures (Figure 1). The lymphoid-like structures are formed in a variety of autoimmune-mediated diseases, such as rheumatoid arthritis or Hashimoto’s thyroiditis. Conglomerates of leukocytes within the adventitia were reported in the early 1970s; however, only in 1997 did Wick et al. (44) name these conglomerates vascularassociated lymphoid tissues (VALTs). These lymphoid structures are formed within advanced atherosclerosis-prone vessels and contain T and B lymphocytes, plasma cells, CD4+ /CD3− inducer (LTi) cells, and some MECA-32+ and HECA-452+ microvessels (9, 86, 89). Follicles located close to the arterial external elastic lamina contain proliferating Ki67+ leukocytes, apoptotic cells, and CD138+ plasma cells, showing local B cell maturation and possible humoral immune response in these structures (86). Whether the VALTs in atherosclerosis are beneﬁcial or proatherogenic is still unclear.

CYTOKINE INVOLVEMENT IN ATHEROSCLEROSIS Cytokines are key players during acute and chronic inﬂammation. The regulation of cytokine production depends on many factors and is tightly regulated during inﬂammation. An excellent review by Tedgui et al. (90) analyzed the cytokine biology in atherosclerosis. Many cytokines, such as TNF-α, IL-1, IL-2, IL-3, IL-6, CXCL8, IL-10, IL-12, IL-15, IL-18, IFN-γ, M-CSF, TGF-β1, TGF-β2, and TGF-β3, are detected within atherosclerosisprone vessels (90). SMCs and ECs produce TNF-α, IL-1, IL-6, CXCL8, and IL-15 and regulate the production of other cytokines in an autocrine and paracrine manner. ECs affect hematopoietic cell proliferation through the production of stem cell factor, IL-3, GMCSF, G-CSF, and M-CSF (90). Under conditions of hyperlipidemia, macrophages produce TNF-α, IL-1, IL-6, IL-12, IL-15, and IL-18 but also the anti-inﬂammatory cytokines IL-10

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and TGF-β. Proatherogenic mast cells generate IL-6 and IFN-γ that are crucial in the induction of mast cell–dependent acceleration of atherosclerosis. Upon activation, platelets shed IL-1β, CD40L, and CXCL4, which may serve as proatherogenic mediators. Cytokines can affect endothelial permeability, the expression of adhesion molecules, SR, lipid metabolism, and proliferation and migration of SMCs and ECs. Cytokines also inﬂuence extracellular matrix composition through the alteration of the expression of matrix metalloproteinase (MMP)-1, -3, -8, -9, and -12 and their inhibitors TIMP-1, -2, and -3 (91). Neovascularization is a proatherogenic process that greatly depends on the local microenvironment of cytokines and growth factors (87). T cell–derived cytokines are likely responsible for plaque neovascularization because many T cells are found within the regions of microvessels surrounding the plaques. Proatherogenic IL-1β, TNF-α, and leptin, as well as vascular endothelial growth factor (VEGF) and placental growth factor (PIGF), accelerate neovascularization. IL-10, CXCL9, CXCL10, and adiponectin may balance the process of neovascularization and play antiatherogenic and antiangiogenic roles.

TNF-α Several studies were conducted to analyze the role of TNF-α in atherogenesis, but until recently results were contradictory. TNF-αdeﬁcient Apoe−/− mice showed a reduction in lesion formation, with a concomitant decrease in VCAM-1, ICAM-1, and CCL2 expression (92). In contrast, mice deﬁcient in TNF-α receptor (TNFR) p55 developed larger lesions compared with controls (93). Because of elevated cholesterol levels in p55-deﬁcient mice, it was difﬁcult to interpret whether TNFR deﬁciency itself resulted in the acceleration of atherosclerosis. Recently, a study with mutant mice clearly identiﬁed the roles of transmembrane and soluble forms of TNF in the context of hyperlipidemia. TNF-α affected the development of atherosclerosis at the fatty streak

stage, and cleavage of TNF was an important step in activating the proatherogenic properties of TNF-α (94).

IL-1 IL-1 stimulation initiates leukocyte adhesion to ECs and transmigration and serves as a local autocrine and paracrine stimulator of other cytokines (90). Studies with blocking IL-1ra antibodies in Apoe−/− mice and with Ldlr−/− transgenic mice that overexpress IL-1ra or that have a deﬁciency in IL-1β clearly show that IL-1 is involved in atherogenesis (90).

IL-2 That IL-2 may serve as a proinﬂammatory, proatherogenic cytokine was shown in experiments with the administration of IL-2 or IL2-blocking antibodies into Apoe−/− mice (95). However, further experiments are needed to analyze the differential role of IL-2 on Tregs and in lipid metabolism.

IL-6 There are two lines of studies that propose a speciﬁc role for IL-6 in atherosclerosis: IL-6 administration into wild-type C57BL/6 mice increases the formation of fatty streaks (96), but Il6−/− Apoe−/− mice show elevated atherosclerosis and decreased leukocyte homing (97, 98). It is important to note that IL-6-dependent regulation of lipid metabolism may have confounded these studies.

IL-12 IL-12 is a key Th1 cytokine that is produced mainly by plaque macrophages and stimulates proliferation and differentiation of NK cells and T cells. IL-12 is detected in the aortas of Apoe−/− mice, and the administration of IL-12 results in enhanced lesion size in Apoe−/− recipients (99). IL-12p40-deﬁcient Il12b−/− Apoe−/− mice have a 52% reduction of plaque area at 30 weeks, but not at 45 weeks of age (100).

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Some interesting details of IL-12 properties come from a study that analyzed CD4+ CD28− T cells in mice. This subset of T cells expresses IL-12 receptor in the absence of antigen stimulation. Upon activation with IL-12, they upregulate their CCR5 expression, chemotaxis, and transendothelial migration toward CCL5 (101). Together, these results support the notion that IL-12 is proatherogenic and proinﬂammatory.

IL-18 IL-18 and IL-12 are involved in the generation of Th1 effector cells. Unexpectedly, administration of IL-18 antibodies accelerates lesion development in Apoe−/− mice, but overexpression of IL-18-binding protein abolishes the proinﬂammatory effects of IL-18, inducing a more stable plaque (90). Il18−/− Apoe−/− mice exhibited reduced expression of I-Ab and IFN-γ, elevated IgG production, and reduced lesions compared with Apoe−/− controls (102). IL-18 seems to enhance atherosclerosis mainly by increasing IFN-γ (103). Interestingly, in Il18−/− Apoe−/− mice, serum cholesterol and triglyceride levels are higher than in Apoe−/− mice, indicating that IL-18 somehow downregulates circulating cholesterol in serum (102).

IFN-γ IFN-γ administration accelerates atherosclerosis in Apoe−/− mice (104). Conversely, IFN-γ receptor–deﬁcient mice on the Apoe−/− (105) or Ldlr−/− background (106) show decreased atherogenesis. T cell–independent IFN-γ secreted by macrophages, NK cells, and vascular cells seems to be sufﬁcient for disease progression. Moreover, IFN-γ can induce atherosclerosis in scid Apoe−/− mice in the absence of detectable leukocytes by acting on SMCs to prime for growth factor–inducible proliferation (107). IFN-γ is involved not only in early but also in late stages of atherosclerosis. Advanced atherosclerotic lesions can be reduced in size and stabilized in composition by IFN-γ inhibition (108). 180

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IFN-α IFN-α is a pluripotent inﬂammatory cytokine typically induced by viral infections. Interestingly, IFN-α produced by aortic plasmacytoid DCs induced a tenfold increase of TNF-related apoptosis-inducing ligand (TRAIL) on CD4+ T cells and enhanced their cytolytic capacity toward SMCs (109). IFN-α also sensitized antigen-presenting cells to pathogen-derived TLR4 ligands by upregulation of TLR4 and intensiﬁed TNF-α, IL-12, and MMP-9 production that led to further plaque destabilization (38). Thus, IFN-α provides a possible link between viral infections and immune-mediated complications of atherosclerosis.

CD40/CD40L T lymphocytes, platelets, ECs, SMCs, macrophages, and DCs express CD40L, whereas CD40 is found on macrophages, ECs, and SMCs from atherosclerosis-prone vessels. The interaction of CD40 with CD40L plays a signiﬁcant role in thrombosis, but it also contributes to the modulation of the immune response in plaques. Treatment with antibodies against CD40L reduces atherosclerosis in Ldlr−/− mice, with a concomitant decrease of macrophages and T cells and a reduction in VCAM-1 expression (110). Further experiments using Cd40lg−/− Apoe−/− mice have demonstrated a proatherogenic role for CD40L in advanced atherosclerosis by promoting lipid core formation and plaque destabilization. Unfortunately, therapeutic effects directed at CD40L failed clinical trials because of an enhanced thrombosis risk. Cd40−/− Ldlr−/− mice showed no reduction in atherosclerotic lesion formation, suggesting a possible alternative ligand for CD40L (111).

ANTI-INFLAMMATORY IL-4 IL-4 is a Th2 cytokine with an unclear role in atherosclerosis. Neither exogenous

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administration of IL-4 into Apoe−/− mice nor IL-4 deﬁciency had any effect on lesion size in mice fed normal or saturated fat diets for 4 weeks (112). However, a long-term diet study in Il4−/− Apoe−/− mice demonstrated a 27% reduction in plaque area at 30 weeks of age and a reduction in aortic arch lesions at 45 weeks of age, compared with Apoe−/− mice (100). Il4−/− mice immunized with HSP-65 showed reduced formation of early atherosclerosis, with concomitant reduction of anti-HSP-65 antibodies and elevated IFN-γ production (113).

IL-5 Immunization with MDA shifts the immune response in atherosclerotic mice toward Th2 and induces signiﬁcant production of natural antibodies of the EO6/T15 type. IgM antibodies like EO6 (74, 75) found in atherosclerosis are structurally and functionally identical to classic natural T15 anti-PC antibodies that are produced by B1 and marginal zone B cells, with concomitant elevation in IL-5 (73). IL-5 further stimulates B1 cells, leading to increased production of these antibodies. Experiments with bone marrow transferred from Il5−/− or Il5+/+ mice suggest an atheroprotective role for IL-5 (73).

IL-10 IL-10 derived from Th2 cells, B cells, monocytes, and macrophages is an important regulator of the balance between Th1 and Th2 responses. Administration of IL-10 delayed atherosclerosis development. Conversely, IL-10-deﬁcient mice showed increased T cell accumulation and IFN-γ production with diminished collagen content in the atherosclerotic vessels (90). Studies with Il10−/− Apoe−/− conﬁrmed the atheroprotective properties of IL-10 at the early stage of atherosclerosis and showed that IL-10 also promotes the stability of advanced plaques (114).

IL-33 IL-33 is expressed in normal atherosclerosis-prone arteries (115).

and The

administration of IL-33 signiﬁcantly reduces atherosclerosis in Apoe−/− mice by mechanisms that involve a switch from a Th1 to a Th2 response, with concomitant increases in IL-4, IL-5, and IL-13, reduction of proinﬂammatory cytokines, and diminished IFN-γ production (115). IL-33 may also neutralize harmful oxLDL through IL-5-dependent production of antibodies against oxLDL (115).

TGF-β TGF-β is produced by several cell types, including ECs, SMCs, macrophages, platelets, and Treg cells. Several studies suggest that TGF-β regulates atherosclerosis through the modulation of SMC and EC phenotypes, as well as by regulating Th1 functions. Introducing blocking antibodies against TGF-β or treatment with soluble TGF-β receptor II accelerates atherosclerosis with signiﬁcant loss of collagen content (90). Apoe−/− mice that express a dominant-negative form of the TGF-β receptor II in T cells, as well as Ldlr−/− irradiated mice that received bone marrow from mice expressing a dominant-negative TGF-β receptor type II under a T cell–speciﬁc promoter, both clearly demonstrated a substantial role for TGF-β in controlling the Th1 response in atherosclerosis (116, 117).

CHEMOKINES IN ATHEROSCLEROSIS Substantial evidence from clinical and experimental research suggests that chemokines and chemokine receptors play critical roles in directing leukocytes into atherosclerosis-prone vessels. Taking into account the chemokine abundance within atherosclerosis-prone arteries and the variety of chemokine receptors on leukocytes, it is clear that a tightly controlled network regulates recruitment, retention, and emigration of leukocytes in the arterial wall (15). CCL2 was the ﬁrst chemokine shown to affect atherosclerosis. CCL2 and its receptor CCR2 are most prominently involved in

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monocyte recruitment from the bone marrow (118) and into the arterial wall (119). Studies with CCL2- and CCR2-deﬁcient mice clearly demonstrate that this pair is mainly involved at the early stages of atherosclerosis (120–123). The important role of platelets is suggested by their capacity to quickly release the proinﬂammatory chemokines CCL5 and CXCL4 to endothelium and thus initiate monocyte and T cell recruitment into the vessel wall (80). CXCL4 can induce activation of ECs by inducing expression of E-selectin, NF-κB activation, and enhanced binding of oxLDL to ECs (124). However, Ccr5−/− Apoe−/− mice lacking CCR5, one of the receptors for CCL5, had no signiﬁcant reduction in early atherosclerosis (125), and Ccr5−/− bone marrow–derived cells affected atherosclerosis only transiently (126). A more recent study found a more than 50% reduction of lesion size in the aortic root and the thoracoabdominal aorta of Apoe−/− Ccr5−/− mice and fewer macrophages and T cells in lesions compared with Apoe−/− mice (127). CXCL8 induces proliferation and migration of SMCs and ECs and affects neovascularization. In the Apoe−/− mouse model, CXCL1 initiated monocyte arrest through the activation of VLA-4 integrin (128). Recently, a unique role for chemokines was documented in the shear stress–dependent modulation of atherosclerotic lesion composition (129). Expression of CCL2, CXCL10, and CXCL1 was detected in low shear stress regions, and exclusive expression of CX3CL1 was observed in the low shear stress regions that had thinner ﬁbrous caps and larger necrotic cores (129). The cytokine MIF is produced by ECs, SMCs, and macrophages in early and advanced atherosclerotic lesions. Binding of MIF to its newly discovered receptor complex of CXCR2 and CD74 resulted in elevated monocyte arrest on atherosclerotic endothelium (130). Studies with Mif −/− Ldlr−/− mice suggest that MIF is involved in atherosclerosis through the regulation of lipid deposition, protease expression, and intimal thickening (131).

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LO: lipoxygenase

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CX3CL1 is expressed in atherosclerotic plaques in a transmembrane form and can initiate the arrest of CX3CR1+ NK cells, subsets of T lymphocytes, and monocytes (15). CX3CL1 can also be shed in an ADAM-17-dependent manner. The cleaved form attracts CX3CR1expressing cells. Several independent studies with Cx3cr1−/− Apoe−/− or Cx3cl1−/− Ldlr−/− mice clearly demonstrate a proatherogenic role for the CX3CL1/CX3CR1 axis in atherosclerosis (15). The only other known transmembrane chemokine is CXCL16, which has dual functions as SR and soluble chemokine and also is involved in atherogenesis (132). CXCL16 is expressed by SMCs, ECs, and macrophages. Cxcl16−/− Ldlr−/− mice had accelerated atherosclerosis, likely because of the lack of CXCL16 function as an SR (132). In contrast, the absence of CXCR6 in Cxcr6GFP/GFP Apoe−/− mice resulted in reduced T cell number within the lesion, dampening the inﬂammatory response at the lesion site, reducing macrophage inﬁltration, and diminishing atherosclerosis (133). CXCL10 is a potent mitogenic and chemotactic factor for SMCs and can modulate the local balance of the effector and regulatory arms of the immune system through the induction of Tregs in aortas (134). Other chemokines, including CCL3, CCL4, CCL11, and CXCL12, are expressed in human and mouse atherosclerotic aortas, but the role of these chemokines in atherosclerosis remains unclear (15).

INFLAMMATION-REGULATING ENZYMES IN ATHEROSCLEROSIS 5-lipoxygenase The 5-lipoxygenase (5-LO) pathway is responsible for the production of leukotrienes, inﬂammatory lipid mediators that have a role in innate immunity but that can also play a proatherogenic role (135). Expression of 5-LO and leukotriene A4 hydrolase in atherosclerotic segments correlates with plaque instability.

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5-LO-deﬁcient Ldlr−/− mice show a dramatic decrease in lesions, and bone marrow transplantation experiments suggest that macrophage 5-LO is mainly responsible for atherogenesis (136).

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12/15-LO The proatherogenic effect of 12/15-LO has been established in 12/15-LO-deﬁcient Apoe−/− mice that showed reduced lesions throughout the whole aorta (137). Unexpectedly, these mice also had diminished plasma IgG autoantibodies to oxidized LDL, which suggests that the 12/15-LO pathway affects not only lipid peroxidation but also the adaptive immune response (137). Overexpression of 12/15-LO in C57BL/6 mice leads to the formation of fatty streak lesions, at least partially through the elevated adhesion of monocytes to endothelium (138). Further experiments demonstrated that 12/15-LO expression in bone marrow–derived cells was responsible for the proatherogenic properties of 12/15-LO in vivo (139). 12/15-LO induces the production of IL-6, TNF-α, and CCL2 and therefore connects the metabolic and immune branches of atherosclerosis (140). By contrast, rabbits overexpressing human 15-LO showed reduced lesion size (141). The reason for this difference may be related to the many products of 12/15-LO, some of which are pro- and other anti-inﬂammatory (142).

Heme Oxygenase -1 Heme oxygenase (HO) catalyzes the ratelimiting step of heme catabolism. The induction of HO-1 reduced monocyte chemotaxis in response to LDL oxidation (143). The absence of HO-1 exacerbated atherosclerosis in HO-1deﬁcient Apoe−/− mice (144), and macrophages expressing HO-1 are crucial players in this process (145). From a therapeutic point of view, it is important to mention that HO-1 is also

involved in antioxidant-dependent protection from atherosclerosis (146). HO: heme oxygenase

Paraoxonases The paraoxonase family consists of three members (PON1, PON2, and PON3) that share structural properties and enzymatic activities, among which is the ability to hydrolyze oxidized lipids in LDL. PON1 prevents oxidation of LDL as well as high-density lipoprotein (HDL), with which PON1 is associated in the serum (147). HDL of Pon1−/− Apoe−/− mice is predisposed to oxidation, and as a consequence lesions in Pon1−/− Apoe−/− mice are larger compared with controls (148). In several other studies using transgenic mice overexpressing PON1, the role of PON1 as inhibitor of lipid oxidation was conﬁrmed (147).

PROINFLAMMATORY MEDIATORS OxLDL According to the oxidation hypothesis of atherosclerosis, oxLDL plays a pivotal role through the induction of foam cell formation, alteration of nitric oxide signaling, initiation of endothelial activation, and expression of adhesion molecules that accelerate leukocyte homing to the site of atherosclerosis (149). One of the key observations that crystallized the important role of oxLDL in atherosclerosis came from a study that showed heparan sulfate–dependent binding of oxLDL to subendothelial matrix (150). Generation of lectin-like oxLDL receptor-1-deﬁcient Olr1−/− Ldlr−/− mice showed reduced atherosclerosis by luminal obstruction and intima thickness (151). OxLDL can also directly affect the migration of monocytes to the aortic wall by switching from CCR2 to CX3CR1 expression using a peroxisome proliferator-activated receptor γ– dependent pathway (152). Autoantibodies to oxLDL are found within normal/nondiseased and atherosclerosis-prone

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AGE: advanced glycation end products

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lesions. IgG autoantibodies to oxLDL are associated with proatherogenic properties, and IgM autoantibodies to oxLDL, including natural antibodies, have been proposed as atheroprotective (73). The mechanism by which the immune response coordinates the production of these two isotypes in atherosclerosis remains to be identiﬁed. Importantly, it is possible to initiate tolerance to oxLDL and MDA-LDL by oral administration of oxLDL or MDALDL before the induction of atherogenesis and to promote generation of oxLDL-speciﬁc CD4+ CD25+ Foxp3+ Treg cells (58).

C-Reactive Protein Elevated plasma C-reactive protein (CRP) is associated with increased risk of atherosclerosis, but the mechanisms have not been fully identiﬁed (153). CRP is found within atherosclerotic plaques close to LDL and macrophages. Recently, several reports demonstrated that CRP can modulate endothelial functions and leukocyte activities. CRP also induced the production of IL-1α, IL-1β, IL-6, CXCL1, and CXCL8 by human monocytes in vitro. In contrast to these proinﬂammatory properties, CRP also displayed anti-inﬂammatory effects through upregulation of liver X receptor-α (153). CRP binds to minimally modiﬁed (mm)LDL and prevents the formation of foam cells from macrophages (154). The functional importance of these observations in vivo and the exact functions of CRP in atherogenesis remain to be investigated.

Advanced Glycation End Products (AGE) Nonenzymatic modiﬁcation of proteins by reducing sugars leads to the formation of AGEs in vivo. These reactions take part during aging and substantially accelerate during cancers, diabetes, and atherosclerosis (155). Although the mechanisms are not fully identiﬁed, the alterations in glucose and lipid metabolism likely lead to the production of excess aldehydes and formation of AGEs. AGEs act directly or via 184

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receptors and participate in the cross-linking proteins of extracellular matrix (155). The receptor for AGE (RAGE) is a member of the immunoglobulin superfamily and is expressed by ECs, SMCs, monocytes, and lymphocytes with enhanced expression in atherosclerotic lesions. Neutralizing AGE by the administration of soluble recombinant RAGE reduced NF-κB induction, VCAM-1 and tissue factor expression, and atherosclerotic lesion burden (156). A critical role for RAGE and its ligands was also demonstrated in RAGE-deﬁcient Apoe−/− mice and in Apoe−/− transgenic mice expressing human dominant-negative RAGE (157). The AGE/RAGE axis has a broad spectrum of effects and elicits oxidative stress, increases endothelial dysfunction, increases production of inﬂammatory cytokines and tissue factor, elevates expression of adhesion molecules, and, through all these mechanisms, accelerates development of atherosclerosis (155).

Reactive Oxygen Species Extensive production of ROS has been implicated in atherosclerosis by inducing the chronic activation of the vascular endothelium and components of the immune system. Vascular endothelial ROS released from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, MPO, xanthine oxidase, lipoxygenases, nitric oxide synthases, and the dysfunctional mitochondrial respiratory chain may play critical roles in ROS generation. In humans, higher expression of NADPH oxidase subunit proteins is associated with increased superoxide (O2 − ) production and severity of atherosclerosis (158). NADPH oxidase-deﬁcient Apoe−/− mice had signiﬁcantly less atherosclerosis compared with Apoe−/− mice (159). Further studies clearly demonstrated that superoxide production from both monocytes/macrophages and vascular cells plays a critical role in atherogenesis (160). One of the mechanisms by which superoxide affects atherogenesis is the activation of SMC mitogenic signaling pathways (160). Platelets also produce ROS, and NADPHinduced superoxide production results in

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enhanced availability of released ADP and ampliﬁed platelet recruitment (161). ROS can be neutralized by antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and others, but ROS production can exceed the scavenging capacity of cellular antioxidant systems, and the resulting oxidative stress can damage lipids, membranes, proteins, and DNA. The mechanisms of regulating antioxidant activity are multidirectional, but it is important to mention that laminar shear stress upregulates a mechanosensitive group of antioxidant enzymes, peroxiredoxins in ECs.

Complement System The complement system plays a central role in innate immunity and also regulates the adaptive response (162). Complement activation is essential to the host’s immune defense, but its uncontrolled or inappropriately targeted activation leads to various diseases such as glomerulonephritis, rheumatoid arthritis, psoriasis, and CVDs (162). Emerging evidence suggests that the complement system plays a role in atherosclerosis, although its exact functions and mechanisms of action remain unclear. Modiﬁed lipoproteins and apoptotic/necrotic cells have been shown to activate the alternative classical complement pathways. Studies using complement-deﬁcient animals have yielded apparently contradictory conclusions. C6 deﬁciency protects against diet-induced atherosclerosis in rabbits (163); however, no difference was observed in diet-induced lesion size in C5-deﬁcient Apoe−/− mice (164) or C3-deﬁcient Ldlr−/− mice (165). The classical pathway of complement activation may be protective because it promotes the clearance of apoptotic cells and immune complexes during atherosclerosis. Indeed, C1q-deﬁcient Ldlr−/− mice have more apoptotic bodies within their plaques and larger atherosclerotic lesions (166). The role of the complement system at the advanced stages of atherosclerosis is not known, but examination of human tissues demonstrates activated complement in

human vulnerable plaques prone to rupture (167).

Heat Shock Protein 60 There are distinct cellular and humoral reactions against microbial HSPs in humans that participate in host defense. However, because of a high degree of sequence homology between microbial and human HSPs, autoimmune responses may be triggered against human HSPs (168). Anti-HSP antibodies elicit production of proinﬂammatory cytokines by macrophages and of adhesion molecules by ECs. Autoantibody levels against HSPs are signiﬁcantly increased in patients with atherosclerosis, and HSP-speciﬁc T cells have been observed within atherosclerotic plaques. Most of the known risk factors for atherosclerosis, such as oxLDL, hypertension, infections, and oxidative stress, evoke increased expression of HSPs in ECs, SMCs, and macrophages. Endothelial HSP60 correlates with site-speciﬁc, ﬂow-dependent atherosclerosis development throughout the aortic tree. Altered wall shear stress after ligation of the left common carotid artery induced rapid production of HSP-60 by ECs at this site (169), which may provide conditions for humoral and cellular reactions to endothelial HSPs in the earliest stages of atherosclerosis. Other HSPs such as HSP-90 might also be involved in atherosclerosis (170).

Toll-Like Receptors There is a signiﬁcant body of evidence that not only metabolic mediators but also bacterial and viral infections might amplify atherosclerosis and worsen the outcome by promoting a proinﬂammatory status of the vessel wall. TLRs are the primary receptors of the innate immune system that recognize highly conserved structural motifs of pathogens. TLR2 and TLR4 are also receptors for HSP-60 (171). Under conditions of hyperlipidemia, TLRs likely participate in the regulation of atherosclerosis. Activation of TLRs induces the production of proinﬂammatory cytokines and nitric oxide in macrophages

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and the induction of DC maturation, leading to the upregulation of costimulatory molecules such as CD80 and CD86. TLR1, TLR2, TLR4, and TLR5 are expressed in atherosclerotic lesions, and TLR4 can be upregulated by oxLDL. Interestingly, atheroprotective laminar ﬂow downregulates TLR2 expression. Mice lacking MyD88, a signaling molecule downstream of most TLRs, showed reduced atherosclerosis (172). Myd88−/− Apoe−/− and Tlr4−/− Apoe−/− mice exhibited reduced atherosclerosis that was associated with reduction in the circulating levels of the proinﬂammatory cytokines IL-12 and CCL2, plaque lipid content, numbers of macrophages, and cyclooxygenase 2 immunoreactivity in plaques (173). TLR2 deﬁciency on non–bone marrow– derived cells resulted in diminished atherosclerosis in Tlr2−/− Ldlr−/− mice, suggesting that an unknown endogenous TLR2 agonist inﬂuenced lesion progression by activating TLR2 in cells that were not of bone marrow origin, most likely ECs (174). Thus, at least TLR2 and TLR4 participate in the inﬂammatory arm of atherosclerosis.

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ASSOCIATION WITH INFLAMMATORY DISEASES Systemic Lupus Erythematosus Atherosclerosis is associated with many chronic inﬂammatory diseases. As we start to appreciate possible similarities between atherosclerosis and autoimmune diseases, we must consider the possible impact of chronic inﬂammation on the acceleration of atherosclerosis. Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving multiple organs that is characterized by autoantibody production (175). In recent years, much attention has been given to the rising incidence of accelerated atherosclerosis and increased risk of CVDs in patients with SLE. Increased production of CCL2, TNF-α, IFN-γ, IL-1, IL-12, and immune complexes, upregulation of adhesion molecules, and increased antibodies to oxLDL may promote atherosclerosis. Gld.Apoe−/− mice that carry an 186

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inactivating mutation in the Fas ligand gene (FasL) develop lupus-like autoimmune disorders (176). The gld.Apoe−/− mice displayed enhanced atherosclerosis compared with Apoe−/− mice, accompanied by an increase in lymphocyte proliferation and autoimmunity. The gld.Apoe−/− mice had high levels of apoptotic material both in tissues and in the circulation. This was due, at least in part, to an impaired ability to scavenge apoptotic debris, suggesting that the synergism between atherosclerosis and SLE can be mediated by impaired apoptotic body clearance (176). Fas-deﬁcient Apoe−/− mice also showed increased production of IgG antibodies against dsDNA and cardiolipin, as well as accelerated atherosclerosis (177).

Metabolic Syndrome and Diabetes Metabolic syndrome is deﬁned as prediabetes, abdominal obesity, elevated LDL cholesterol, and increased blood pressure that signiﬁcantly correlates with CVD. Despite evidence for a tight correlation between atherosclerosis and metabolic syndrome, mechanisms by which these diseases accelerate each other are not well identiﬁed and very little is known about the underlying basis for differential susceptibility to vascular injury in patients with diabetes. Diabetes-accelerated atherosclerosis is observed in type 1 and type 2 diabetic patients, but it is not known whether atherosclerosis is induced through the same mechanisms (178). In type 1 diabetes, hyperglycemia generally occurs in the absence of elevated blood lipid levels, whereas type 2 diabetes is frequently associated with dyslipidemia. Few animal models are available to study diabetesaccelerated atherosclerosis (179). To dissect the role of lipids, glucose, and insulin in atherosclerosis, investigators generated Ldlr−/− mice that express a lymphocytic choriomeningitis virus glycoprotein transgene under control of the insulin promoter (180). Diabetic mice on regular chow diet, in the absence of the lipid abnormalities, developed atherosclerosis with preferential accumulation of macrophages within the

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aortas. Western diet–fed diabetic mice showed advanced lesions, characterized by extensive intralesional hemorrhage, suggesting that hyperlipidemia induces a formation of more advanced lesions (180). Mice expressing human aldose reductase that may multiply toxic effects of glucose metabolism showed advanced atherosclerosis compared with controls (181). Thus, glucose or products of glucose metabolism are sufﬁcient to induce atherosclerosis in hyperlipidemic conditions. Type 2 diabetes is much more prevalent than type 1 diabetes and is often preceded by the metabolic syndrome, but it is even more difﬁcult to generate an appropriate mouse model (179). Hyperinsulinemia and hyperglycemia with dyslipidemia induced accelerated atherosclerosis in Apoe−/− mice fed a regular chow diet and lacking the leptin receptor (182) and in leptin-deﬁcient mice on the Apoe−/− or Ldlr−/− background (179). Although these models showed elevated atherosclerosis, the increased lipid levels in experimental groups compared with control made the interpretations of these results difﬁcult. Adipose tissues release cytokines that regulate not only body weight homeostasis, but also insulin resistance that in turn inﬂuences atherosclerosis (183). Several reports suggest that adipose tissues are active regulators of inﬂammation through the production of adipokines, proinﬂammatory cytokines, and CRP that can affect immune response, induce endothelial dysfunction, increase oxidative stress, and thus accelerate atherosclerosis (183).

ANTI-INFLAMMATORY DRUGS AND MEDIATORS Statins Statins inhibit 3-hydroxyl-3-methylglutaryl coenzyme A reductase, an enzyme crucial to cholesterol synthesis. They reduce total and LDL cholesterol as well as triglycerides and slightly increase HDL cholesterol and reduce the risk for CVD and stroke (184). Although

the clinical beneﬁts of statins are mediated in large part through lipid modulation, emerging evidence supports the existence of other mechanisms of action. The beneﬁcial inﬂuences may include modiﬁcation of endothelial function, increased plaque stability, reduced thrombus formation, and, in particular, dampened inﬂammatory pathways. Statins activate peroxisome proliferative activated receptors (185) and thus, indirectly, control lipid and glucose metabolism, vascular inﬂammation and thrombosis, and NF-κB-dependent activation of SMCs and monocytes. The level of CRP can be regulated in an LDL-independent manner by statins, but the mechanisms of this inﬂuence as well of CRP’s role in atherogenesis are not clear (186). Clinical data show decreased levels of IL-6, CXCL8, and CCL2 after treatment with simvastatin. Pretreatment of human monocytes with statins induced downregulation of IL-1, CCL3, and CCL4 and of IL-18, CCR1, and CCR2 (184). Statins also inhibit elevated expression of both ICAM-1 and lymphocyte function–associated antigen 1 (LFA-1) and may reduce leukocyte adhesion and retention within the aortic wall (184).

High-Density Lipoprotein Efﬂux of cholesterol from peripheral tissues into plasma, then to the liver and bile, is termed reverse cholesterol transport (187). HDLs mediate most reverse cholesterol transport and thus inﬂuence the amount of cellular cholesterols under normal and pathogenic conditions. Several studies with knockout mice for ApoA-1, SR-B1, or ABCA1 dissected the role and importance of reverse cholesterol transport and highlighted HDL’s functions in this process (187). The role of ABCG1 in atherogenesis is less clear because conﬂicting results have been reported. HDL also has antioxidant, anti-inﬂammatory, antiapoptotic, and vasodilatory properties. Recently, investigators have also appreciated that HDLs can lose their usual atheroprotective properties through speciﬁc chemical and structural alterations and can play a proinﬂammatory role by alteration of reverse cholesterol

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transport, enhanced oxidation of LDLs, and increased vascular inﬂammation (188).

CONCLUSIONS

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Our understanding of atherosclerosis has progressed remarkably over the past few years. The discovery of subsets of inﬂammatory and resident monocytes, M1 and M2 macrophages, NKT cells, and Tregs opened new perspectives on the role of inﬂammation and immune responses in atherosclerosis. New data suggest an important role for chemokine and chemokine receptors in atherosclerosis and highlight a network of cytokines that modulate the immune

response and inﬂammation in the aortic wall. All phases of atherosclerosis are regulated by inﬂammatory mechanisms that provide overlapping networks of pathways involved in the regulation of immune cell functions, activation of endothelium, and alteration of metabolic parameters. Increasing evidence suggests that components of the immune system may alter lipid metabolism and thus affect atherosclerosis in yet another way. More work is needed to understand the effects of statins on inﬂammation and the immune system. All these data will help to identify potential therapeutic targets for the prevention and treatment of atherosclerosis and other CVDs.

SUMMARY POINTS 1. Atherosclerosis is a complex chronic inﬂammatory disease that affects large- and mediumsize arteries, inducing atherosclerotic plaques and alterations of the phenotypes of vascular cells. 2. An early step in the development of atherosclerosis is the retention of LDLs in the arterial wall. 3. Monocyte recruitment into aortas and formation of foam cells are hallmarks of atherosclerosis. Recruitment of immune cells into the aortas is likely modulated by adhesion molecules and chemokines. 4. There are some candidates for possible autoantigens during atherosclerosis, including HSP-60 and oxLDLs. 5. The immune response in atherosclerosis-prone conditions is predominantly Th1-biased. 6. Monocytes, macrophages, DCs, subsets of T cells, NK cells, NKT cells, neutrophils, platelets, and mast cells likely play proatherogenic roles. 7. Tregs and B cells (through the production of natural antibodies) suppress inﬂammation during atherosclerosis. 8. Chronic inﬂammation produces inﬂammatory mediators such as modiﬁed LDLs, ROS, and AGE that accelerate vascular inﬂammation and atherosclerosis. 9. Many autoimmune diseases such as systemic lupus erythematosus and diabetes accelerate atherosclerosis development, probably through the defective clearance of apoptotic cells.

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review. 188

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ACKNOWLEDGMENTS This work was supported by NIH grants HL 58108 and 55798 and by the American Heart Association Scientist Development Grant 0730234N. Because of space constraints, it was not possible to cite all of the relevant original papers and many excellent reviews. We apologize to the authors whose important work could not be included in the list of references.

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6. First demonstrated monocyte adhesion to the aortic endothelium and formation of foam cell lesions in areas of enhanced permeability of aortic intima during atherosclerosis development.

14. Established a dominant role of VCAM-1 but not ICAM-1 in the initiation of atherosclerosis.

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41. Perrin-Cocon L, Coutant F, Agaugue S, Deforges S, Andre P, Lotteau V. 2001. Oxidized lowdensity lipoprotein promotes mature dendritic cell transition from differentiating monocyte. J. Immunol. 167:3785–91 42. Mafﬁa P, Zinselmeyer BH, Ialenti A, Kennedy S, Baker AH, et al. 2007. Images in cardiovascular medicine. Multiphoton microscopy for 3-dimensional imaging of lymphocyte recruitment into apolipoprotein-E-deﬁcient mouse carotid artery. Circulation 115:e326–28 43. Hansson GK, Holm J, Jonasson L. 1989. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am. J. Pathol. 135:169–75 44. Wick G, Romen M, Amberger A, Metzler B, Mayr M, et al. 1997. Atherosclerosis, autoimmunity, and vascular-associated lymphoid tissue. FASEB J. 11:1199–207 45. Paulsson G, Zhou X, Tornquist E, Hansson GK. 2000. Oligoclonal T cell expansions in atherosclerotic lesions of apolipoprotein E-deﬁcient mice. Arterioscler. Thromb. Vasc. Biol. 20:10–17 46. Rossmann A, Henderson B, Heidecker B, Seiler R, Fraedrich G, et al. 2008. T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor repertoire. Exp. Gerontol. 43:229–37 47. Robertson AK, Hansson GK. 2006. T cells in atherogenesis: for better or for worse? Arterioscler. Thromb. Vasc. Biol. 26:2421–32 48. Zhou X, Paulsson G, Stemme S, Hansson GK. 1998. Hypercholesterolemia is associated with a T helper (Th) 1/Th2 switch of the autoimmune response in atherosclerotic apo E-knockout mice. J. Clin. Invest. 101:1717–25 49. Buono C, Binder CJ, Stavrakis G, Witztum JL, Glimcher LH, Lichtman AH. 2005. T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses. Proc. Natl. Acad. Sci. USA 102:1596–601 50. Nofer JR, Bot M, Brodde M, Taylor PJ, Salm P, et al. 2007. FTY720, a synthetic sphingosine 1 phosphate analogue, inhibits development of atherosclerosis in low-density lipoprotein receptor-deﬁcient mice. Circulation 115:501–8 51. Keul P, Tolle M, Lucke S, von Wnuck LK, Heusch G, et al. 2007. The sphingosine-1-phosphate analogue FTY720 reduces atherosclerosis in apolipoprotein E-deﬁcient mice. Arterioscler. Thromb. Vasc. Biol. 27:607–13 52. Sakaguchi S, Ono M, Setoguchi R, Yagi H, Hori S, et al. 2006. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 212:8–27 53. Mallat Z, Gojova A, Brun V, Esposito B, Fournier N, et al. 2003. Induction of a regulatory T cell type 1 response reduces the development of atherosclerosis in apolipoprotein E-knockout mice. Circulation 108:1232–37 54. it-Oufella H, Salomon BL, Potteaux S, Robertson AK, Gourdy P, et al. 2006. Natural regulatory T cells control the development of atherosclerosis in mice. Nat. Med. 12:178–80 55. Gotsman I, Grabie N, Gupta R, Dacosta R, MacConmara M, et al. 2006. Impaired regulatory T-cell response and enhanced atherosclerosis in the absence of inducible costimulatory molecule. Circulation 114:2047–55 56. Groyer E, Nicoletti A, it-Oufella H, Khallou-Laschet J, Varthaman A, et al. 2007. Atheroprotective effect of CD31 receptor globulin through enrichment of circulating regulatory T-cells. J. Am. Coll. Cardiol. 50:344–50 57. Taleb S, Herbin O, it-Oufella H, Verreth W, Gourdy P, et al. 2007. Defective leptin/leptin receptor signaling improves regulatory T cell immune response and protects mice from atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 27:2691–98 58. van Puijvelde GH, van Es T, van Wanrooij EJ, Habets KL, de Vos P, et al. 2007. Induction of oral tolerance to HSP60 or an HSP60-peptide activates T cell regulation and reduces atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 27:2677–83 59. Elhage R, Gourdy P, Brouchet L, Jawien J, Fouque MJ, et al. 2004. Deleting TCRαβ+ or CD4+ T lymphocytes leads to opposite effects on site-speciﬁc atherosclerosis in female apolipoprotein E-deﬁcient mice. Am. J. Pathol. 165:2013–18 60. Whitman SC, Ramsamy TA. 2006. Participatory role of natural killer and natural killer T cells in atherosclerosis: lessons learned from in vivo mouse studies. Can. J. Physiol. Pharmacol. 84:67–75 www.annualreviews.org • Immune and Inflammatory Mechanisms of Atherosclerosis

43. Demonstrated presence of activated T cells within atherosclerotic plaques close to HLA-DR+ SMCs and macrophages and suggested possible antigen presentation during atherosclerosis.

44. Proposed the term vascular-associated lymphoid tissues (VALT).

49. Clearly demonstrated a proatherogenic role for the Th1 response in atherosclerosis and showed that Th1 cells regulate production of atheroprotective EO6 antibodies.

54. Showed that naturally arising CD4+ CD25+ regulatory T cells suppress atherosclerosis development by reducing infiltration of T cells and macrophages into plaques, increasing lesional collagen content and switching to an anti-inflammatory cytokine profile.

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75. Demonstrated that many autoantibodies to oxLDL share genetic and structural similarity with antibodies against common infectious pathogens, suggesting molecular mimicry between epitopes of oxLDL and microbes.

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Primary B Cell Immunodeﬁciencies: Comparisons and Contrasts Mary Ellen Conley,1,2 A. Kerry Dobbs,2 Dana M. Farmer,2 Sebnem Kilic,3 Kenneth Paris,4 Soﬁa Grigoriadou,5 Elaine Coustan-Smith,6 Vanessa Howard,2 and Dario Campana1,6 1

Department of Pediatrics, University of Tennessee College of Medicine, Memphis, Tennessee 38163

2

Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105; email: [email protected], [email protected], [email protected], [email protected]

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Department of Pediatrics, Uludag University, Faculty of Medicine, Bursa, 16059 Turkey; email: [email protected]

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Department of Pediatrics, Children’s Hospital of New Orleans, New Orleans, Louisiana 70118; email: [email protected]

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Department of Immunology, Barts and The London NHS Trust, London, EC1A 7BE, UK; email: soﬁ[email protected]

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Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105; email: [email protected], [email protected]

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Key Words

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

X-linked agammaglobulinemia, hyper-IgM syndrome, common variable immunodeﬁciency, Btk, TACI

This article’s doi: 10.1146/annurev.immunol.021908.132649 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0199$20.00

Abstract Sophisticated genetic tools have made possible the identiﬁcation of the genes responsible for most well-described immunodeﬁciencies in the past 15 years. Mutations in Btk, components of the pre-B cell and B cell receptor (λ5, Igα, Igβ), or the scaffold protein BLNK account for approximately 90% of patients with defects in early B cell development. Hyper-IgM syndromes result from mutations in CD40 ligand, CD40, AID, or UNG in 70–80% of affected patients. Rare defects in ICOS or CD19 can result in a clinical picture that is consistent with common variable immunodeﬁciency, and as many as 10% of patients with this disorder have heterozygous amino acid substitutions in TACI. For all these disorders, there is considerable clinical heterogeneity in patients with the same mutation. Identifying the genetic and environmental factors that inﬂuence the clinical phenotype may enhance patient care and our understanding of normal B cell development.

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INTRODUCTION

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The term primary B cell immunodeﬁciencies encompasses a heterogeneous group of disorders that share the marked reduction or absence of serum immunoglobulins. In the past, we thought of primary B cell immunodeﬁciencies either as single-gene defects of the immune system or as multifactorial disorders, inﬂuenced by a combination of susceptibility genes. However, recent studies have taught us that even patients with the same single-gene defect may demonstrate striking variability in clinical and laboratory ﬁndings (1, 2). Although the speciﬁc mutation in the gene of interest may account for some of this variability (3–5), modifying genetic factors, the age of the patient, environmental exposures, and other factors play a role as well. In the outbred human population, it is clear that the lines between monogenetic and polygenetic disorders are often blurred (6, 7). The abnormal genes that are primarily responsible for antibody deﬁciencies, or that function as susceptibility genes, may be intrinsic to the B cell lineage (8, 9), may encode signal transduction molecules made by T cells (10, 11), or, conceivably, may be derived from myeloid cells or the stromal cells that provide the essential microenvironment for B lineage cells. Identifying the genes responsible for immunodeﬁciency and the modifying factors may help clarify the regulatory requirements for normal B cell development and the underlying basis for some common disorders, such as autoimmunity. All antibody deﬁciencies are associated with an increased susceptibility to infection with encapsulated bacteria, particularly Streptococcus pneumoniae and Haemophilus inﬂuenza (12, 13– 16). The infections seen in affected patients are those typically associated with these two organisms. Bronchitis and pneumonia are common and often lead to chronic lung disease. Small children usually have recurrent otitis, whereas sinusitis predominates in adults (17, 18). Giardia infections are also common in all types of antibody deﬁciencies (13, 19, 20). Other infections tend to be more limited to a subset of anti-

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body deﬁciencies. Regardless of the speciﬁc diagnosis, all patients with antibody deﬁciencies are treated with gammaglobulin replacement. This therapy is impressively successful, but it is also expensive, costing approximately $50,000 per year for an average-sized adult. In the past 10 years, there has been a shift away from monthly intravenous administration of gammaglobulin in a hospital or clinic setting toward weekly self-administration of subcutaneous gammaglobulin. Most patients feel that the more consistent levels of serum IgG and the convenience offer distinct advantages (21, 22). There are three major categories of antibody deﬁciencies: (a) defects in early B cell development, (b) hyper-IgM syndromes (also called class switch recombination defects), and (c) common variable immunodeﬁciency (CVID). Distinguishing between the last two categories may be difﬁcult. Patients in both groups generally have a marked reduction in serum IgG and IgA. The serum IgM is usually markedly elevated in patients with defects in class switch recombination and is often very low in CVID, but it may be normal, or close to normal, in patients with either disorder (13, 20, 23). Both hyper-IgM syndromes and CVID have been reviewed recently (13, 16, 24–27) and are not considered in great detail here.

DEFECTS IN EARLY B CELL DEVELOPMENT Defects in early B cell development are characterized by the onset of recurrent bacterial infections in the ﬁrst 5 years of life, profound hypogammaglobulinemia, markedly reduced or absent B cells in the peripheral circulation, and (in the bone marrow) a severe block in B cell differentiation before the production of surface immunoglobulin-positive B cells. Mutations in Btk, the gene responsible for X-linked agammaglobulinemia (XLA), account for approximately 85% of affected patients (28). Approximately half of the remaining patients have mutations in genes encoding components of the pre-B cell receptor (pre-BCR) or BCR, including μ heavy chain (IGHM ); the signal

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transduction molecules Igα (CD79A) and Igβ (CD79B ); and λ5 (IGLL1), which forms the surrogate light chain with Vpre-B (9, 29–35). A small number of patients with defects in BLNK, a scaffold protein that assembles signal transduction molecules activated by cross-linking of the BCR, have been reported (30, 36). Btk is expressed in myeloid cells and platelets, as well as B cells (8, 37–39); BLNK is expressed in B cells and monocytes (40, 41); and the remaining genes are B cell speciﬁc.

X-Linked Agammaglobulinemia XLA is often considered the prototype immunodeﬁciency. It was one of the ﬁrst immunodeﬁciencies described, and it was certainly the ﬁrst immunodeﬁciency for which a laboratory ﬁnding (agammaglobulinemia) explained the clinical symptoms and dictated successful therapy (subcutaneous gammaglobulin). In 1952, Bruton (12) reported the case of an 8-yearold boy with multiple episodes of pneumococcal sepsis associated with the complete absence of the serum globulin fraction as detected by protein electrophoresis. Additional patients were soon described (42, 43). When agammaglobulinemia was seen in children, it occurred predominantly in boys and often followed an X-linked pattern of inheritance (42, 43). By contrast, affected adults were almost equally divided between males and females, and a clear pattern of inheritance was rarely obvious (44– 48). The adult-onset disorder came to be known as CVID. In the early 1970s, it was shown that patients with XLA had markedly reduced numbers of B cells in the peripheral circulation, whereas the number of B cells was usually normal in the adults with CVID (49–52). In 1993, two groups reported that XLA resulted from mutations in a cytoplasmic tyrosine kinase called Btk or Bruton’s tyrosine kinase (8, 37).

Btk Btk is a member of a family of cytoplasmic tyrosine kinases, called Tec kinases, that includes Tec, Itk, Rlk, and Bmx, as well as Btk (53–56).

These enzymes are predominantly expressed in hematopoietic cells; in fact, most cell lineages contain more than one family member. B cells and platelets express Btk and Tec; T cells express Itk, Rlk, and Tec; and myeloid cells, including mast cells, express Btk, Tec, Itk, and Rlk. Family members (which are activated by growth, differentiation, or survival signals) are characterized by a C-terminal kinase domain preceded by SH2 and SH3 domains, a prolinerich region, and an NH2-terminal PH (pleckstrin homology) domain. Immediately after Btk was identiﬁed, several studies showed that it was activated through a variety of cell surface molecules, including the BCR and pre-BCR (57–59) and the IL-5 and IL-6 receptors on B cells (60, 61), the highafﬁnity IgE receptor on mast cells (62), and the collagen receptor glycoprotein VI on platelets (39, 63, 64). Recently, there has been a great deal of interest in the role of Btk in signaling through CXCR4 on B cells (65) and the Tolllike receptors (TLRs) on myeloid cells and B cells (66–70). With activation, Btk moves to the inner side of the plasma membrane, where it is phosphorylated and partially activated by a src family member (71) (Figure 1). Btk then undergoes autophosphorylation (72). Activated Btk and PLCγ2 bind to the scaffold protein BLNK via their SH2 domains, allowing Btk to phosphorylate PLCγ2 (73). This leads to calcium ﬂux and activation of the MAP kinases ERK and JNK (74). In addition, Btk phosphorylates several transcription factors and can be found in the nucleus (75, 76). The block in B cell differentiation in both humans and mice that lack Btk provides strong support for the importance of Btk in signaling through the pre-BCR and BCR. It is not clear that signaling through any of the other receptors or the migration of Btk into the nucleus contributes to the pathophysiology of XLA. Affected patients have normal numbers of platelets and myeloid cells. Most patients with XLA lead active lives (18), which often include participation in contact sports. However, unusual bruising or bleeding has not been www.annualreviews.org • Primary B Cell Immunodeficiencies

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Figure 1 Signal transduction through the pre-B cell receptor. With activation, there is clustering of the signal transduction molecules Igα and Igβ. Their ITAM motifs are phosphorylated by a src family member, shown here as lyn. Syk is then activated by binding to the phosphorylated ITAM motifs. Activated Syk phosphorylates multiple tyrosine residues in the scaffold protein BLNK. Lyn also phosphorylates Btk and CD19. Phosphorylated CD19 serves as a docking site for phosphatidylinositol 3-kinase (PI3K), which produces PIP3. PIP3 acts as a docking site for the PH domains of Btk and PLCγ2. The SH2 domains of Btk and PLCγ2 bind to phosphorylated tyrosines in BLNK, which allows Btk to phosphorylate PLCγ2. Phosphorylated tyrosine residues that act as docking sites for SH2 domains are shown as red circles. NFAT, NF-κB, and AP1 are transcription factors.

reported. This suggests that the absence of Btk does not have a major impact on platelet function. It is more difﬁcult to determine the importance of Btk in mature B cell and myeloid function. Comparing the clinical ﬁndings in patients with mutations in Btk to those in patients with defects that are limited to signaling through the BCR, such as μ heavy chain or Igα, will help clarify whether Btk has a broader role in B cell function.

Clinical Signs and Symptoms in XLA Patients with XLA are usually healthy in the newborn period but have the onset of recurrent bacterial infections between 3 and 18 months of age (14, 17, 77). In the current era, the mean 202

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age at diagnosis in North America is 3 years, but the median is 26 months (17). Most patients are recognized to have immunodeﬁciency when they are hospitalized for a severe infection such as sepsis, meningitis, or pneumonia with empyema (pus in the pleural cavity). Notably, many have had an earlier hospitalization for a common viral infection, such as croup, diarrhea, or RSV (respiratory syncytial virus) pneumonia (17). These infections are not generally considered worrisome in patients with XLA. As many as one-third of patients are evaluated for immunodeﬁciency when they are hospitalized for a dramatic constellation of ﬁndings, including (a) pyoderma gangrenosum, perirectal abscess, cellulitis, or impetigo; (b) pseudomonas or staphylococcal sepsis; and (c) neutropenia. This

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presentation is particularly common in patients who are recognized to have XLA at less than 1 year of age (17). Pseudomonas and staphylococcus are commonly seen in patients with neutropenia (78) but are rarely signiﬁcant problems in patients with XLA after diagnosis and the initiation of gammaglobulin therapy. Some XLA patients who develop sepsis owing to these organisms have a history of a viral infection immediately preceding the dramatic presentation (17). In young children, neutropenia and bone marrow suppression are sometimes seen after viral infections (79). We hypothesize that the dramatic presentation in patients with XLA is initiated by a viral infection that results in neutropenia. When one couples this ﬁnding with the observation that XLA patients have an increased rate of hospitalization for common viral infections in infancy, one can speculate that the lack of natural antibody makes these patients unusually vulnerable to viral infections in infancy. Once T cell immunity develops, most viral infections are tolerated without problems. Many patients with XLA acquired hepatitis C from contaminated gammaglobulin in the late 1980s (80–82). However, these patients had fewer problems with hepatitis C than patients with CVID, and most handled the infection as well as immunocompetent individuals who received other contaminated blood products. Enteroviral infections are the exception to the rule. It has been recognized for over 30 years that vaccine-associated polio, coxsackie, and echo viral infections can cause serious problems in a subset of patients with XLA (83–86). Interestingly, not all XLA patients who acquire these infections develop severe or progressive disease (87). Before vaccine policy changed from live to killed polio vaccine in 1997, many boys with XLA were given live polio vaccine before they were recognized to have antibody deﬁciency. Most had no unusual problems. There are adult patients with XLA who had wild-type polio with minimal sequelae many years before they were known to have XLA (17, 88). The modifying

factors that confer susceptibility to severe enteroviral infections in patients with XLA have not been identiﬁed. In addition to problems with S. pneumoniae, H. inﬂuenza, and Giardia, patients with XLA and CVID have an increased incidence of pneumonias, joint infections, and prostatitis owing to infections with mycoplasmas and ureoplasmas (89, 90). A small number of adolescents and adults with XLA have developed slowly progressive vasculitis and/or cellulitis of the lower extremities owing to infection with rare subspecies of Helicobacter (91). The basis for the unusual susceptibility to these organisms is not clear.

Laboratory Findings in XLA XLA is a leaky defect in B cell development. Almost all children with mutations in Btk have measurable amounts of serum immunoglobulin and a few B cells in the peripheral circulation (92, 93). The number of B cells that can be detected tends to decrease with age (5, 92). This probably reﬂects the normal decrease in B cell production that is seen with aging (94). The B cells in patients with XLA have a distinctive phenotype that can be used to help support the diagnosis in a patient with reduced numbers of B cells. Although the intensity of CD19 expression is relatively homogeneous in normal controls, it is low and variable in patients with XLA. By contrast, surface IgM expression is variable in normal controls but high and homogeneous in patients (Figure 2). Btk is expressed in monocytes and platelets, as well as B cells. This facilitates diagnosis, as over 90% of mutations in Btk (including one-third of all amino acid substitutions) are associated with the absence of Btk in monocytes (95). Bone marrow studies in patients with XLA demonstrate a strong block in differentiation or proliferation at the pro-B cell to pre-B cell transition (96–98). In normal children, between 10% and 25% of CD19+ cells in the bone marrow are pro-B cells, as deﬁned by the expression of CD19, TdT, and CD34 and the absence of cytoplasmic or surface μ heavy chain. www.annualreviews.org • Primary B Cell Immunodeficiencies

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CD22 Figure 2 B cell phenotype in patients with primary B cell immunodeﬁciencies. Ficoll density–separated peripheral blood lymphocytes were stained with PE-labeled CD19 and FITC-labeled isotype control, anti-IgM, CD38, CD21, or CD22. Shown are cells from a healthy control ( ﬁrst column from left), an 11-year-old patient with a premature stop codon (R255X) in Btk (second column), a 15-year-old patient with a hypomorphic mutation (G137S) in Igβ (third column), and a 5-year-old patient with a large deletion of the μ constant region on one allele and a two base pair deletion (AA del in codon 168) in exon 2 of μ heavy chain on the other allele ( fourth column). The number of gated events shown is 17,000 to 19,000 in the healthy control sample and 100,000 to 150,000 in the patient samples. Figure adapted from Reference 33, with the permission of American Association of Immunologists, Inc., copyright 2007.

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Pre-B cells, CD19+ /CD34− /TdT− cells that have cytoplasmic μ heavy chain but no surface IgM, comprise 35–60% of CD19+ cells, and mature B cells that express CD19 and surface IgM comprise 20–40% of the bone marrow B lineage cells. By contrast, in patients with defects in Btk, 75–90% of the CD19+ cells bear a pro-B cell phenotype, and less than 10% have a typical pre-B cell phenotype. Furthermore, patients with mutations in Btk have an unusual population of CD19+ cells that appear to be stalled between the pro-B cell and pre-B cell stage of differentiation. These cells, which constitute 5–10% of the total CD19+ cells in patients with XLA, continue to express CD34 and TdT, but they also express cytoplasmic μ heavy chain (Figure 3) (98).

ClgM

Mutations in Btk Over 600 different mutations in Btk have been identiﬁed (99, 100). Single base pair substitutions, or the insertion or deletion of less than ﬁve base pairs, account for more than 90% of these mutations. The remaining mutations include large deletions, duplications, inversions, complex combinations of insertions and deletions, and retrotransposon insertions (101, 102). Several factors contribute to this striking variability. First, similar to other X-linked disorders that are lethal without medical intervention, XLA is maintained in the population by new mutations (28). As these new mutations occur independently, they can involve multiple sites throughout the gene.

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TdT Figure 3 Bone marrow cells from patients with defects in B cell development were stained for CD19, cytoplasmic μ heavy chain, and TdT and then analyzed by ﬂow cytometry. The cells shown were within the CD19+ gate. (Top row) Cells from a healthy 6-year-old control, a patient with a premature stop codon in Btk (W588X), and a patient with an amino acid substitution in Btk (C506F). (Bottom row) Cells from patients with defects in μ heavy chain: one patient with the codon 168 frameshift mutation and two brothers with the alternative splice defect at codon 433. The stalled pro-B cells in the patients with mutations in Btk are seen in the upper right-hand corner, and the pre-B cell-like cells in the patients with defects in μ heavy chain are seen in the lower left-hand corner.

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Second, Btk is highly conserved. Human and murine Btk are 98% identical in amino acid sequence, suggesting minimal tolerance for any alteration in sequence. Our laboratory has identiﬁed 186 different mutations in Btk in 226 unrelated families (99). No single mutation accounts for more than 3% of the total. In many families, it is possible to identify the source of the new mutation in Btk. The mother of a patient with sporadic XLA has an 80% chance of being a carrier, but the maternal grandmother is a carrier only 25% of the time (28). These percentages, which are similar to that seen in other X-linked immunodeﬁciencies (103), can be explained by the fact that most new mutations occur in male gametes (104–106). In our studies on XLA, it is often possible to show that the allele bearing the mutation in Btk came from the unaffected maternal grandfather or great-grandfather (28). We have identiﬁed two families with two alterations in Btk. In one family, two affected brothers had an amino acid substitution (Y418H) near the ATP binding site and a premature stop codon (K625X) in the carboxyterminal portion of the kinase domain. Their mother was heterozygous for both alterations; however, their healthy maternal grandfather had the amino acid substitution at codon 418 but did not have the premature stop codon at codon 625 (107). Analysis of polymorphic markers ﬂanking Btk clearly demonstrated that the mutant allele in the affected boys was inherited from their maternal grandfather without crossovers, indicating that the second alteration had arisen in the sperm that gave rise to the mother or during the in utero development of the mother. To determine if the amino acid substitution in the 58-year-old grandfather had a deleterious effect, we examined his serum immunoglobulin concentrations, titers to vaccine antigens, and peripheral blood B cells. The serum IgG and IgA were within the normal range (690 mg dl−1 and 85 mg dl−1 , respectively), but the serum IgM was slightly low (36 mg dl−1 , with the normal adult male range being 48– 263 mg dl−1 ). Titers to vaccine antigens, in-

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cluding pneumococcus, were normal; however, the number of CD19+ B cells in the peripheral circulation was only 0.85% (6–20% is considered normal). Monocytes from this man and his affected grandson were analyzed by ﬂow cytometry for expression of Btk. No Btk was seen in the monocytes of the child with both alterations in Btk; however, cells from the grandfather had normal amounts of Btk (107). A Y418H mutation had been reported in a patient with typical XLA; therefore, it was important to determine the functional consequences of this alteration. Btk− cells from the chicken B cell line DT40 were transfected with either wild-type or Y418H Btk and then stimulated with anti-IgM. The cells bearing the Y418H mutation consistently showed a 15– 25% decrease in calcium ﬂux and IP3 production at 0.5 min when compared with cells that received the wild-type Btk vector (107). These ﬁndings suggest that even a mild reduction in Btk function can result in a decreased number of B cells. Furthermore, a reduced number of B cells is the most consistent feature in XLA. In a second family, a boy with XLA, his two cousins, and his maternal grandfather had two amino acid substitutions in Btk. In the ﬁrst alteration, the wild-type isoleucine at codon 305, within the SH2 domain, was replaced with a serine, and in the second alteration, the wildtype glycine was replaced with alanine at codon 556 in the kinase domain. Neither of these alterations has been described in other patients. Monocytes from both the patient and his grandfather had normal amounts of Btk as analyzed by ﬂow cytometry. Perez de Diego et al. (108) recently reported studies in a third family in which the affected boy had two amino acid substitutions in Btk. One alteration, an arginine to histidine at codon 641 in the kinase domain, has been reported in several other patients with XLA (99, 109). The second alteration, an alanine to valine at codon 230 in the SH3 domain, had not been reported previously. The boy’s mother was heterozygous for both alterations. However, his maternal grandmother, two aunts, and two cousins had only the A230V alteration. One

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of the healthy male cousins with the A230V alteration had 13% CD19+ B cells, indicating that this alteration is a polymorphism that does not affect the function of Btk. The A230V alteration is the only reported polymorphism in Btk that changes the amino acid sequence but not the function. It has been seen only in this family. The occurrence of two alterations in Btk in these three families is unexpected. Because XLA is uncommon, occurring with a frequency of ﬁve to ten cases per million births (110), and because most mutations have occurred relatively recently, one must assume that two rare events altered the same segment of DNA. This raises the issue of factors that might predispose a segment of DNA to mutation. In two of the families described above, analysis of multiple family members showed that the two alterations occurred independently on the same allele. This made us wonder if there were features of the Btk locus that might inﬂuence the development of new mutations. We addressed this question by analyzing four single nucleotide polymorphisms at the Btk locus. The ﬁrst two were in intron 1, and the last two were in the 3 untranslated region, 30– 35 kb distal to exon 1. The Btk haplotype of 47 unrelated males with XLA was compared with that of their unaffected fathers. Two haplotypes accounted for 74% of the individuals, and both haplotypes were seen with equal frequency in the patients and their fathers. Of the two families in which we identiﬁed two alterations in Btk, one family had the alterations on one of the common haplotypes. In the other family, the alterations were on an uncommon haplotype that was seen in two patients but in none of the fathers. These preliminary results do not rule out the possibility of local characteristics of the DNA that make it more vulnerable to mutation. Future studies may examine additional single nucleotide polymorphisms and expand the haplotype analysis to sites as far as 1 megabase away. It may be that minor variations in DNA sequence inﬂuence chromatin structure and therefore susceptibility to mutation.

Genotype/Phenotype Correlation The great diversity in Btk mutations makes it more difﬁcult to examine genotype/phenotype correlations. Furthermore, objective measurements of disease severity are not deﬁned easily. We chose to focus on age at diagnosis, the plasma IgM, and the number of B cells in the peripheral circulation (5). Mutations were divided into two broad categories, mild and severe. Mild mutations were amino acid substitutions and splice defects that occur at sites in the consensus sequence that are conserved but not invariant. These mutations conceivably allow the production of some Btk. Even amino acid substitutions that ablate the kinase activity may be associated with some function as a scaffold protein, provided by the PH, SH3, and SH2 domains (111). All the remaining mutations were considered severe, including premature stop codons, frameshift mutations, splice defects found at invariant sites in the splice consensus sequence (the ﬁrst two and last two base pairs of each intron), large deletions and duplications, and complex mutations. In an analysis of 110 patients from 94 unrelated families, the mild mutations were associated with later age at diagnosis ( p = 0.04) and a higher number of B cells in the peripheral circulation ( p = 0.09). However, the marker showing the best correlation with mild mutations was higher plasma IgM ( p < 0.001) (5). Although the age at diagnosis did not correlate with either the percentage of circulating B cells or the plasma IgM, the percentage of B cells and the plasma IgM correlated with each other ( p < 0.001). When the patients with amino acid substitutions were divided into those whose monocytes were positive for Btk and those whose monocytes were negative, the Btk+ patients were slightly older at diagnosis and had slightly higher mean plasma IgM than the patients who were Btk− , but both groups differed from the patients with severe mutations. Amino acid substitutions resulting in unstable proteins may have some residual function. Similar ﬁndings have been described by others. Plebani et al. (3) noted that certain amino

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acid substitutions in Btk were associated with higher concentrations of serum immunoglobulins at the time of diagnosis. Lopez-Granados et al. (4) analyzed 54 patients with proven mutations in Btk, from 40 unrelated families, using a system similar to ours to classify severe versus mild mutations. The age at diagnosis, the concentrations of serum immunoglobulins at diagnosis, and the percentage of CD19+ B cells all correlated with the severity of mutation. However, the speciﬁc mutation in Btk clearly is not the only factor that inﬂuences the severity of disease. Environmental factors may play a role, but modifying genetic factors likely wield a stronger inﬂuence. When considering modifying genetic factors, one can see that polymorphic variants in components of the BCR signal transduction pathway are obvious candidates. Both IgM and λ5 are highly polymorphic (112, 113). However, it is not clear which components of this pathway act as limiting factors. One might expect that polymorphic variants in the Btk family member Tec might inﬂuence the severity of disease. Mice that are mutant in Tec as well as Btk have a more severe phenotype than mice that are deﬁcient in Btk alone (114), indicating that Tec, which is activated by many of the same signals as Btk (115), may compensate for Btk when the latter is mutant. However, we did not ﬁnd polymorphic variants in Tec that could explain clinical or laboratory variability (5). Polymorphic variants in molecules that enhance signaling through the BCR, such as CD19, or dampen signaling, such as CD22, might impact the number of B cells or the concentration of IgM. Furthermore, some modifying genetic factors may depend on the speciﬁc type of mutation. For example, polymorphic variants in the splicing apparatus might be expected to affect splice defects but not amino acid substitutions.

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Autosomal Recessive Agammaglobulinemia Starting in the 1970s, several reports described females with a clinical disorder that was indis208

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tinguishable from XLA (116–118). The affected girls had an early onset of disease, profound hypogammaglobulinemia, and less than 1% of the normal number of B cells in the peripheral circulation. This suggested that there were autosomal recessive forms of the disease. The identiﬁcation of Btk as the gene responsible for XLA made it possible to exclude this diagnosis in some families and spurred a search for the genes that might cause autosomal recessive agammaglobulinemia.

Defects in μ Heavy Chain If the most important role for Btk is its involvement in signaling through the pre-BCR and BCR, then other genes required for this pathway would be strong candidates for unidentiﬁed forms of agammaglobulinemia. Using a combination of homozygosity mapping and candidate gene analysis, in 1996 we showed that mutations in μ heavy chain (IGHM ) cause agammaglobulinemia and a clinical picture that is similar to that seen in XLA (9). Twenty-six families with mutations in μ heavy chain have been reported to date (9, 29, 30, 112, 119). All the reported mutations result in the complete absence of CD19+ B cells in the peripheral circulation, with a detection threshold of 0.01%. Although there is considerable overlap, the patients with mutations in μ heavy chain tend to have a more severe phenotype than that seen in patients with mutations in Btk (112). They are recognized to have immunodeﬁciency at a mean age of 11 months rather than 35 months in patients with XLA, and they have a higher incidence of enteroviral infection and pseudomonas sepsis with neutropenia. These ﬁndings indicate that the small amount of immunoglobulin produced by patients with XLA has some protective value. These ﬁndings also imply that the enteroviral infections and neutropenia in patients with XLA result from hypogammaglobulinemia rather than requirements for Btk in myeloid cells. The spectrum of mutations seen in patients with defects in μ heavy chain is quite different from that seen in patients with XLA. Between

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30% and 50% of mutations are large deletions that remove between 70 and 700 kb of DNA, including JH and DH regions, as well as all μ constant region exons (30, 112). Some deletions also result in the loss of some VH segments and/or heavy chain constant segments. In the 17 families in which we have identiﬁed mutations in μ heavy chain, two mutations have been seen in several unrelated families with agammaglobulinemia. One mutation, a two base pair deletion at codon 168 in exon 2, has been seen in four unrelated families, two from Spain and two from Mexico (112). In all these families, the mutation is on the same uncommon μ heavy chain haplotype, indicating that the patients share a common ancestor. By contrast, the other mutation, a single base pair substitution in codon 433, at the −1 position of the alternative splice site, has been seen on three different haplotypes in six unrelated families, indicating that it is a recurrent mutation (112). The guanine to adenine substitution at the −1 position of the alternative splice site has three effects. First, it changes the amino acid sequence of the secretory form of μ heavy chain from glycine to serine at codon 433; second, it replaces the negatively charged glutamic acid with the positively charged lysine at the same site in the membrane form of μ heavy chain; and, ﬁnally, because the base pair substitution is at a site that is conserved but not invariant within the splice consensus sequence, it is predicted to markedly impair but not ablate the production of transcripts for the membrane form of μ heavy chain. The effects of the base pair substitution at codon 433 on B cell development were evaluated in bone marrow samples from two brothers with this mutation. Studies from two patients with other mutations in μ heavy chain were analyzed for comparison. One of these patients had the codon 168 frameshift mutation on one allele and a large deletion on the other allele; the other patient had an amino acid substitution at an essential cysteine (codon 412) in exon 4 on one allele and a large deletion on the other allele (9). In both teenagers with the codon 433 mutation, 65–83% of the CD19+ cells in the

bone marrow were pro-B cells (Figure 3). The stalled pro-B cells, cytoplasmic μ+ /TdT+ cells, which have been identiﬁed in patients with mutations in Btk, were not seen. Between 15% and 30% of the CD19+ cells had a pre-B cell phenotype manifested by the low-intensity expression of cytoplasmic μ heavy chain and the absence of CD34 or TdT. Mature, surface Ig+ cells were not seen. The other two patients with defects in μ heavy chain also had a small percentage of CD19+ /TdT− /CD34− cells; however, the percentage was lower (5–10% of the CD19+ cells). Schiff et al. (29) also noted that a patient with a homozygous frameshift mutation in exon 1 of μ heavy chain had a small number of CD19+ /CD34− /TdT− cells in the bone marrow. The identity of the CD19+ /CD34− /TdT− cells in all the patients with defects in μ heavy chain is not clear. The two patients with frameshift mutations in the amino terminal portion of μ heavy chain should not be able to make any cytoplasmic or surface pre-BCRs. Expression of a pre-BCR is considered essential for B cells to progress beyond the pro-B cell stage of differentiation. In our patient with a frameshift mutation, the CD19+ /CD34− /TdT− cells were characterized in more detail. These cells were positive for CD22 but negative for CD20, CD21, and CD37. The TdT− cells from one of the patients with the alternative splice defect were positive for cytoplasmic Vpre-B and Igα and negative for CD117. The observation that the patients with the alternative splice site defect had a few more of these pre-B-like cells, compared with other patients with mutations in Btk or μ heavy chain, made us question if some μ heavy chain was being produced. We therefore analyzed the cDNA from the bone marrow of the two brothers with the codon 433 mutation. PCR (polymerase chain reaction primers expected to amplify transcripts for the membrane form of μ heavy chain were used to amplify cDNA from both patients. The results demonstrated three products (Figure 4). The sequencing of these products indicated that the largest could be www.annualreviews.org • Primary B Cell Immunodeficiencies

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Figure 4 Transcripts for the membrane form of μ heavy chain in a patient with a defect at the alternative splice site. (a) A RT PCR was used to amplify cDNA from the Daudi B cell line (column 1), Jurkat T cells (column 2), control peripheral blood lymphocytes (column 3), or from the bone marrow of a patient with the alternative splice site defect (column 4 ). Transcripts for membrane μ (top panel ) and secretory μ (bottom panel ) are shown. (b) A semiquantitative PCR was conducted to estimate the amount of correctly spliced membrane μ transcripts in the patient with the splice defect. Transcripts from the bone marrow of a normal control (column 1), two patients with mutations in Btk (columns 2 and 3), and a patient with the alternative splice defect (column 4 ) are shown. The cDNA from the normal control was diluted 1:10 (column 5 ) and 1:100 (column 6 ) to allow comparison. The amount of correctly spliced message in the sample from the patient with the alternative splice defect was approximately 1% of the control.

attributed to the use of a cryptic splice site (GTGAG) 136 base pairs downstream of the authentic alternative splice site and 75 base pairs downstream of the stop codon for the secretory form of μ heavy chain. This transcript encodes the secretory form of μ heavy chain with an amino acid substitution, glycine to serine, at codon 433 (at the alternative splice site position). Ferrari et al. (119) studied a different patient with the alternative splice defect and identiﬁed this transcript but not the other two. The smallest PCR product showed the use of a cryptic splice site (GTATG) 173 base pairs upstream of the authentic splice site. This alteration would result in a frameshift mutation and a premature stop codon four amino acids downstream of the cryptic splice site. The protein encoded by this transcript would lack a transmembrane domain. The third product represented a correctly spliced message encoding the membrane form of μ heavy chain with the substitution of lysine for the wild-type glutamic acid at codon 433. The wild-type glutamic acid at this site is conserved in mice, rabbits, and 210

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camels, and an aspartic acid is seen at a homologous position of the membrane form of μ heavy chain in ducks and sharks. The site of the amino acid substitution is 13 amino acids proximal to the transmembrane domain. It forms part of the conserved extracellular stalk that permits the dimerization of μ heavy chain and binding to the signal transduction molecules Igα and Igβ. The substitution of the highly conserved, negatively charged glutamic acid at codon 433 of the membrane form of μ heavy chain with the positively charged lysine might be expected to inﬂuence cell surface expression of μ heavy chain or signaling through the BCR. We tested this possibility by recreating normal or mutant BCRs in Jurkat T cell lines. Two retroviral vectors—one containing GFP (green ﬂuorescence protein), λ light chain, and either normal or codon 433 mutant μ heavy chain and the other containing YFP (yellow ﬂuorescence protein), Igα, and Igβ—were used to transduce Jurkat cells. Six to 10 days after transduction, GFP+ /YFP+ cells were sorted and placed back into culture. The cultured cells were stained for

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Figure 5 Expression of a wild-type or mutant BCR in Jurkat T cells. Jurkat cells were transduced with two retroviral vectors, one expressing YFP, Igα, and Igβ and the other expressing GFP, λ light chain, and either wild-type membrane μ heavy chain or μ heavy chain with the alternative splice (AS) site mutation (E433K). Empty vectors were used as a control. (a) Cells transduced with empty vectors, wild-type vectors, or vectors expressing mutant BCR were sorted to obtain populations with equal amounts of YFP and GFP. These cells demonstrated equal amounts of surface IgM, indicating that the amino acid substitution did not impair membrane expression of μ heavy chain. (b) The cells shown in panel a were cultured with anti-CD3 or anti-IgM. Cells expressing the wild-type or mutant BCR secreted approximately equal amounts of IL-2, suggesting that the mutation did not impair signal transduction.

surface expression of IgM 8 to 30 days after the sort. When we gated on cells that were equally positive for GFP and YFP, the cells bearing the normal and mutant BCR expressed comparable amounts of surface IgM (Figure 5), indicating that the mutation did not impair cell surface expression of μ heavy chain. The ability of the mutant BCR to signal was tested by culturing the Jurkat cells bearing the normal or mutant BCR for 24 h with anti-CD3 or anti-IgM and

measuring IL-2 released into the supernatant. Approximately equal amounts of IL-2 were produced by cells bearing the normal or mutant BCR. These ﬁndings indicate that the small amount of membrane μ heavy chain produced in the patients with the alternative splice defects enhanced the transition of pro-B cells to the pre-B cell stage of differentiation, but it was insufﬁcient to support the expansion or further differentiation of pre-B cells. www.annualreviews.org • Primary B Cell Immunodeficiencies

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Meffre et al. (120) examined VDJ rearrangements in a patient with a frameshift mutation in exon 1 of μ heavy chain and found that the CDR3 regions were longer than those seen in controls, indicating that expression of a preBCR inﬂuences the immunoglobulin repertoire. Furthermore, light chain rearrangement could be detected in the μ heavy chain–deﬁcient patients, and the kappa repertoire was skewed toward 5 Vks and 3 Jks, suggesting that in the absence of an effective pre-BCR, continued light chain rearrangement occurs (120).

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Defects in λ5, Igα, Igβ, and BLNK A small number of patients with defects in λ5 (IGLL1), Igα (CD79A ), Igβ (CD79B ), or BLNK have been reported (31–36). These patients generally have clinical ﬁndings that are indistinguishable from those seen in patients with mutations in Btk. Similar to patients with defects in μ heavy chain, patients with other forms of autosomal recessive agammaglobulinemia tend to have the onset of severe infections within the ﬁrst year of life. However, there are exceptions. One of the two patients studied with defects in λ5 was recognized to have immunodeﬁciency after his second hospitalization for pneumococcal pneumonia at 29 years of age (M.E. Conley, D.M. Farmer, A.K. Dobbs, and K. Paris, unpublished observations). Significant enteroviral infections and pseudomonas sepsis with neutropenia were seen in patients with Igα or BLNK deﬁciency. One child with Igα deﬁciency had wild-type or vaccineassociated polio at 12 months of age (M.E. Conley and V. Howard, unpublished observations). A second child with Igα deﬁciency had progressive weakness and a dermatomyositislike syndrome, ﬁndings typical of enteroviral infection (32). The older brother of one of the patients with BLNK deﬁciency died of pseudomonas sepsis and neutropenia at 16 months of age (36). It is likely that he also had BLNK deﬁciency. We have analyzed B cell number and phenotype in two patients with λ5 deﬁciency. One patient, who has a premature stop codon on one 212

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allele and a proline to leucine amino acid substitution at codon 142 on the other allele, has been studied several times between 4 and 15 years of age. He has never had more than 0.06% circulating CD19+ cells, and in recent years he has had less than 0.02% CD19+ cells (35). These cells have surface IgM and CD19 expression that is similar to that seen in healthy individuals. The other patient, who has a single base pair deletion, a guanine deletion in codon 85, was ﬁrst analyzed at 35 years of age and had less than 0.01% CD19+ cells (M.E. Conley, D.M. Farmer, K.A. Dobbs, K. Paris, unpublished observations). The B cell phenotype was evaluated in two patients with mutations in BLNK, an 8-year-old girl with a homozygous premature stop codon in exon 123 (M.E. Conley and S. Kilic, unpublished observations) and a 20-year-old man with a homozygous splice defect. The girl had 0.01% CD19+ cells in the peripheral circulation, and, by analyzing nearly 500,000 events, we showed that the small number of B cells had a phenotype that was similar to that seen in patients with mutations in Btk. The cells expressed variable and slightly dimmer CD19, but also expressed high levels of surface IgM. Bone marrow from both patients showed a cell distribution that was similar to that seen in patients with mutations in Btk; both patients showed an easily identiﬁed population of stalled pro-B cells. Two additional patients with mutations in BLNK have been noted, but the precise mutations and phenotypic characteristics of these patients have not been reported (30). Our laboratory has evaluated three females with homozygous null mutations in Igα (31). The ﬁrst had an adenine to guanine base pair substitution at the invariant −2 position of the acceptor splice site for intron 2; the second had a guanine to thymine base pair substitution at codon 48, leading to the replacement of the wild-type glutamic acid with a premature stop codon; and the third had a guanine-cytosine deletion that spanned codons 68 and 69. All these mutations occurred upstream of the transmembrane domain. Blood and bone marrow studies done on the ﬁrst two patients, who were

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both small children at the time, showed less than 0.01% CD19+ cells in the peripheral circulation and a block at the pro-B to pre-B cell transition that was indistinguishable from that seen in patients with null mutations in μ heavy chain. Blood and bone marrow samples were not available from the third patient. Wang et al. (32) reported a fourth patient with a null mutation in Igα, a splice defect in the donor site for intron 2, but no details were available about the laboratory phenotype. Ferrari et al. (34) recently described a boy with a base pair substitution in codon 80 of Igβ, resulting in a premature stop codon (Q80X). This alteration, which is upstream of the transmembrane domain, is a null mutation, and the affected patient was reported to have less than 1% CD19+ cells in the blood and a complete block at the pro-B cell to pre-B cell stage of differentiation. We also identiﬁed a patient with a defect in Igβ, a 15-year-old girl who had a homozygous amino acid substitution at codon 137 (33). The alteration at this site, which is immediately downstream of the cysteine that forms the disulﬁde bridge with Igα, is the replacement of the wild-type glycine with serine. This glycine is conserved not only in Igβ, but also in Igα, from humans, mice, dogs, and cattle. The patient with the G137S mutation in Igβ had a small number of B cells in the peripheral circulation (0.08% CD19+ cells) (33). These B cells showed striking similarities and differences when compared with those seen in patients with mutations in Btk (Figure 2). B cells from both demonstrated variable intensity of CD19 expression, had increased expression of CD38, and had decreased expression of CD21. However, the B cells from the patient with the Igβ mutation showed decreased or absent expression of surface IgM, whereas those from patients with mutations in Btk show increased expression of surface IgM. These ﬁndings suggest that the alteration in Igβ inﬂuenced the ability of the BCR to reach the cell surface. They also support the hypothesis that the phenotype of the B cells in patients with XLA could be attributed to defective signaling through the BCR.

To examine the ability of the G137S mutant Igβ to bring the BCR to the cell surface, we transfected Jurkat T cells to produce a wildtype or mutant BCR. With transient transfection, there was no difference in the intensity of surface IgM in the cells that had either the wild-type or mutant Igβ. By contrast, with stable transduction (using the retroviral viral vectors described above), there was consistently less surface IgM in cells that contained the mutant Igβ (33). This study provides another example of the severe consequences of subtle decreases in the BCR signal transduction pathway.

HYPER-IgM SYNDROMES (CLASS SWITCH RECOMBINATION DEFECTS) In the early 1960s, several groups described patients with recurrent infections and elevated β2 macroglobulin (121, 122) (the term IgM did not come into use until the mid-1960s) but decreased serum gammaglobulin. These patients were said to have dysgammaglobulinemia, and many, but not all, were boys with neutropenia and the early onset of disease. The term hyperIgM syndrome was ﬁrst used to describe this group of patients in 1974 (123). It is now obvious that not all patients with the genetic disorders that come under this category have elevated IgM. Instead, the most consistent feature of these disorders is a defect in class switch recombination, and Durandy et al. (25) proposed the term class switch recombination defects to describe them. Patients with recurrent bacterial infections and normal or elevated serum IgM but low serum IgG, IgA, and IgE are considered to have hyper-IgM syndrome or class switch recombination defects. Patients with mutations in CD40 ligand (also called CD154, gene symbol CD40LG ) account for approximately 65% of patients with defects in class switch recombination (10, 124– 127). As a group, these patients are sicker than those with early defects in B cell development. Median age at diagnosis is less than 12 months, and more than half the patients have opportunistic infections and/or neutropenia (20, 23). www.annualreviews.org • Primary B Cell Immunodeficiencies

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The opportunistic infections (which include pneumocystis pneumonia and infections with cytomegalovirus or cryptosporidium) are generally attributed to the failure to initiate the normal cross talk between CD40 ligand–expressing T cells and CD40-expressing macrophages and dendritic cells. Interestingly, affected patients may have recurrent episodes of pneumocystis pneumonia (23), indicating that the patients have a defect in the memory T cell response, as well as the primary response. European patients with CD40 ligand deﬁciency have a high incidence of sclerosing cholangitis owing to infection with cryptosporidium (19.6%) (20). This complication occurs in North American patients, but it is less common (6%) (23). Four patients with null mutations in CD40 have been reported (24, 128). These patients had a clinical phenotype that was indistinguishable from that seen in those with defects in CD40 ligand. This indicates that neither CD40 nor CD40 ligand has additional ligands or receptors. Approximately 10–15% of patients with defects in class switch recombination have autosomal recessive mutations in AICDA, which encodes the B cell–speciﬁc enzyme AID (activation-induced cytosine deaminase) (129– 131). AID is transiently and selectively expressed in germinal center B cells in response to stimulation through CD40 and cytokines (132, 133). It initiates both class switch recombination and somatic hypermutation by deaminating cytosine residues in VH regions and switch regions in actively transcribed immunoglobulin genes (134, 135). The resulting uracil residues are then deglycosylated and removed by an enzyme called uracil-DNA glycosylase (UNG ) (25, 136–138). The nicks in DNA are then converted to double-strand breaks, which are processed by mismatch repair proteins and proteins involved in nonhomologous end joining (133). Patients with AID mutations may be recognized as having immunodeﬁciency in the ﬁrst 5 years of life, but more than half are older at the time of diagnosis and the initiation of therapy (129, 130). In addition to problems with infections (which may be quite severe), these

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patients have enlarged lymph nodes and a high incidence of autoimmune disorders (139). Notably, markedly enlarged lymph nodes are not as common in CD40 ligand deﬁciencies. The speciﬁc mutation in AICDA does inﬂuence the phenotype. Patients with mutations in the 3 part of the gene, the part encoding the nuclear localization signal, have impaired class switch recombination but not somatic hypermutation (140). A small number of patients with heterozygous premature stop codons at residues 186 or 190 in the 3 part of AICDA have been identiﬁed (141). These patients have a milder disease. They are usually not evaluated for immunodeﬁciency until they are adolescents or adults, and they may have asymptomatic relatives who share the same heterozygous mutation. Their serum IgM is usually mildly increased, IgG is low, IgA is variable, and serum IgE is absent. Somatic hypermutation is normal in these patients. The normal somatic hypermutation combined with defective class switch recombination in patients with mutations in the carboxy-terminal region of AID suggests that this portion of the molecule has a function that is speciﬁc to class switch recombination. The difference between the autosomal recessive and autosomal dominant forms may reﬂect the amount of stable protein that is produced. Because AID functions as a tetramer, a stable truncated form of the protein may have a dominant negative function. Imai et al. (137) described three unrelated patients with autosomal recessive defects in UNG. These patients were clinically similar to those who had mutations in AICDA; two had lymphadenopathy, and one had autoimmune disease. Laboratory studies showed a severe defect in class switch recombination and a skewed pattern of somatic hypermutation. Almost all the mutations were transitions (guanine to adenine or cytosine to thymine), whereas in controls transitions composed only 65% of mutations. It is highly likely that there is at least one additional single-gene defect causing autosomal recessive hyper-IgM syndrome. Peron et al. (142) described a group of patients with

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recurrent respiratory infections and normal or elevated serum IgM, but low serum IgG, IgA, and IgE. Detailed studies in a subset of these patients, including two brothers and the child of consanguineous parents, showed normal activation of AID and normal production of AID-initiated double-strand DNA breaks. Fibroblast cell lines from the affected patients demonstrated increased radiation sensitivity, suggesting a defect in DNA repair. It is not clear that all the children with recurrent infections, normal or elevated serum IgM, and low IgG, IgA, and IgE will have single-gene defects of the immune system. This phenotype is common in adults who are given the diagnosis of CVID.

COMMON VARIABLE IMMUNODEFICIENCY (CVID) All clinical immunologists would agree that the term CVID includes a heterogeneous group of disorders (13, 26, 27). Typically, affected patients have the onset of recurrent infections after the ﬁrst 10 years of life; they have normal or low serum IgM and low IgG and IgA with poor production of antibody to vaccine antigens (13, 16, 26, 48). Autoimmune manifestations are common and may be more difﬁcult to control than the immunodeﬁciency. The number of B cells in the peripheral circulation is usually within the normal range but may be very low. Most patients have very low numbers of CD27+ switched memory B cells (143). However, there are exceptions to each of these features. CVID should be considered a diagnosis of exclusion (144). Malignancies, congenital infections, drug reactions, and single-gene defects of the immune system (145–147) can all masquerade as CVID. Most patients with what are considered single-gene defects of the immune system are evaluated for immunodeﬁciency in the ﬁrst 5 years of life because of recurrent or persistent infections (14, 20). By contrast, patients with CVID, which is generally thought to be multifactorial in etiology, are more likely to have the onset of disease in adulthood (13, 16, 26). It is not clear why patients with CVID have a de-

layed onset of vulnerability. It may be that some of them have had problems with infections from early childhood, but the problems were not severe enough to arouse concern. However, other patients adamantly deny having had excessive infections as children. Did these patients lose their immunity? Have they acquired inappropriate suppression of antibody production? Do they have an accelerated exhaustion of the immune system? Over the years, studies evaluating the clinical and laboratory ﬁndings in patients with CVID have suggested many different answers to these questions (143, 148– 151), but thus far the answers have not proven to be satisfying, and there are no animal models that clearly replicate the ﬁndings in CVID. Approximately 10–20% of patients with CVID have a family history of autoimmunity or disorders of antibody production (152, 153). Both autosomal dominant and autosomal recessive patterns of inheritance have been reported (11, 154–156). In some family members, the serum IgM, IgG, or IgA is elevated rather than decreased. IgA deﬁciency is particularly common in relatives of patients with CVID. Early attempts to identify the genetic variations that contribute to CVID demonstrated that certain HLA haplotypes were more common in both CVID and IgA deﬁciency (157–162). Because the HLA locus is complex (including genes for complement factors C2 and C4, TNF-α, and mismatch repair genes, as well as genes for histocompatibility antigens), it has been difﬁcult to pin down the speciﬁc genetic changes that confer vulnerability. Recent studies have shown that there are polymorphic variants in the mismatch repair gene Msh5 (MSH5 ), which is encoded in the central MHC class III region (163). Msh5 and its heterodimeric partner, Msh4, help resolve the DNA breaks that occur as part of class switch recombination. CVID patients with these polymorphic variants show extended microhomology regions at switch joints (163). However, these polymorphic variants are not more common in CVID patients compared with controls, making their role in the pathogenesis of CVID uncertain. www.annualreviews.org • Primary B Cell Immunodeficiencies

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Defects in ICOS and CD19

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Homozygous mutations in ICOS or CD19 are clearly the cause of disease. In 2003, Grimbacher et al. (11) identiﬁed four patients, two sibling pairs, whose T cells failed to express ICOS after activation. Genomic studies revealed a 1.8-kb deletion that removed exons 2 and 3 of ICOS in all the patients. Later studies described ﬁve additional patients with the same mutation on the same haplotype, indicating that all nine patients shared a common ancestor (164). ICOS, a member of the CD28 CTLA4 family, is transiently expressed on activated T cells and acts as a positive costimulatory molecule (165, 166). Its ligand, B7RP-1, is a member of the B7 family; it is expressed constitutively on B cells and in response to stimulation on antigen-presenting cells (167, 168). Knockout mice that fail to express ICOS have decreased serum IgG1, IgG2, and IgE but normal or elevated serum IgM and IgG3 (169– 171). Failure to produce IgG1 in these mice could be overcome by CD40 stimulation (169). Although seven of the nine patients with mutations in ICOS had the onset of recurrent infections at 15–28 years of age (which is typical of CVID), the remaining two were less than 5 years old at the time of diagnosis (164). These two patients had an older sibling with the ICOS mutation, which probably prompted an early evaluation of recurrent infections. None of the patients has been reported to have autoimmune disease. Four of the nine patients had serum IgM concentrations that were within the normal range, and one had normal serum IgA. B cell numbers were low or borderline low in all except the two youngest patients. Long-term follow up of these patients will indicate whether B cell numbers and immunoglobulin concentrations decline with age. Mutations in CD19 also result in an autosomal recessive form of hypogammaglobulinemia with similarities to CVID. Six patients from four unrelated families have been identiﬁed (172, 173; M.E. Conley, A.K. Dobbs, D.M. Farmer, J-L. Casanova, unpublished re-

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sults). One patient had a splice defect on one allele and a large deletion on the other (173). The remaining ﬁve patients had three different homozygous frameshift mutations within the cytoplasmic domain of CD19. All the mutations resulted in normal numbers of circulating B cells, as identiﬁed by expression of CD20; however, there was minimal or no CD19 identiﬁed on the B cell surface. CD19 is normally expressed throughout B cell differentiation as part of a signaling complex that includes CD21, CD81, and CD225 (174). Studies done in CD19 knockout and transgenic mice indicate that CD19 regulates basal signal transduction thresholds in resting B cells (175). It does this by amplifying src family activation following BCR ligation. Elevated CD19 expression is associated with autoantibody production (175). These ﬁndings raise questions about the effects of decreased CD19 expression on B cells from patients with mutations in Btk. The six patients described above include three siblings with a frameshift mutation who were not recognized to have immunodeﬁciency until they were 35–49 years old. However, all three had a history of frequent infections starting in childhood (172). The remaining three patients were found to have hypogammaglobulinemia at 5–12 years of age. All the patients had low serum IgG, and most, but not all, had low IgM and IgA. No autoimmunity has been reported. Van Zelm et al. (172) examined four of the patients with CD19 defects in great detail. They found normal amounts of CD19 transcripts in the B cells from the patients with frameshift mutations but detected minimal amounts of intracellular protein, suggesting that the frameshift mutations did not result in nonsense-mediated decay of the message, but instead decreased translation or survival of the protein. Surface expression of CD21 on the B cells was decreased, but CD81 and CD225 expression was normal. Other cell surface markers (including IgM, IgD, CD22, CD38, and CD40) were normally expressed. CD27+ memory B cells were present, but in reduced numbers (1–6% of B cells compared with 17–28% in

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controls). Analysis of the switch memory B cells showed normal somatic hypermutation. B cells from the four patients showed decreased calcium ﬂux after cross-linking of the BCR. The primary IgG response to rabies immunization was at the low end of the normal range, whereas the secondary response was clearly below normal.

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Alterations in TACI Heterozygous mutations in the TNF receptor family member TACI (transmembrane activator and calcium-modulating cyclophilin ligand interactor) can be found in up to 10% of patients with CVID (156, 176–179). However, the relationship between TACI and CVID is complex. Large population studies indicate that approximately 1–2% of healthy controls have one of the amino acid substitutions found in patients with CVID (178, 179). On the basis of Hardy-Weinberg law (180, p. 147), we may reasonably assume that 1/10,000 individuals are homozygous for these amino acid substitutions (0.01 × 0.01). The prevalence of CVID in the population has been estimated to be 1/25,000 (156). Taken together, these ﬁgures indicate that over 90% of people who are heterozygous or homozygous for one of the amino acid substitutions associated with CVID do not have infections severe enough to elicit an evaluation for immunodeﬁciency. In almost all the patients with CVID and heterozygous alterations in TACI, the alteration is an amino acid substitution. Premature stop codons and frameshift mutations have been seen in individuals who were compound heterozygotes and had an amino acid substitution on the other allele; however, only a single patient with a premature stop codon on a single allele has been reported (164). These ﬁndings suggest that the amino acid substitutions in TACI function as dominant-negative mutations. This can be explained by the fact that TACI forms trimers prior to ligand interaction. Using transfection of 293T cells, Garibyan et al. (181) demonstrated that proteins bearing the amino acid substitution seen most fre-

quently in CVID, C104R, can assemble with wild-type TACI, but they are unable to signal appropriately. The serum IgG concentration in patients with TACI abnormalities is often borderline low, and the serum IgM may be within normal range (176, 177). However, autoimmunity, particularly thrombocytopenia and splenomegaly, is more common in this group of patients (156, 177). It is not clear why some individuals with particular heterozygous alterations in TACI have disease and others do not. Salzer et al. (156) and Waldrup et al. (182) examined HLA haplotypes in patients with TACI alterations to determine if these patients were more likely to have HLA haplotypes previously associated with disease. Although the numbers are small, the two susceptibility factors do not appear to cosegregate. The functional data using the C104R mutation and the higher prevalence of TACI amino acid substitutions in CVID patients compared with controls indicate that alterations in TACI can function as susceptibility genes; however, the relatively high prevalence of these amino acid substitutions in healthy donors demonstrates that these alterations are not diseasecausing mutations.

CONCLUDING REMARKS Tremendous progress has been made in the identiﬁcation and characterization of genes responsible for immunodeﬁciencies in the past 15 years. For some of the classic X-linked immunodeﬁciencies, such as XLA and X-linked hyper-IgM syndrome, hundreds of different mutations have been described in the causative genes. Careful analysis of the functional consequences of some of these mutations can provide new insight into the requirements for normal B cell development. Further areas of investigation will include a better understanding of susceptibility genes and modifying genetic factors, as well as the identiﬁcation of the mutant genes in patients who do not appear to have defects in the genes already associated with immunodeﬁciency. www.annualreviews.org • Primary B Cell Immunodeficiencies

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SUMMARY POINTS 1. Mutations in Btk, the gene responsible for XLA, account for approximately 85% of patients with early onset of infection, profound hypogammaglobulinemia, and markedly reduced or absent B cells. 2. XLA is a leaky defect in B cell development. Most patients do have a small number of B cells in the peripheral circulation. Those B cells have a distinctive phenotype. 3. The speciﬁc mutation in Btk is only one factor that inﬂuences the severity of disease in XLA. Annu. Rev. Immunol. 2009.27. Downloaded from arjournals.annualreviews.org by University of Bergen UNIVERSITETSBIBLIOTEKET on 01/13/09. For personal use only.

4. Mutations in μ heavy chain account for approximately 5% of patients with defects in early B cell development. All the reported mutations have been associated with the complete absence of B cells in the peripheral circulation. 5. A small number of mutations in other components of the pre-BCR or the scaffold protein BLNK have been identiﬁed. All the known mutations in Igα have been null mutations resulting in the complete absence of B cells in the blood. Some mutations in λ5, Igβ, and BLNK have been associated with a very small number of B cells in the blood. The B cells seen in BLNK deﬁciency and in a patient with a hypomorphic Igβ mutation have a phenotype similar to that seen in patients with mutations in Btk. 6. Patients with hyper-IgM syndrome or defects in class switch recombination may have mutations in CD40 ligand (65% of patients), CD40 (<1%), AID (20%), or UNG (<1%). Mutations in AID can cause an autosomal recessive or a milder autosomal dominant form of disease. 7. The predisposing genetic factors that are associated with CVID are unknown in the majority of affected patients. A very small number of patients have homozygous defects in ICOS or CD19. 8. Some heterozygous amino acid substitutions in TACI act as susceptibility genes for CVID. These polymorphisms are seen in healthy controls, but they are more common in patients with CVID, occurring in up to 10% of patients.

FUTURE ISSUES 1. Although most genes responsible for defects in early B cell development or hyper-IgM syndrome have been identiﬁed, there are still some patients with these clinical disorders who do not have defects in the described genes. What are the best approaches for determining the nature of the defect in these patients? 2. Knowing the genetic etiology of a particular immunodeﬁciency allows more informed genetic counseling and lays the groundwork for gene therapy. Are there other ways in which this information can beneﬁt the patient? Can we ﬁnd ways to compensate for the genetic defect? 3. Are there modifying genetic factors that inﬂuence the severity of all primary B cell immunodeﬁciencies, or are there disease-speciﬁc modifying factors?

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4. Large cooperative studies may make it possible to determine if there are one or many modifying genetic factors that dictate whether an individual with a heterozygous alteration in TACI will have immunodeﬁciency.

DISCLOSURE STATEMENT

Annu. Rev. Immunol. 2009.27. Downloaded from arjournals.annualreviews.org by University of Bergen UNIVERSITETSBIBLIOTEKET on 01/13/09. For personal use only.

The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

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Review in Advance first posted online on December 8, 2008. (Minor changes may still occur before final publication online and in print.)

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The Inﬂammasomes: Guardians of the Body ¨ Tschopp2 Fabio Martinon,1 Annick Mayor,2 and Jurg 1

Harvard School of Public Health, Department of Immunology and Infectious Diseases, Boston, Massachusetts 02115

2

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland; email: [email protected]

Annu. Rev. Immunol. 2009. 27:229–65

Key Words

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

adjuvanticity, danger signal, inﬂammation, innate immunity, NOD-like receptors

This article’s doi: 10.1146/annurev.immunol.021908.132715 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0229$20.00

Abstract The innate immune system relies on its capacity to rapidly detect invading pathogenic microbes as foreign and to eliminate them. The discovery of Toll-like receptors (TLRs) provided a class of membrane receptors that sense extracellular microbes and trigger antipathogen signaling cascades. More recently, intracellular microbial sensors have been identiﬁed, including NOD-like receptors (NLRs). Some of the NLRs also sense nonmicrobial danger signals and form large cytoplasmic complexes called inﬂammasomes that link the sensing of microbial products and metabolic stress to the proteolytic activation of the proinﬂammatory cytokines IL-1β and IL-18. The NALP3 inﬂammasome has been associated with several autoinﬂammatory conditions including gout. Likewise, the NALP3 inﬂammasome is a crucial element in the adjuvant effect of aluminum and can direct a humoral adaptive immune response. In this review, we discuss the role of NLRs, and in particular the inﬂammasomes, in the recognition of microbial and danger components and the role they play in health and disease.

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INTRODUCTION AND OVERVIEW

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Pattern-recognition receptor (PRR): membrane receptor expressed by cells of the immune system to identify molecules associated with microbial pathogens or cellular stress Pathogen-associated molecular pattern (PAMP): highly conserved microbial structure that is essential for microbial survival and is detected by host innate immune receptors Danger signal: signal released by injured or damaged tissues that trigger an innate immune response Toll-like receptor (TLR): membrane receptor involved in innate immune sensing

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Vertebrates have evolved two complementary systems to detect and clear pathogens: the innate and the adaptive immune systems. The innate immune system is the ﬁrst one to be activated by pathogens (1) and is usually sufﬁcient to clear the infection. However, when the innate immune system is overwhelmed, it triggers and directs the adaptive arm, thus activating speciﬁc B and T cells for pathogen clearance. Receptors expressed by B and T cells are generated through somatic gene rearrangement and hypermutation. This process enables the generation of a virtually inﬁnite repertoire of antigen receptors, allowing the adaptive immunity to speciﬁcally recognize any type of microorganism. In contrast, innate immunity is characterized by its ability to recognize a wide range of pathogens such as viruses, bacteria, and fungi, but through a limited number of germlineencoded receptors called pattern-recognition receptors (PRRs) (2, 3). PRRs are expressed by many cell types including macrophages, monocytes, dendritic cells (DCs), neutrophils, and epithelial cells, and they allow the early detection of pathogens directly at the site of infection. PRRs recognize conserved microbial signatures (4) termed pathogen-associated molecular patterns, or PAMPs (see below). Once activated, the innate immune system initiates the inﬂammatory response by secreting cytokines and chemokines, inducing the expression of adhesion and costimulatory molecules in order to recruit immune cells to the site of infection and to trigger the adaptive immune response. Pathogens can rapidly evolve and, in principle, could avoid detection by the innate immune system by simply altering the targeted PAMPs. By doing so, the pathogen would not only escape the recognition by the innate immune system but also avoid the adaptive immune response. However, the immune system has evolved to recognize PAMPs that are essential for the viability of microbes and are

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thus less prone to modiﬁcations. PAMPs can be of diverse origins; sugars, ﬂagellin, and the cell wall components peptidoglycan (PGN) and lipopolysaccharide (LPS) are all recognized by the innate immune system. The model proposed by Charles Janeway based on PAMP recognition is, however, too simplistic. Indeed, if PAMPs activate the immune pathway, how does the immune system distinguish pathogenic microorganisms from commensal and other non-pathogenic bacteria? To answer this question, Matzinger (5, 6) suggested that the activation of the innate immune system is not only based on the recognition of PAMPs but also relies on the presence of danger signals or danger-associated molecular patterns (DAMPs) released by injured cells. These two models seem completely opposed, but several reports now show activation of innate immunity by host molecules. Indeed among others, mammalian dsDNA (7) and uric acid crystals (8) activate an inﬂammatory response. The release of DAMPs is a common event, as tissue damage and cell lysis are often associated with infections and lead to the release of host molecules. Recognition of these DAMPs by the immune system not only allows the sensing of an ongoing infection and subsequent recruitment of more immune cells, but also can initiate the repair of the damaged tissue (9). It seems then that the innate immune pathway not only scans the cellular environment for signs of invading pathogens, but also recognizes the damage caused by them. PAMPs are recognized by PRRs that are either cytoplasmic, membrane-bound, or secreted; the most intensely studied are the Toll-like receptors (TLRs). These receptors are expressed by many cell types including mononuclear, endothelial, and epithelial cells. Once activated by PAMPs, the TLRs induce different signaling cascades depending on the adaptor protein, ultimately leading to the activation of the transcription factors NF-κB, AP-1, and interferon-regulatory factor (IRF)3. TLR activation results in the production of antimicrobial peptides, inﬂammatory cytokines and chemokines, tumor necrosis factor

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(TNF)-α, and costimulatory and adhesion molecules, as well as in the upregulation of major histocompatibility complexes (MHCs). As one given pathogen does not trigger only one speciﬁc TLR, but rather a set of TLRs leading to the expression of different proteins depending on the nature of the activated TLRs, the immune system is instructed on the type of the invading microorganism and mounts the most appropriate response to ﬁght it. Excellent reviews on the biology of TLRs have been published recently (10–12). TLRs are therefore not the focus of this review. Recently, two other families of PRRs were described: the NLRs (NOD-like receptors) and the RLHs (RIG-like helicases). Unlike TLRs, these families consist of soluble proteins that survey the cytoplasm for signs that advertise the presence of intracellular invaders. Two RNA helicases, namely RIG-I and MDA5 (13–15), were identiﬁed as cytoplasmic, viral RNA sensors. Upon viral stimulation of the two RLHs, NF-κB and IRF3/7 are activated and, in turn, induce the transcription of type I interferon (IFN). Based on the CARD (caspase recruitment domain) homology with RIG-I or MDA5, the CARD adaptor (Cardif, also known as IPS1, MAVS, or VISA) that induces IFN-β was identiﬁed (16–18). Both the CARD domain and the mitochondrial localization of Cardif are required to induce NF-κB and IRF3/7 activation (16, 18). Studies on TLR signaling pathways and the analysis of key TLR-deﬁcient mice revealed that TLRs could not be the only innate immune receptors responsible for cytokine production. Indeed, computational analysis of the genome identiﬁed the NLR proteins. NLR proteins are intracellular LRR (leucine-rich repeat)-containing proteins that resemble plant disease–resistance genes. The characterization of these NLRs has advanced greatly in recent years, underscoring their essential roles in innate immunity. In particular, cytoplasmic complexes called inﬂammasomes, in which the scaffolding and sensing proteins are members of the NLR family, have been found to be central platforms of innate immunity.

In this review, we examine the remarkably important and emerging functions of inﬂammasomes as guardians of the body. We begin by describing the general molecular nature of the inﬂammasome complexes and the known pathways that activate them. We then highlight the current understanding of the function of this pathway—its role in orchestrating host defenses and in the pathogenesis of inﬂammatory diseases.

THE NLR FAMILY In recent years, the central role of the NLR family has become increasingly appreciated (19, 20). NLRs form central molecular platforms that organize signaling complexes such as inﬂammasomes and NOD signalosomes. Most NLRs are expressed in the cytosol. Structurally, NLRs are multidomain proteins with a tripartite architecture containing a C-terminal region characterized by a series of LRRs, a central nucleotide domain termed the NACHT domain (also referred to as NOD domain), and an N-terminal effector domain (Figure 1). The LRR domain has been implicated in ligand sensing and autoregulation of NLRs, yet the precise mechanism of how NLR LRRs sense their ligands is largely unknown. The LRR is a widespread structural motif of 20–30 amino acids with a characteristic pattern rich in the hydrophobic amino acid leucine. LRR domains are formed by tandem repeats of a structural unit consisting of a β strand and an α helix, and are organized in such a way that all the β strands and the helices are parallel to the same axis, resulting in a nonglobular, horseshoe-shaped molecule with the curved β parallel sheet lining the inner circumference and the α helices lining the outer circumference (21). These modules are associated with a wide range of functions including a role as pathogen sensors in various innate immune receptors such as TLRs and NLRs. TLRs contain LRRs that recognize or sense the presence of a wide range of PAMPs including LPS, lipoproteins, ﬂagellin, and RNA from bacteria or viruses. They are believed to sense directly www.annualreviews.org • The Inflammasomes

NOD-like receptor (NLR): cytosolic protein involved in innate immune sensing RIG-like helicase (RLH): cytosolic helicase involved in innate immune sensing of nucleic acids Inflammasome: molecular complex involved in the activation of inﬂammatory caspases resulting in the processing of immature proIL-1β and proIL-18 into their mature forms NOD signalosome: complex that is assembled upon oligomerization of NOD1 or NOD2 that activates RIP2 and triggers NF-κB activation

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NOD-like receptors NALP1 CARD FIIND

NALP3 NALP4-14

NAIP

NOD1 NOD3 NOD4

NOD2

NLRX1/ NOD5

CIITA

IPAF

Plant NLR

LRR

WD40

NAD

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APAF1

NBARC

NACHT PYD

NALPs

AD

BIR

CARD

IPAF/NAIP

TIR

NODs

Figure 1 Domain organization of representative NOD-like receptors (NLRs). NLRs are characterized by three distinct domains: the ligand-sensing leucine-rich repeats (LRRs); the NACHT domain, which is responsible for the capacity of NLRs to oligomerize; and the effector domain, which can be a pyrin domain (PYD), CARD, or BIR domain. Most NLRs also contain a NACHT-associated domain (NAD) C-terminal of the NACHT domain. NLRs are divided into two large subfamilies: the 14 members of the PYD-containing NALP clan and the ﬁve members of the NODs and CIITA. IPAF and the BIR-containing NAIP form the remaining NLR members. For comparison, the structural organization of a plant NLR-like gene and APAF1 are shown. (Abbreviations: CARD, caspase recruitment domain; PYD, pyrin domain; FIIND, function to ﬁnd; NACHT, domain conserved in NAIP, CIITA, HET-E and TP1; NAD, NACHT-associated domain; BIR, baculovirus IAP repeat; AD, activation domain; NALP, NACHT, LRR, and PYD-containing protein; CIITA, MHC class II transcription activator; IPAF, ICE-protease-activating factor.)

Apoptosome: oligomeric structure that is assembled when APAF1 interacts with cytochrome c released from mitochondria; triggers apoptosis by activating caspase-9 STAND family of NTPases: Subfamily of AAA+ NTPases that includes NLRs AAA+ NTPases: superfamily of ATPases associated with a variety of cellular activities and characterized by their extended P-loop ATPase domain capable of forming donut-shaped oligomers

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or indirectly their activating PAMPs. Doublestranded RNA and lipopeptides have recently been shown to bind TLR3 and TLR1/TLR2 complexes, respectively, whereas LPS-induced TLR4 activation is presumed to be indirect and to involve binding of LPS to MD2 (22, 23). In contrast, no experimental data have convincingly demonstrated a direct interaction between the LRRs of NLRs and their respective activators, suggesting that sensing of pathogens and other signals by NLRs may be indirect. The NACHT domain, which is central to all NLRs, has similarity to the NB-ARC motif of the apoptotic mediator APAF1. APAF1 performs its cellular function through the formation of a caspase-9 activating, heptameric platform termed an apoptosome. The NBARC domain is responsible for dATP/ ATP-dependent oligomerization of APAF1 upon cytochrome c binding, a process that initiates apoptosis. Both the NACHT and NB-ARC Martinon

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domains belong to the recently deﬁned STAND family of NTPases (24). Oligomerization has been reported for several STAND family proteins, as well as in other related AAA+ NTPases. Similarly, it is believed that the crucial step in NLR activation lies in the oligomerization of the NACHT domain, thereby forming active, high molecular weight complexes that characterize inﬂammasomes and NOD signalosomes (25, 26). NLR subfamilies differ in their N-terminal effector domains, which mediate signal transduction to downstream targets, leading to activation of inﬂammatory caspases by inﬂammasomes or NF-κB by NOD signalosomes. The vast majority of NLRs harbor a death-fold domain at the N terminus, which is either a CARD or a pyrin domain (PYD) (Figure 1, Table 1). The death-fold domain superfamily was originally described in proapoptotic signaling pathways and, in addition to CARD and

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Table 1

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subfamilya

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NOD-like receptors (NLRs)

NLR NALPs

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Commonly used nomenclature human

mouse

NALP1

Other names and aliases DEFCAP, NAC, CARD7, NLRP1

Structure PYD-NACHT-NAD-LRRFIIND-CARD

NALP1a

NACHT-NAD-LRR-FIINDCARD

NALP1a

NACHT-NAD-LRR-FIINDCARD

NALP1a

NACHT-NAD-LRR-FIINDCARD

NALP2

Pypaf2, NBS1, PAN1, NLRP2 NALP2

NALP3 (Cryopyrin)

PYD-NACHT-NAD-LRR PYD-NACHT-NAD-LRR

Pypaf1, CIAS1, NLRP3

PYD-NACHT-NAD-LRR

Cias1, Pypaf1, Mmig1

PYD-NACHT-NAD-LRR

Pypaf4, PAN2, RNH2, NLRP4

PYD-NACHT-NAD-LRR

NALP4a

Nalp-η, NALP9D

PYD-NACHT-NAD-LRR

NALP4b

Nalp-γ, NALP9E

PYD-NACHT-NAD-LRR

NALP4c

Nalp-α, Rnh2

PYD-NACHT-NAD-LRR

NALP4d

Nalp-β

PYD-NACHT-NAD-LRR

NALP4e

Nalp-ε

PYD-NACHT-NAD-LRR

NALP4f

Nalp-κ, NALP9F

PYD-NACHT-NAD-LRR

NALP3 NALP4

NALP4g NALP5 NALP5 NALP6

PYD-NACHT-NAD-LRR Pypaf8, Mater, PAN11, NLRP5

PYD-NACHT-NAD-LRR

mater, Op1

NACHT-NAD-LRR

Pypaf5, PAN3, NLRP6

PYD-NACHT-NAD-LRR

NALP6

PYD-NACHT-NAD-LRR

NALP7

Pypaf3, NOD12, NLRP7

PYD-NACHT-NAD-LRR

NALP8

PAN4, NOD16, NLRP8

PYD-NACHT-NAD-LRR

NALP9

NOD6, NLRP9

PYD-NACHT-NAD-LRR

NALP9a

Nalp-θ

PYD-NACHT-NAD-LRR

NALP9b

Nalp-δ

PYD-NACHT-NAD-LRR

NALP9c

Nalp-ζ

PYD-NACHT-NAD-LRR

PAN5, NOD8, Pynod, NLRP10

PYD-NACHT-NAD

Pynod

PYD-NACHT-NAD

NALP11

Pypaf6, NOD17, NLRP11

PYD-NACHT-NAD-LRR

NALP12

Pypaf7, Monarch1, RNO2, PAN6, NLRP12

PYD-NACHT-NAD-LRR

NALP10 NALP10

NALP12

PYD-NACHT-NAD-LRR

NALP13

NOD14, NLRP13

PYD-NACHT-NAD-LRR

NALP14

NOD5, NLRP14

PYD-NACHT-NAD-LRR

Nalp-ι, GC-LRR,

PYD-NACHT-NAD-LRR

NALP14

(Continued )

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(Continued )

NLR

Commonly used nomenclature

subfamilya IPAF/NAIP

human

mouse

Ipaf

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Structure

CARD12, CLAN, NLRC4

CARD-NACHT-LRR

Ipaf

CARD12, CLAN

CARD-NACHT-LRR

BIRC1

BIR3x-NACHT-LRR

NAIPa

Birc1a, NAIP1

BIR3x-NACHT-LRR

NAIPb

Birc1b, Naip-rs6, NAIP2

BIR3x-NACHT-LRR

NAIPc

Birc1c, Naip-rs5, NAIP3

BIR3x-NACHT-LRR

NAIPd

Birc1d, Naip-rs2, NAIP4

BIR3x-NACHT-LRR

NAIPe

Birc1e, Naip-rs3, NAIP5

BIR3x-NACHT-LRR

NAIPf

Birc1f, Naip-rs4, NAIP6

BIR3x-NACHT-LRR

NAIPg

Birc1g, NAIP7

BIR3x-NACHT-LRR

CARD4, CLR7.1

CARD-NACHT-NAD-LRR

CARD4

CARD-NACHT-NAD-LRR

CARD15, CD, BLAU, IBD1, PSORAS1, CLR16.3

CARD2x-NACHT-NAD-LRR

CARD15

CARD2x-NACHT-NAD-LRR

CLR16.2, NLRC3

CARD-NACHT-NAD-LRR

NAIP

NODs

Other names and aliases

Ipaf

NOD1 NOD1 NOD2 NOD2 NOD3 NOD3 NOD4

CARD-NACHT-NAD-LRR NOD27, CLR19.3, NLRC5

NOD4 NOD5 (NLRX1) NOD5(NLRX1) CIITA CIITA

CARD-NACHT-LRR CARD-NACHT-LRR

NOD9, CLR11.3, NLRX1

X-NACHT-LRR

BC034204

X-NACHT-LRR

MHC2TA, C2TA

(CARD)-AD-NACHT-NAD-LRR

C2TA

(CARD)-AD-NACHT-NAD-LRR

a NLRs are divided into two large subfamilies: the 14 members of the PYD-containing NALP clan and the ﬁve members of the NODs and CIITA. IPAF and the BIR-containing NAIP form the remaining NLR members.

PYD, includes the death domain (DD) and the death effector domain (DED) (27). The deathfold domain is characterized by six α helices that are tightly packed in a Greek key fold and form trimers or dimers with other members of the same subfamily. In most known cases a DD interacts with a DD, a CARD with a CARD, a DED with a DED, and a PYD with a PYD. These four domains are frequently found in pathways that lead to the activation of caspases or that activate the transcription factor NF-κB. The observation that every death-foldcontaining family member is able to interact with another partner harboring the same domain was instrumental in the identiﬁcation of major signaling pathways involved in apoptosis and immunity. The death fold acts as a molec234

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ular velcro that bridges receptors to adaptors and effector proteins. Similarly, the N-terminal PYD or CARD present in NLRs recruits PYDor CARD-containing molecules to signaling platforms.

NLR Subfamilies Structurally and functionally, NLRs are divided into subfamilies. In this review, we use the nomenclature that is most commonly used and highlight the various NLR subfamilies. An overview of the family members and their historical or alternative nomenclature is listed in Table 1. The NLR family emerged almost ten years ago with the cloning of NOD1 and NOD2 and the subsequent identiﬁcation of

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the PYD domain (28–31). Based on phylogenetic distribution, we distinguish three different NLR subfamilies, all characterized by a speciﬁc molecular structure (29, 32) (Figure 1). NALPs represent the largest NLR subfamily and have 14 genes identiﬁed in humans (29) (Table 1). Some of them, such as NALP1, NALP2, and NALP3, were shown to be the central scaffold of caspase-1-activating complexes known as inﬂammasomes. NALP proteins harbor a NACHT and a LRR and are characterized by an N-terminal PYD domain. Interestingly, the LRR region within NALPs is organized in the genome in a very conserved and precise manner. NALP LRR regions are formed by tandem repeats of exons of exactly 171 nucleotides and are deﬁned completely by a preserved intron-exon structure. Each exon encodes one central LRR and two halves of the neighboring LRRs (33). The phasing and position of the introns are consistent with rapid and efﬁcient exon ampliﬁcation during evolution. This particular modular organization possibly allows extensive alternative splicing of the LRR region without disturbing the three-dimensional fold of the region and, as a consequence, maximizing variability in the ligand-sensing unit. Evolutionarily, IPAF and NAIP group together and are well separated from other NLRs. IPAF contains an N-terminal CARD, whereas NAIP has three BIR domains, which are often found in proteins involved in apoptosis such as the IAP family of caspase inhibitors. Both IPAF and NAIP are involved in the formation of inﬂammasomes, either alone or in combination with one another. The third class of NLRs includes the remaining CARD-containing NLRs such as NOD1, NOD2, NOD4, and CIITA. This clade also contains a slightly separated group with NOD3 and NOD5/NLRX1 (19, 32). NOD5/NLRX1 does not have a deﬁned Nterminal domain, whereas at least one splice variant of CIITA has been reported to harbor a CARD. NOD1 and NOD2 activate the transcription factor NF-κB, a major regulator of inﬂammatory responses. CIITA regulates the

transcriptional regulation of genes encoding MHC II (34). NOD5/NLRX1 is recruited to the outer membrane of mitochondria (35). The function of NOD5/NLRX1 is still controversial. One study suggested that NOD5/NLRX1 interacts and negatively regulates the antiviral pathway involving the CARD-containing adapter MAVS/CARDIF/IPS-1/VISA (36), whereas another study proposed that NLRX1 promotes the production of reactive oxygen species (ROS) (37). NOD3 and NOD4 have no identiﬁed functions yet.

NLR Expression Patterns and Gene Regulation Expression patterns of most NLRs in various cell populations and tissues have not yet been studied in detail. Nevertheless, the importance of NLRs in defense strategies of the body is supported by the fact that several NLRs are expressed in cells and tissues that have a role in immunity such as phagocytes. Some NLRs are also critical in epithelial cells, which form the ﬁrst barrier of defense against bacteria in human tissues and express NOD1, NOD2, NALP3, and NAIP (20, 38, 39). NALP1 is widely expressed, whereas NALP3 is found mainly in immune cells, epithelial cells, and osteoblasts (40). NAIP and IPAF are expressed in the brain and in macrophages and macrophage-rich tissues such as spleen, lung, and liver (41, 42). The expression of some NLRs seems to be highly restricted; for example, NALP5, NALP8, NALP4, NALP7, NALP10, and NALP11 are mainly expressed in germ cells and preimplantation embryos (43). Most NLRs may be induced by other branches of the innate immunity as part of a regulatory network. TLR stimulation, for example, increases the expression of NLRs, such as NOD1, NOD2, and NALP3, possibly reﬂecting enhancement of NLR responses after TLR stimulation (44).

NLRs: Lessons from Evolution Ever since the discovery of NLRs in mammals, the similarity of these genes with a family of www.annualreviews.org • The Inflammasomes

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plant genes involved in immune defenses has cross-fertilized NLR research (28). The plant genes, known as R-genes (R for resistance), are crucial for the immune defense of plants against bacteria, fungi, viruses, and other pathogens. The largest known class of R-genes structurally resembles mammalian NLRs. They have a C-terminal LRR, a central oligomerization module related to the NB-ARC subtype of STAND domains, and an N-terminal effector domain that is generally either a coiled-coil domain or a TIR domain (45). The TIR domain is a well-known recruitment domain involved in the TLR and IL-1R family of immune mediators. There are more than 150 NLR-like R-genes in Arabidopsis (46). Many of these genes have been implicated in sensing pathogens. The vast repertoire of NLRs in plants is believed to be the result of a complex host-pathogen race that promoted the evolution of speciﬁc NLR genes that genetically interact with speciﬁc avirulence genes from distinct pathogens (47). Although plant NLR-like proteins are functionally and structurally similar to mammalian NLRs, there is no evidence for a common evolutionary origin. It is more likely that these innate immune sensors are an example of convergent evolution. The NLR structure (based on a C-terminal LRR sensing unit, a STAND oligomerization module, and an N-terminal recruitment domain) probably originated independently through evolution, which emphasizes key molecular constraints required to design successful innate immune systems (48, 49). Supporting the convergent evolution idea, no NLR-like proteins have been found in insects. In the animal kingdom, the ﬁrst evolutionarily conserved NLRs are observed in the echinoderm sea urchin. The observation by Mechnikov of phagocytosis in echinoderms is considered to be a critically deﬁning moment that led to the original concept of immune defenses and, more speciﬁcally, innate immunity (50). More than one century after these experiments, the sequencing of the sea urchin genome provided us with another appreciation of the complexity and general conservation of innate immune system mechanisms

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(51, 52). The sea urchin genome contains 222 TLRs and 203 NLRs. Interestingly, most of the NLRs are expressed in the gut, suggesting that gut-related immunity is a likely driving force behind the expansion of this family in echinoderms (51). These ﬁndings highlight NLRs as well as TLRs as evolutionarily important immune genes that preceded the acquisition of the adaptive immune system in vertebrates. In nonmammalian vertebrates such as zebraﬁsh, three distinct families of NLRs have been identiﬁed (53, 54). The ﬁrst subfamily is related to the NOD subclass of mammalian NLRs and contains orthologs of NOD1, NOD2, NOD3, and NOD4. The second subfamily resembles the NALPs and contains at least six genes. Finally, the last subfamily has the highest similarity with the NACHT domain of NOD3 and has expanded in teleost ﬁsh into several hundreds of predicted genes. Most of these genes encode a PYD domain at the N terminus (similar to the one that characterizes NALPs). Interestingly, some of these NLRs have a C-terminal extension following the LRR that contains a PRY-SPRY domain. The PRY-SPRY domain is also found in human Pyrin, a PYDcontaining regulator of the inﬂammasome (see below). The precise role of the PRY-SPRY in Pyrin or in nonmammalian, vertebrate NLRs is unknown; however, this ﬁnding further highlights the role of Pyrin and possibly other PRY-SPRY-containing proteins in the regulation of NLRs (55, 56). Extensive diversiﬁcation of the NLRs occurred also within the mammalian lineage; this is particularly true for NALPs that mainly evolved through gene duplication events. Some NALPs such as NALP2 and NALP7 in humans are clearly paralogs, whereas others, such as NALP4 and NALP9, expanded only in the mouse (Table 1). A similar evolutionary trend was followed by NAIP in mice, where the locus expanded to seven NAIP genes (33). All these observations indicate that the NLR repertoire within a species, and across vertebrates, is large. Some of these NLRs have undergone lineage-speciﬁc ampliﬁcation, such as NOD3-related NLRs in zebraﬁsh or NALPs in

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mammals. Genes involved in interactions with pathogens are likely to diversify by undergoing lineage-speciﬁc expansion (57), reﬂecting the adaptive dynamics of a species to new environments with emerging pathogens.

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NOD Signalosomes NOD1 was one of the ﬁrst NLRs to be described (58, 59). Both NOD1 and NOD2, once activated, recruit and engage the kinase RIP2 through CARD-CARD interactions. Oligomerization of RIP2 in the NOD signalosome results in the activation of the transcription factor NF-κB (60). Recently, the CARD-containing protein CARD9 has also been found to interact with NOD2 and RIP2 and to be involved in the activation of JNK and p38 by NOD2 (61). Both NOD1 and NOD2 detect muropeptides released from bacterial PGNs. NOD1 and NOD2 sense distinct PGN structures. Whereas NOD2 detects muramyl dipeptides (MDP), the largest motif common to Gram-negative and Gram-positive bacteria, NOD1 detects meso-diaminopimelic acid (meso-DAP), which is mainly found in Gram-negative bacteria (62, 63). Both PGNs are degradation products of bacterial cell wall components released by intracellular or phagocytosed bacteria. Pathogens that do not reach the intracellular compartment of the host cell may use specialized secretion systems to inject the PGN fragment into the host cytosol. Helicobacter pylori, for example, elicits NOD1 activation by delivering PGN fragments into the host cell trough a mechanism that requires a functional type IV secretion system (64). NOD1 and NOD2 are crucial innate immune receptors in epithelial cells, where they are important to control infection via the gastro-intestinal route, for example, by H. pylori and Listeria monocytogenes (64, 65). Importantly, mutations in NOD2 have been associated with Crohn’s disease, a form of inﬂammatory bowel disease. Most NOD2 mutations in these patients affect the LRR region of NOD2 and are believed to disrupt the protein’s ability to sense bacteria (66). This probably confers a

loss of tolerance toward commensal bacteria or allows the proliferation of pathogenic bacteria in the gut. Gain-of-function mutations in the NACHT domain of NOD2 have been shown to be responsible for Blau syndrome, a rare autoinﬂammatory disorder starting in childhood and characterized by skin rashes, uveitis, and joint inﬂammation (67). More recently, NOD2 has been suggested to play a role in the activation of some types of inﬂammasomes (68, 69). These ﬁndings are discussed in more detail below.

INFLAMMASOMES The term inﬂammasome was coined to describe a high molecular weight complex that activates inﬂammatory caspases and the cytokine IL1β (70). Inﬂammasome is assembled from the word inﬂammation—to reﬂect the function of this complex—and the sufﬁx “some” from the Greek soma meaning body, which is frequently used in cell biology to deﬁne entities or molecular complexes such as proteasome, liposome, ribosome, etc. Importantly, the term inﬂammasome was also chosen to highlight structural and functional similarities with another well-known caspase-activating complex, the apoptosome, a molecular platform that triggers apoptosis (71).

Inflammasomes Lead to Activation of Inflammatory Caspases Caspases are proteases produced in cells as catalytically inactive zymogens and usually undergo proteolytic processing during activation (72). The subset of caspases that cleave substrates during apoptosis are known as executioner caspases. These executioner caspases (caspase-3, -6, and -7 in mammals) are generally activated by the initiator caspases such as caspase-8, caspase-10, caspase-2, or caspase-9. Initiator caspases harbor an N-terminal deathfold domain (CARD or DED in mammals) that is required for the activation of their C-terminal catalytic region. The mechanism of activation of initiator caspases depends on the engagement and activation of platforms such as the www.annualreviews.org • The Inflammasomes

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INFLAMMATORY CASPASES In mammals, the inﬂammatory caspases include human and murine caspase-1, human and murine caspase-12, murine caspase-11, and the two caspase-1-related human caspases, caspase-4 and caspase-5 (33, 204). Inﬂammatory caspases in mammals have a CARD domain followed by a domain containing the catalytic residue cysteine. These caspases are termed inﬂammatory because the main substrates of caspase-1 identiﬁed to date are cytokines (such as IL-1β, IL-18, and possibly IL-33) that are crucial mediators of the inﬂammatory response. Caspase-1 was the ﬁrst caspase to be discovered in mammals, but only recently have the pathways (inﬂammasomes) leading to its activation been discovered (70). Although both human caspase-5 and mouse caspase-11 have been associated with caspase-1 activation, no speciﬁc substrates have been described for them. Caspase-5 is recruited by the C-terminal CARD of NALP1, suggesting that it may be involved in the activity of inﬂammasomes harboring NALP1. Caspase-12 and caspase-4 are activated by endoplasmic reticulum (ER) stress, an adaptive response that copes with protein overload in the ER. However, the function of these inﬂammatory caspases upon ER stress is unclear (75). Moreover, caspase-12 appears to be an inhibitor of the inﬂammasome, possibly by interfering with caspase-1 activation, a process that has been associated with susceptibility to sepsis (209).

death-inducing signaling complex (DISC) for caspase-8 and -10, the PIDDosome for caspase2, and the apoptosome for caspase-9 (73). These platforms integrate cellular signals, recruit initiator caspases via their death-fold domain, and promote dimerization of the caspases, which all lead to the formation of an active enzyme proﬁcient enough to initiate speciﬁc signaling cascades (74). Inﬂammasomes activate a class of caspases known as inﬂammatory caspases (25, 75) (see side bar, Inﬂammatory Caspases). An increasing number of studies highlight the importance and complexity of inﬂammatory caspase activation.

Prototypical Inflammasomes Although the biochemistry and diversity of inﬂammasomes are still poorly understood, we

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distinguish three prototypes of inﬂammasomes: The NALP1 inﬂammasome, the NALP3 inﬂammasome, and the IPAF inﬂammasome. For several NALPs, there is evidence for their roles as scaffolding proteins of inﬂammasomes (76). It is assumed that the PYD of NALPs interacts and recruits the adaptor ASC (apoptosisassociated speck-like protein containing a caspase recruitment domain) via PYD-PYD interaction (Figure 2). ASC contains an Nterminal PYD and a C-terminal CARD and is an essential component for inﬂammasome formation (70, 77). The CARD domain within ASC binds and recruits caspase-1 to the inﬂammasome. NALP1 has a C-terminal extension that harbors a CARD, which was shown to recruit caspase-5 (70) or a second caspase-1 (26). Other NALPs do not have the NALP1 C-terminal extension; instead, CARDINAL (a protein very similar to the NALP1 C terminus) interacts with other inﬂammasomes such as the NALP3 or NALP2 inﬂammasome (78). Neither CARDINAL nor NALP1 are highly conserved in mice. The NALP1 locus in mice contains three paralogs that have no functional PYD, whereas CARDINAL is not present in the mouse genome at all. It is therefore possible that NALP1 genes fulﬁll CARDINAL functions in mice. IPAF has an N-terminal CARD and directly recruits caspase-1 (41) (Figure 2). Basic mechanisms implicated in the activation of NLRs are also involved in inﬂammasome assembly. IPAF and NALP3 selectively bind ATP/dATP, and nucleotide binding is necessary for oligomerization of the NACHT domain (79, 80). Both IPAF and NALP3 bind SGT1 and HSP90 (81, 82), two proteins whose plant orthologs were previously shown to regulate and interact with plant NLRs. In mammals, as in plants, the activity of the HSP90-SGT1 complex is essential for NALP3 activation, probably for keeping the inﬂammasome inactive but competent for activation. HSP90 has been suggested to act upstream of NALP1 inﬂammasome activation by anthrax lethal toxin (83).

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Although the IPAF, NALP3, and NALP1 inﬂammasomes form prototypical inﬂammasome complexes, recent genetic evidence suggests that other NLRs, such as NAIP or NOD2, may be involved in forming inﬂammasomes or in modulating their activity. Exactly how and at what step these proteins are connected to the formation of inﬂammasomes is unknown. It is also unclear whether multiple NLRs may assemble as heterocomplexes to form competent inﬂammasomes (84, 85). Thus, the complexity and diversity of inﬂammasomes may turn out to be considerable (86).

NALP3 inflammasome

INFLAMMASOMES AS SENSORS OF DANGER

Inflammasome

Although inﬂammasomes are emerging more and more as key players in inﬂammatory and immune responses, a growing number of studies reveal their function in the sensing of a controversial signal: danger. It is well known that a major function of the immune system is to differentiate self from nonself—to respond to self with tolerance and to mount an immune response against nonself. Innate immunity, for example, detects PAMPs from microbes, including pathogens. There is also evidence that innate immunity is able to discriminate pathogenic microbes from nonpathogenic microbes or commensals; but this raises the question of how the immune system interprets the microbial environment allowing the discrimination between nonpathogenic and pathogenic microbes (6). Matzinger and colleagues (87), to account for some unsolved questions of the self-from-nonself model, promoted an alternative hypothesis (the danger hypothesis), suggesting that it is the presentation of an antigen in the context of a danger signal that triggers an efﬁcient immune response, not simply the foreignness of the antigen. This led to multiple studies that identiﬁed molecules and signals released by damaged or stressed tissues and that trigger or modulate the immune response (88, 89). Both the self-from-nonself model and the danger model may synergize to determine the quality and extent of the in-

NALP3

IPAF inflammasome NALP3

IPAF

ASC Caspase-1

IPAF

Caspase-1 CARD

CARD

ASC Caspase-1

Caspase-1

ProIL-1β IL-1β

Figure 2 Structural organization of the typical NALP3 and IPAF inﬂammasomes. The core structure of the NALP3 inﬂammasome is formed by NALP3, the adaptor ASC, and caspase-1. PYD-PYD and CARD-CARD homotypic interactions are crucial for the recruitment and activation of either the adaptor ASC or the inﬂammatory caspases (left panel ). IPAF recruits caspase-1 directly via CARD-CARD interactions (right panel ). The leucine-rich repeats of NALP3 or IPAF are proposed to sense the activating signals leading to the oligomerization of the NACHT domain and initiating the formation of the donut-shaped inﬂammasome. Based on the structure of the apoptosome, the caspases and IL-1β processing activity most likely face toward the inside of the donut (lower panel ).

nate immune response (90). Although the danger hypothesis was ﬁrst proposed in mammals, evidence for such a type of immune response was ﬁrst documented in plants. Plant immunity relies on an effector-triggered immunity that mainly detects pathogen-driven modiﬁcations, stress, or danger signals in the infected host cell (91). Similarly, the mammalian NLR NALP3 was found to be involved in sensing danger signals. www.annualreviews.org • The Inflammasomes

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Cell Disruption Activates the Inflammasome

ings demonstrating that various danger signals and stimuli that activate the NALP3 inﬂammasome can trigger potassium efﬂux, thereby lowering the cytosolic potassium concentration of stimulated cells (93, 95) (Figure 3).

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Early observations that cell lysis in a hypotonic buffer can lead to the processing of proIL-1β provided an important model system that permitted the identiﬁcation of caspase-1 as the protease responsible for the processing of proIL-1β (92). Using this cell-free system, investigators noticed that caspase-1 activation was restricted to a few cell types, such as the monocytic cell line THP-1. A similar assay subsequently allowed the ﬁrst biochemical identiﬁcation and characterization of an inﬂammasome complex (70). After disruption of cellular integrity, the inﬂammasome is spontaneously formed. Assembly can be inhibited by complementing the cell extracts with potassium levels that mimic normal levels found in the cytosol of healthy cells (over 70 mM) (70, 93, 94). The observation that subphysiological amounts of potassium are required for spontaneous inﬂammasome formation suggests that the inﬂammasome may sense drops in potassium levels. This possibility is supported by recent ﬁnd-

Bacterial toxins A. hydrophila (Aerolysin) Dinoﬂagellates (Maitotoxin) L. monocytogenes (LLO) S. aureus

Extracellular ATP serves as a danger signal that alerts the immune system by binding to the purinoreceptor P2X7, thereby activating NALP3 and caspase-1 (8, 96–98). Although ATP is emerging as an important modulator of inﬂammation (99), the critical role of extracellular ATP as a danger signal is unclear. The amount of extracellular ATP that is required to activate macrophages in vitro is relatively high (2 to 5 mM), and, moreover, in vivo most of the extracellular ATP may be rapidly hydrolyzed by ectonucleotidases (100). ATP is released from cells as a consequence of cell damage and/or cellular stress. In endothelial as well as in epithelial cells, ATP release is triggered by nonlytic, mechanical stimuli as diverse as

K+ eﬄux ? ROS

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PAMPs Viral DNA MDP

Sensing Extracellular ATP

NOD2, NALP1?

Danger signals MSU CPPD Alum Asbestos

Large particles

ATP Skin irritants UV

NALP3 inflammasome

Figure 3 Multiple NALP3 inﬂammasome activators trigger cellular signals, such as potassium efﬂux and reactive oxygen species, that eventually activate an inﬂammasome dependent on caspase-1, ASC, and NALP3. Note that muramyl dipeptide (MDP) activation of caspase-1 may also require the NOD-like receptors NOD2 and NALP1. (Abbreviations: CPPD, calcium pyrophosphate dihydrate crystals; MSU, monosodium urate crystals; PAMP, pathogen-associated molecular pattern; UV, ultraviolet light.) 240

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compression, hydrostatic pressure changes, and hypotonic shock (101). ATP release can also occur through secretory organelles that store large amounts of ATP (102). Adrenal medullary chromafﬁn granules, which may be released upon physical or psychological stress, have concentrations of ATP around 100 mM, whereas platelet-dense granules contain concentrations of ATP that can reach 500 mM (100 times more than the cytosolic concentration). Similarly, secretion of insulin-containing granules from pancreatic β cells releases the ATP pool stored in the granules (103). ATP can also be released from microbial ﬂora and pathogens. Salivary histatins, for example, trigger ATP efﬂux from Candida albicans, increasing extracellular ATP, a mechanism that may contribute to the antifungal properties of these proteins (104). Exposure of cells to extracellular ATP activates caspase-1 (105). Several studies have demonstrated that ATP-induced caspase-1 activation and subsequent IL-1β maturation requires activation of the purinoreceptor P2X7 in combination with another type of channel, the pannexin-1 channel (106). Interestingly, pannexin-1, besides its role as a gap junction protein, can act as a speciﬁc ATP release channel (107). This suggests an amplifying mechanism for P2X7-mediated inﬂammasome activation via pannexin-1, which is indeed observed, at least in vitro (108). The generation of ASC-deﬁcient mice demonstrated that ATP-mediated caspase-1 activation requires ASC and it was therefore probably dependent on the activation of a NALP protein (77, 109). This hypothesis was conﬁrmed in studies using NALP3-deﬁcient mice (8, 96–98), demonstrating that extracellular ATP can act as a danger signal to activate the NALP3 inﬂammasome and promote caspase-1 activation and IL-1β maturation. Although extracellular ATP has been shown to be involved in inﬂammatory conditions, as in asthmatic airway inﬂammation (110), the physiological signiﬁcance of extracellular ATP-mediated NALP3 inﬂammasome activation still remains to be demonstrated in vivo.

Uric Acid: A Danger Signal Involved in Gout In addition to ATP, cells release other danger signals to activate the immune system. In a seminal paper, the Rock laboratory (111) puriﬁed, from the supernatant of dying cells, a low molecular fraction that could trigger adjuvanticity in vivo and identiﬁed uric acid as the active compound of that fraction. Uric acid is the end product of the cellular catabolism of purines and is present at near saturating amounts in body ﬂuids and at much higher concentration in the cytosol of healthy cells. It is believed that extracellular uric acid coming in contact with the high levels of free sodium present in the extracellular environment nucleates and forms monosodium urate (MSU) crystals. MSU is considered to be the biologically active structure that is responsible for the adjuvantic effect of uric acid. Therefore, formation of this danger signal is the result of a multistep process that starts with the release of uric acid. The biological activity of MSU, including its adjuvanticity, depends on the activation of the NALP3 inﬂammasome and the production of IL-1, but not on TLRs (8, 112, 113). MSU stimulates the NALP3 inﬂammasome to produce active IL-1β (8), and macrophages from mice deﬁcient in components of the inﬂammasome, such as caspase-1, NALP3, and ASC, have a highly reduced crystal-induced IL-1β activation capacity. The in vivo relevance of uric acid signals has been addressed in a follow-up study by Rock and colleagues (114), who showed that elimination of uric acid reduced the generation of cytotoxic T cells to an antigen in transplanted syngeneic cells and the proliferation of autoreactive T cells in a transgenic diabetes model. It has also been suggested that erythrocytes infected with Plasmodium parasites accumulate high levels of the uric acid precursor hypoxanthine, which is released and converted into uric acid upon rupture of the erythrocytes, a process that results in inﬂammation (115). Uric acid release also occurs in DCs incubated in the presence of alum (aluminum hydroxide),

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Adjuvant: substance that enhances the capacity of an antigen to stimulate the immune system

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which is the most widely used adjuvant in human vaccines (116). Interestingly, alum was recently found to be a direct inﬂammasome activator (113, 117–119) (see below). High levels of circulating uric acid (hyperuricemia) has been associated with various inﬂammatory diseases, including multiple sclerosis, hypertension, and cardiovascular diseases (120). Hyperuricemia and MSU formation are strongly linked to gout. The autoinﬂammatory disease gout is characterized by arthropathies generated by the inﬂammatory reaction to MSU in the joints and periarticular tissues (121). In a model of MSU crystalinduced peritonitis in mice, impaired inﬂammation is found in inﬂammasome-deﬁcient mice or mice deﬁcient in the IL-1 receptor (IL-1R) (8, 112), suggesting that the inﬂammatory response in gout is dependent on the inﬂammasome. The importance of IL-1 in the pathology of gout is supported by promising preliminary clinical trials in patients with acute gout. Patients responded positively to the injection of the IL-1R antagonist IL-1ra (122, 123). Similarly, patients with pseudogout, an inﬂammatory disease caused by the deposition of calcium pyrophosphate dihydrate crystals (CPPD), another type of pathogenic microcrystal that activates the NALP3 inﬂammasome (8), responded well to treatment with the IL-1ra (124).

Silica and Asbestos and Inflammation in the Lung Alveolar macrophages reside in the respiratory surfaces, one of the major boundaries between the body and the outside world. These cells are phagocytes that play an important role in host defenses against microorganisms and remove particles such as dust. Silica and asbestos dust are particularly strong inﬂammation inducers in the lungs (125). Macrophages can dissolve MSU, but are not able to efﬁciently eliminate microparticles of silica and asbestos. Inhaling ﬁnely divided crystalline silica or asbestos dust in very small quantities over time can lead to inﬂammatory conditions known as silicosis and asbestosis, respectively (126). As the dust be242

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comes lodged in the lungs, continuous irritation ensues, resulting in chronic inﬂammation that favors the development of cancer. This, in particular with asbestos, is associated with the development of malignant mesotheliomas. Similar to what was found for MSU, asbestos and silica microparticles activate the NALP3 inﬂammasome (127–129). It is signiﬁcant to note that pulmonary inﬂammation is greatly reduced in NALP3-deﬁcient mice after in vivo inhalation of asbestos or silica (127, 128).

Aluminum Particles: An Inflammasome-Dependent Adjuvant Vaccine adjuvants are exogenic preparations that boost the immune response to achieve protective immunity. Most adjuvants activate innate immune receptors such as TLRs (130). More recently, NLRs and inﬂammasomes were found to respond to speciﬁc adjuvants. Alum has been the most widely used adjuvant in human vaccination for more than half a century (131). Among the early observations on the adjuvantic effect of alum was that immunization with this adjuvant led to an increase in antigen-induced T cell proliferation, apparently resulting from the augmented production of IL-1 (132). AntiIL-1 antibodies are able to inhibit an antigenspeciﬁc T cell proliferative response after immunization with alum adjuvant, but not with Freund’s complete adjuvant (133). The ability of alum to activate caspase-1 and produce active IL-1β and IL-18 was demonstrated in vitro (134). Alum-induced caspase-1 activation depends on the NALP3 inﬂammasome (113, 117–119). Mice deﬁcient in NALP3, ASC, or caspase-1 fail to mount a signiﬁcant antibody response to antigen immunization with alum adjuvants (113, 117, 119), conﬁrming that the NALP3 inﬂammasome is a key player that links the innate immune system with the adaptive immune system.

Inflammasomes and Inflammation in the Skin The skin is the body’s ﬁrst line of defense against external threats and serves as an

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effective barrier against ordinary environmental intrusions. As such, the skin is often damaged by various insults and proﬁcient in mounting efﬁcient immune responses. Ultraviolet irradiation, for example, was recently shown to activate the NALP3 inﬂammasome and promote IL-1β maturation in keratinocytes (135). A role of the inﬂammasome in the skin was also found in contact hypersensitivity, an inﬂammatory disease caused by irritant chemicals that penetrate the skin surface and that induce a T cell–mediated immune response (136). This response is divided into two phases. The ﬁrst is a sensitization phase in which the sensitizing chemical acts both as an adjuvant and as a foreign hapten. Uptake of the chemical by skin-resident antigen-presenting cells (APCs) and their migration to draining lymph nodes ensues, promoting T cell priming. Reexposure to the chemical deﬁnes a second phase, also known as the elicitation phase. Here, challenge with the corresponding antigen triggers the activation of primed T cells. The innate immune module of the sensitization phase depends on the presence of functional caspase-1, IL-1β, and IL-18 (137–140), suggesting a potential involvement of the inﬂammasome. The role of the inﬂammasome was conﬁrmed in ASC- and NALP3-deﬁcient mice that showed an impaired contact hypersensitivity response to the irritants trinitrophenylchloride (TNPCl) (97), trinitrochlorobenzene (TNCB), and dinitroﬂuorobenzene (DNFB) (141). In these mice, transfer of primed T cells results in a normal contact hypersensitivity, suggesting that only the sensitization phase requires NALP3 and ASC. Interestingly, DNFB promotes the release of IL-1β in a caspase-1-dependent manner in primary keratinocytes as well as in a DC line, suggesting that the inﬂammasome may either detect such compounds directly or, more likely, may detect some danger signals released or produced by these irritants (142). On the contrary, dinitrothiocanobenzene (DNTB) is unable to activate the inﬂammasome and, as such, fails to induce a strong immune response in vivo despite the fact that DNTB is competent in inducing the effector phase if the re-

lated antigen DNFB (that activates the inﬂammasome) was used for sensitization (143). Together, these ﬁndings suggest that the inﬂammasome can bridge danger signals triggered by the irritant effect of sensitizing chemicals with the activation of IL-1β and IL-18, thus promoting efﬁcient activation of the adaptive immune system.

ROS, the Common NALP3 Activator?

Antigen-presenting cells (APCs): cells that process antigens and have special molecules (MHC) that bind the processed antigens and display them on the cell surface for T cells to recognize

Most danger signals described to activate the NALP3 inﬂammasome trigger similar intracellular changes that may converge on a common mechanism of NALP3 activation (Figure 3). Potassium efﬂux, the induction of frustrated phagocytosis, and ROS production are the most striking features associated with NALP3 activators (127). Large particles and crystals such as MSU, alum, asbestos, and silica can induce the so-called frustrated phagocytosis at the surface of the cell, provoking the formation of cytoskeletal ﬁlaments (144). Inhibition of cytoskeletal ﬁlament generation with the pharmacological agents cytochalasin D or colchicine disrupts the ability of particles to trigger IL-1β activation (8, 113, 127), suggesting that the process of phagocytosis or frustrated phagocytosis is involved in NALP3 activation. Interestingly, colchicine (145) was one of the ﬁrst anti-inﬂammatory drugs identiﬁed for the treatment of gout by the Greeks more than ﬁfteen centuries ago (146) and is still used in modern medicine to treat inﬂammatory diseases. ROS production occurs quickly upon exposure of macrophages with silica or asbestos dust (127, 128, 147, 148). Similarly, MSU and alum produce ROS (113, 127). Both ATP and the toxin nigericin (that do not require phagocytosis to activate the inﬂammasome) activate ROS (149). A cellular redox imbalance also occurs upon cellular stimulation with the skin sensitizer DNCB, and ROS are also induced by UV (150). Along this line, knockdown of NAPDH subunits or the use of antioxidants inhibit inﬂammasome activation induced by alum, MSU, ATP, nigericin, asbestos, and silica (93, 113, 127, 149). It is therefore reasonable to www.annualreviews.org • The Inflammasomes

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Receptor for advanced glycation end product (RAGE): involved in the sensing of HMGB1, a danger signal released by injured cells

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(receptor for advanced glycation end product). However, NLRs are localized in the cytosol and thus are specialized in sampling PAMPs and danger signals that ultimately reach or affect this particular cellular compartment. Although microbes may reach the cytosol of cells during their life cycles, degradation products from phagocytosed bacteria and viruses may also be present in the cytosol and contribute to NLR and inﬂammasome activation (154). By deﬁnition, PAMPs represent molecules vital for microbial survival and are therefore unlikely to vary in their structures because any major change would be detrimental. Examples of PAMPs are bacterial structural components, such as LPS and PGNs, or viral nucleic acids. PAMPs are signatures that deﬁne classes of microbes and, as such, are critical in alerting the immune system. In addition to detecting PAMPs, inﬂammasomes also detect toxins and signals that are restricted to certain pathogens (155) (Figures 3 and 4). It is tempting to hypothesize that these signals may orchestrate speciﬁc innate immune responses as a result of a unique host-pathogen coevolution maximizing ﬁtness for both the pathogen and the host. The pathogen may beneﬁt from virulence to promote spreading replication and survival, whereas the host evolves to cope with the infection (156).

propose that ROS are either directly sensed by NALP3 or indirectly sensed through cytoplasmic proteins that modulate inﬂammasome activity. ROS production by hydrogen peroxide activates DCs in a similar way to TLRs (151) and activates the inﬂammasome (127). ROS production is a well-known, highly conserved signal involved in damage and stress sensing. ROS are also important players in innate immune responses in plants (152). Arabidopsis mutants that contain disruptions of NADPH oxidases fail to generate a full oxidative burst in response to infection by bacterial and fungal pathogens (48). Interestingly, plant potassium efﬂux has been linked to ROS production at the membrane (152). Potassium efﬂux has also been implicated in NADPH activation in granulocytes (153). It is therefore possible that potassium efﬂux by NALP3 activators may be involved in ROS generation.

INFLAMMASOMES AS SENSORS OF PATHOGENS A key function of the innate immune system is the recognition of invading microbes. Responses to extracellular PAMPs and some extracellular danger signals are mediated by membrane receptors such as TLRs and RAGE

Pathogen Shigella flexneri Salmonella typhimurium Pseudomonas aeruginosa Legionella pneumophila

T3SS

Virulence factor

Co-factors

?

ASC

Flagellin

ASC

Flagellin

NAIP5

T3SS

IPAF inflammasome

T3SS T4SS

IPAF Caspase-1

Figure 4 Gram-negative pathogens secrete factors such as ﬂagellin and possibly other virulence factors through type III (T3SS) or type IV (T4SS) secretion systems to trigger an inﬂammasome dependent on caspase-1 and IPAF. Genetic studies have demonstrated that caspase-1 activation in this context may also require the adaptor ASC or the NOD-like receptor protein NAIP. Whether NAIP and ASC contribute to the formation of IPAF inﬂammasomes directly or indirectly is unknown. 244

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Inflammasome Activation by PAMPs Inﬂammasomes respond to immunomodulatory PAMPs, mainly bacterial PGNs and nucleic acids. PGNs are structural units of cell walls common to all bacteria (157). Degradation of PGNs leads to the release of several structural units including MDP. MDP is sensed in the cytosol by the NLR NOD2, which activates NF-κB. MDP also activates caspase-1 and IL-1β (158) via NALP3 in human monocytes, suggesting that NALP3 is an additional MDP sensor (69, 159, 160). The strength of the immune response to MDP varies greatly and depends on the animal species and genetic background. Mice are much less sensitive than humans, guinea pigs, or rats to these PGNderived peptides; moreover, C57BL/6 mice are less sensitive than BALB/c mice (161, 162). Interestingly, both NF-κB and IL-1β activation are greatly enhanced by MDP in the presence of the protein synthesis inhibitor cycloheximide (CHX), demonstrating that a CHX-sensitive pathway may affect MDP internalization or its presentation to NLRs (69). Genetic studies in mice have shown that IL-1β activation by MDP requires both NOD2 and NALP3, suggesting that both these NLRs may cooperate either indirectly or directly as part of the same molecular complex (69). This ﬁnding is consistent with the observation that monocytes from Crohn’s disease patients who have functional mutations in the NOD2 gene fail to activate IL-1β upon MDP stimulation (163). Moreover, MuckleWells patients that harbor a gain-of-function mutation in the NALP3 gene overproduce IL-1β upon stimulation with MDP (159). Similarly, a probable gain-of-function mutation in NOD2 in the mouse leads to increased IL-1β production upon stimulation of macrophages with MDP (164). NOD2 has also been suggested to play a role, together with NALP1, in MDP-induced caspase-1 activation, further suggesting that multiple NLRs may cooperate and synergize to mount host defenses (68, 165). TLR9 and the intracellular protein DAI have been identiﬁed as sensors for DNA resulting in the triggering of a type I IFN response

(166, 167). Similar to DAI, the inﬂammasome is capable of sensing cytosolic DNA (168), although this is unlikely to be direct. Infection of a monocytic cell line or mouse macrophages with adenoviruses and herpesviruses leads to activation of caspase-1 and IL-1β. NALP3- and ASC-deﬁcient mice display reduced innate inﬂammatory responses to infection with adenovirus. Inﬂammasome activation also occurs as a result of transfected cytosolic bacterial, viral, and mammalian (host) DNA; however, in this case sensing is dependent on ASC only and not on NALP3. It is also independent of TLRs and IRFs (168). Studies have also identiﬁed RNA as an activator of NALP3 (98, 169), although this study could not be conﬁrmed by other groups.

Pore-Forming Bacterial Toxins Activate the NALP3 Inflammasome Many bacterial pathogens produce toxins that contribute to virulence by modifying host responses. Bacterial toxins are generally either enzymes or pore-forming proteins (170). Most of the bacterial toxins that activate NALP3 are pore-forming toxins. These toxins are released by bacteria in a soluble form and subsequently polymerize into a ring-like structure forming a pore in the membrane of the host cell. Pore formation triggers ionic imbalance; in particular, potassium efﬂux and calcium inﬂuxes are observed. These two processes are common danger signals and are frequently associated with NALP3 inﬂammasome activation (155). Among pore-forming toxins, α-toxin from Staphylococcus aureus and aerolysin from Aeromonas hydrophila are potent activators of the NALP3 inﬂammasome (96, 171). Similarly, listeriolysin O (LLO), a toxin released by Listeria monocytogenesis, activates caspase-1 in an ASCand NALP3-dependent manner (96, 172). Interestingly, ivanolysin O, a LLO-related cytolysin that exhibits quite similar function regarding the contribution to the escape of Listeria from the phagosome into the cytosol, is unable to restore caspase-1 activation in LLOdeﬁcient strains, suggesting that these toxins may engage speciﬁc signals beyond their ability

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to form pores (173). The release into the cytosol of ﬂagellin during Listeria infection activates the IPAF inﬂammasome (but not the NALP3 inﬂammasome, see below), highlighting the ability of pathogens to engage speciﬁc and multiple inﬂammasomes (174).

Anthrax Lethal Toxin Activates NALP1

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Bacillus anthracis is the causative agent of anthrax and depends for its virulence on the secretion of factors that form functional toxins. Anthrax lethal toxin (LeTx) is one of the major toxins produced by B. anthracis and is believed to be responsible for causing death in systemic anthrax infections. Macrophages from inbred mice are either susceptible or resistant to cell death in response to LeTx. This trait difference has been mapped to a locus on chromosome 11 and is associated with a polymorphism in the nalp1b gene that impinges on caspase-1 activation (175). How LeTx activates NALP1 and the role of the inﬂammasome in anthrax pathology are still unknown. Murine NALP1b does not contain a PYD; hence, it is not clear whether it requires ASC or dimerization with another NALP for caspase-1 recruitment. On the other hand, NALP1b possesses a CARD and a region related to CARDINAL. It is therefore possible that this CARD-containing region is able to activate caspase-1 in an ASC-independent manner, as it was suggested for human NALP1 in vitro (26). Activation of caspase-1 by LeTx requires binding, uptake, and endosome acidiﬁcation to mediate translocation of lethal factor (a functional subunit of LeTx) into the host cell cytosol. Interestingly, catalytically active lethal factor activates caspase-1 by a mechanism involving proteasome activity and potassium efﬂux (176–178).

IPAF Inflammasome Activation by Injected Virulence Factors IPAF and NAIP5 have been involved in the detection of virulence factors from Gram246

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negative bacteria. Recognition of Salmonella typhimurium and Shigella ﬂexneri activates the IPAF inﬂammasome that requires the ASC adaptor (77, 179). The role of ASC in the IPAF inﬂammasome is still unclear, but ASC may stabilize or facilitate caspase-1 recruitment to IPAF. Alternatively, IPAF may cooperate with a yet to be deﬁned NALP to activate caspase-1. In contrast to S. typhimurium and S. ﬂexneri, Legionella pneumophila requires two murine NLRs, NAIP5 and IPAF, for inﬂammasome formation, but does not require ASC (180– 182). The role of NAIP5 in IPAF inﬂammasome activation is unknown. It has been suggested that defective NAIP5 signaling renders macrophages permissive to L. pneumophila despite caspase-1 activation, suggesting that NAIP5 may have additional functions beyond its role in IPAF and caspase-1 activation (183). Unlike the seven NAIP genes found in the murine genome, humans only harbor one copy. Consistent with ﬁndings in the mouse, knockdown of NAIP or IPAF in human cell lines leads to enhanced susceptibility to L. pneumophila (38). IL-18 production and protection against Anaplasma phagocytophilum, a neutrophilic obligate bacteria that causes human anaplasmosis, relies on ASC and caspase-1 and partially on IPAF, but not on NALP3 (184). Similarly, Pseudonomas aeruginosa speciﬁcally activates the IPAF inﬂammasome (185–187). Most Gram-negative pathogens that activate IPAF require the type III secretion system (T3SS) or the type IV secretion system (T4SS) to inject into the host cell IPAF-activating virulence factors, mainly ﬂagellin. These bacterial injection machines span the two membranes from the bacteria and the host cell membrane (84). Polymers of ﬂagellin form the ﬂagella, a structure anchored to the bacterial cell wall that enables bacterial motility. Flagellin is a well-known activator of host innate immunity through its capacity to trigger TLR5 activation. In this context, ﬂagellin is considered a PAMP as it is a vital, evolutionarily conserved element of mobile bacteria. In the context of IPAF activation, ﬂagellin could be interpreted as a

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virulence factor, as ﬂagella formation is not required and ﬂagellin release in the cytosol is apparently the result of an active process dependent on T3SS or T4SS injection systems (84). Interestingly, S. ﬂexneri, a pathogen that does not have a ﬂagella and apparently does not express ﬂagellin, still requires the T3SS for IPAF-induced caspase-1 activation, indicating that ﬂagellin may not be the only T3SS virulence factor used by pathogens to activate IPAF inﬂammasomes (188). How ﬂagellin or other virulence factors engage IPAF inﬂammasomes as well as the precise function of ASC or NAIP in detecting these signals and activating caspase-1 are fascinating questions that need to be solved in future studies.

INFLAMMASOME REGULATORS Little is known about the mechanisms that regulate inﬂammasome activity. Inﬂammasome, caspase-1, and IL-1β activation are best performed in cells that express all the components at high concentrations and in active forms. Most cells do not express all the components required for inﬂammasome activation, necessitating prior stimulation or sensitization. THP-1 cells, for example, require differentiation of this monocytic cell line into a macrophage-like cell (70). Similarly, mouse macrophages are generally primed with LPS, and the response to MDP may require incubation with the protein synthesis inhibitor CHX (69). The pathogen Francisella tularensis ﬁrst triggers type I IFN activation to enable ASC-dependent inﬂammasome activation (189). These experimental ﬁndings hint of synergisms, feedback loops, and checkpoints that ultimately control inﬂammasome activation and orchestrate the physiological inﬂammatory response. Of particular interest are negative feedback loops that are crucial for the resolution phase of inﬂammation. NFκB for example, a well-known proinﬂammatory transcription factor, is also a crucial player in the down-modulation of the inﬂammatory response including inﬂammasome activation (190). Although we know little at the physiological level, investigators have identiﬁed vari-

ous proteins that may interfere with inﬂammasome assembly and inﬂammatory caspase activation. Based on the modular structure of these proteins, we can distinguish two major types of inﬂammasome regulators: those containing a CARD domain and those with a PYD domain (Figure 5).

Pyrin Domain–Containing Inflammasome Regulators PYD-containing regulators are believed to interfere with PYD-PYD interaction between NALPs and the adaptor ASC. These PYD regulators include Pyrin, POP1, POP2, and viral PYDs (vPYDs). POPs and the poxviral gene product M13L-PYD (also known as vPYD) are short proteins that contain mainly a PYD (191). Poxviruses deﬁcient in vPYD produce an enhanced activation of caspase-1 and secretion of IL-1β, further strengthening the idea that inﬂammasomes (that sense viral DNA) are important players in immunity against viruses (192–194). Similarly, POP1 and POP2 modulate inﬂammasome activity probably by disrupting ASC-NALP interactions (191, 195). The absence in the mouse genome of both POP1 and POP2 make the evaluation of their physiological importance in vivo challenging. Pyrin was initially identiﬁed as the product of the MEFV gene, which is mutated in patients with familial Mediterranean fever (FMF) (196), a hereditary autoinﬂammatory syndrome characterized by episodic fever and serosal or synovial inﬂammation. Targeted disruption of Pyrin in mice causes increased endotoxin sensitivity and enhanced caspase-1 activation (197). Most of the mutations in Pyrin in FMF patients affect the C terminus, which harbors a PRY-SPRY domain. The function of this domain, that is partially absent in the mouse, is not clear, but a role in the regulation of the inﬂammasome and caspase-1 was proposed (55, 56, 197). The PYD of Pyrin interacts with the PYD of ASC (197), suggesting that it may be involved in blocking the recruitment of ASC and inﬂammasome formation. However, artiﬁcial overexpression of Pyrin can also be www.annualreviews.org • The Inflammasomes

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NALP3 inflammasome

IPAF inflammasome

Pyrin POP1, POP2 vPYDs NALP3

Bcl-2 Bcl-XL IPAF

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Caspase-1 Caspase-1

ASC

PYD CARD

Caspase-12

PRY-SPRY BBox

Pi9, vCrmA, Flightless-I

Iceberg Pseudo-ICE INCA

Figure 5 Inﬂammasome activity is inhibited by PYD-containing proteins that interfere with ASC and NALP interaction and CARD-containing proteins that disrupt caspase-1 interaction with IPAF or ASC. Bcl-2 and Bcl-XL proteins have been suggested to inhibit NALP oligomerization (165). Caspase-1 activity can be directly blocked by Flightless-I (259), and the serpin protease inhibitor 9 (Pi9) (260) or cowpox virus-encoded inhibitor of caspase-1, vCrmA (261).

proinﬂammatory. This led Fernandes-Alnemri et al. (198) to propose an alternative hypothesis, namely that Pyrin, like NALP3, assembles an inﬂammasome with ASC and caspase-1. On the other hand, FMF is mainly considered to be an autosomal recessive autoinﬂammatory disorder, an observation more consistent with the notion that Pyrin loss-of-function may cause the aberrant inﬂammation in these patients by allowing inﬂammasome hyperactivation (199). PSTPIP1, a Pyrin-interacting protein, is mutated in PAPA syndrome (pyogenic arthritis, pyoderma gangrenosum, and acne), an autoinﬂammatory disease associated with overproduction of IL-1β (200). Moreover, mutations in a mouse-related protein, PSTPIP2, cause a macrophage-dependent autoinﬂammatory syndrome, further delineating the importance of Pyrin and inﬂammasome regulation in autoinﬂammatory disorders (201, 202) (see below). 248

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CARD Domain-Containing Regulators A small family of inﬂammasome regulators harbor a CARD that is highly similar to the CARD of caspase-1. These proteins most likely emerged from successive gene duplications and include in humans iceberg, INCA, COP, and caspase-12 (33, 203, 204). Through CARDCARD interactions these proteins presumably inhibit processing of proIL-1β by preventing recruitment and/or activation of the caspase by the adaptor ASC or IPAF. Except for caspase12, most of these proteins are not present in the mouse or rat genomes, again highlighting considerable differences in the regulation of the inﬂammasome in diverse species. Caspase12 is present in two main polymorphic variants in human, resulting in the production of either a truncated protein containing the Nterminal CARD domain (CARD-only) or a fulllength variant molecule (caspase-12L) (205).

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The full-length variant of caspase-12, which is the less frequent allele, conﬁned to the population of African descent, is linked to hyporesponsiveness to LPS-induced production of cytokines (205, 206). Genetic studies have predicted that the polymorphism generating the caspase-12 short variant was driven by positive selection to complete ﬁxation in the human genome approximately 60,000–100,000 years ago (207, 208). Loss of the caspase-12 C terminus may have conferred a selective beneﬁt, possibly by increasing sepsis resistance in human populations that experienced emergent infectious diseases as geographic expansion occurred in association with increases in human population size and density. In line with this hypothesis, caspase-12-deﬁcient mice clear bacterial infection more efﬁciently than do wild-type littermates and have an enhanced production of proinﬂammatory cytokines, including IL-1β and IL-18 (209). Caspase-12 was proposed to be a decoy caspase that blocks caspase-1 activation resulting in enhanced vulnerability to bacterial infection and septic mortality, similar to cFLIP (a decoy caspase-8-like protein), which regulates caspase-8-mediated apoptosis. However, contrary to FLIP, the fulllength variant of caspase-12 has autoproteolytic activities, a mechanism that may regulate its anti-inﬂammasome properties or may underline the possibility that the full-length variant of caspase-12 cleaves speciﬁc substrates yet to be identiﬁed (210).

INFLAMMASOME SIGNALING AND DISEASE ASSOCIATIONS Although inﬂammasomes are involved in both pathways of pathogen and danger signal sensing, their function converges in the activation of inﬂammatory caspases (mainly caspase-1), which have few known substrates, primarily IL1β, IL-18, and possibly IL-33. The complexity of inﬂammasome assembly contrasts with the reductionist vision of its main role as a trigger of IL-1β and IL-18 maturation. Moreover, IL-1β, IL-18, and IL-33 are related cy-

tokines that initiate a MyD88-dependent signaling pathway, very similar to the pathway engaged by TLRs. Therefore, pathogen sensing by inﬂammasomes can be interpreted as an indirect activation of a TLR-like receptor (IL-1R or IL-18R), a scenario that is comparable to the activation mechanism of the Toll receptor in Drosophila (19). Both mammalian and Drosophila signaling pathways involve the activation of, in mammals, cytokines IL-1β and IL-18 and, in Drosophila, Sp¨atzle through proteolytic processing, which is initiated by microbial sensors that engage and activate speciﬁc proteases. Although IL-1-processing inﬂammasomes in mammals sense pathogens in the cytosol, the proteases that activate Sp¨atzle are localized and activated in the hemolymph of the fruit ﬂy. With this in mind, the complexity and diversity of inﬂammasomes may reﬂect the multitude of pathogens and danger signals that are detected in speciﬁc locations, whereas the conservation of TLRs and IL-1β/IL-18 receptor signaling cascades emphasizes the importance of MyD88 signaling in innate immunity (19). The discovery of speciﬁc activators that signal through an inﬂammasome-IL-1β pathway brought new interest to the biology of IL-1β. As new caspase-1 substrates are being uncovered, new inﬂammasome functions will emerge beyond its role in the maturation of IL-1β, IL-18, and possibly IL-33 (171, 211– 213).

Inflammasomes, Inflammation, and Inflammatory Diseases Originally identiﬁed as the endogenous pyrogen, exogenous IL-1β triggers fever in experimental animals (214). In addition to fever, IL-1β has multiple other effects on the central nervous system. These include induction of slow-wave sleep, anorexia, and inﬂammatory pain hypersensitivity, typically associated with infections or injury (214, 215). The important role of IL-1β and the inﬂammasome in inﬂammation and fever is strongly supported by genetic evidence that links the inﬂammasome to

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Diseases associated with inflammasome activity

Disease

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Clinical features

Gene mutated

Etiologic agent

Inflammasome involvement

Anakinra response

Familial cold autoinﬂammatory syndrome (FCAS)

Fever, arthralgia, cold-induced urticaria

NALP3

overactive

yes

Muckle-Wells syndrome (MWS)

Fever, arthralgia, urticaria, sensorineural deafness, amyloidosis

NALP3

overactive

yes

Chronic infantile neurological cutaneous and articular syndrome (CINCA, NOMID)

Fever, severe arthralgia, urticaria, neurological problems, severe amyloidosis

NALP3

overactive

yes

Familial Mediterranean fever (FMF)

Fever, peritonitis, pleuritis, amyloidosis

Pyrin

overactive

partial

Pyogenic arthritis, pyoderma gangrenosum, and acne syndrome (PAPA)

Pyogenic sterile arthritis

PSTPIP1

overactive

yes

Hyperimmunoglobulin D syndrome (HIDS)

Arthralgia, abdominal pain, lymphadenopathy

Mevalonate kinase

to be demonstrated

yes

Tumor necrosis factor receptor-1-associated syndrome (TRAPS)

Fever, abdominal pain, skin lesions

TNF-R1

to be demonstrated

yes

Systemic juvenile idiopathic arthritis (SOJIA)

Chronic joint inﬂammation

unknown

to be demonstrated

yes

Adult-onset Still’s disease (AOSD)

Arthralgia, fever

unknown

to be demonstrated

yes

Behcet’s disease

Arthralgia, uveitis, ulcers

unknown

to be demonstrated

yes

Schnitzler’s syndrome

Urticaria, fever arthralgia

unknown

to be demonstrated

yes

Gout

Metabolic arthritis, pain

uric acid (MSU)

activated

yes

Pseudogout

Arthritis

CPPD

activated

yes

Contact dermatitis

Urticaria

irritants

activated

unknown

Fever syndrome

Fever

NALP12

unknown

unknown

Hydatidiform mole

Hydatid mole

NALP7

unknown

unknown

Vitiligo

Skin depigmentation, automimmunity

NALP1

unknown

unknown

a family of hereditary periodic fevers (HPFs) (216) (Table 2). HPFs are heritable disorders characterized by unexplained and recurrent episodes of fever and severe inﬂammation. These patients suffer from rashes and serosal and synovial inﬂammation with varying degree of neurological involvement. HPFs are part of the expanding family of so-called autoinﬂammatory diseases that differ from au-

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toimmune disorders in that evidence for adaptive immunity components such as autoreactive T cells or immunoglobulins to self-antigens is lacking (217). Familial cold autoinﬂammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and chronic infantile cutaneous neurological articular syndrome (CINCA), also termed neonatal-onset multisystem inﬂammatory disease (NOMID), are all caused by

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mutations in the third exon of NALP3 (218, 219). These diseases have overlapping characteristics and form a clinical continuum. FCAS patients have the less severe symptoms that generally include fever (often triggered by temperature changes), arthralgia, and skin rashes. MWS patients may, in addition, develop amyloidosis together with deafness. NOMID patients have the most severe symptoms that may lead to very serious neurological impairment caused by chronic polymorphonuclear meningitis. The disease-causing mutations in MWS, NOMID, and FCAS are believed to confer gain-of-function to NALP3 leading to a hyperactive inﬂammasome. Consistent with this model, increased secretion of IL-1β is observed in macrophages from patients with disease-associated NALP3 mutations (78, 220). Moreover, overexpression of mutant NALP3 proteins induces spontaneous IL-1β secretion (221). IL-1β is therefore the likely major mediator of inﬂammation in this disease. This model is supported by studies demonstrating that treatment of these patients with an inhibitor of IL-1 (IL1-ra) leads to a very striking and dramatic improvement of symptoms in all three conditions (222–225). Genes responsible for other autoinﬂammatory diseases may also be linked to inﬂammasome activity (Table 2). FMF is an HPF characterized by recurrent inﬂammation of serosal surfaces and is associated with mutations in the PYD-containing inﬂammasome regulator Pyrin (220). The inﬂammatory disease PAPA is another condition in which impaired IL-1β regulation is caused by mutations in PSTPIP1(CD2BP), a protein that binds to Pyrin and may be involved in scaffolding inﬂammasome components to the cytoskeleton (200). Other autoinﬂammatory diseases such as gout, pseudogout, silicosis, and asbestosis are associated with aberrant activation of NALP3. In these cases, aberrant inﬂammasome activation is not caused by an inherited mutation in inﬂammasome components or regulators, but by chronic exposure to inﬂammasome activators such as MSU, CPPD, and inﬂammationinducing dust (226).

Inflammasomes, Adjuvanticity, and Autoimmune Diseases Adjuvants are materials that enhance the immune response to an antigen. Several different types of adjuvants exist, ranging from mineral salts such as alum to oil-based emulsions such as incomplete Freund’s adjuvant. On a molecular basis, adjuvants are believed to boost antigen presentation by APCs such as macrophages or DCs. More efﬁcient antigen presentation can be achieved by adding, for example, TLR agonists, which activate the APCs, promote the upregulation of costimulatory molecules, improve antigen presentation, and enhance cytokine induction. Unfortunately, TLR agonists may be too toxic to be used in human vaccines; moreover, immune responses to an antigen can also occur in the absence of TLR signaling (227). Similar to TLR agonists, IL-1β has adjuvant properties. When mice are immunized with protein antigens together with IL-1β, serum antibody production is enhanced. In humans, IL-1 also promotes the expansion of IL-17-secreting memory CD4+ T cells (228, 229). Interestingly, many of the inﬂammasome activators have adjuvant properties; these include MDP [whose role in enhancing immune responses has been known for more than three decades (230)], MSU (111), and alum (131). Alum deﬁnes particle materials based on aluminum salt precipitates that are the most widely used adjuvants in human vaccines. Alum activates a Th2-biased immunity with elevated Th2-dependent antibody isotypes IgG1 and IgE (131). Similar to MSU, alum triggers an inﬂux of neutrophils when injected in vivo, and both have similar adjuvant properties resulting (in presence of an antigen) in an efﬁcient adaptive immune response (8, 113, 117). Importantly, both MSU and alum depend on an intact inﬂammasome including NALP3, ASC, and caspase-1 to trigger a Th2-biased response (113, 117, 119). Similarly, silicosis triggered by silica dust (another NALP3 activator) is also characterized by a Th2 response (231). These ﬁndings demonstrate the importance of the inﬂammasome in linking innate immunity to

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Cytokines

Alum

?

Antigen

IL-1β

IL-18

IL-33

Cytokines IL-1β IL-18

Inﬂammasome

2

1

IL-33?

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?

ANTIG EN PRES ENTING CELL

B CELL

3

Th2 cytokines

An ti MHgenC

Cos t mo imula lec tory ule s

T CELL

Figure 6 Model of inﬂammasome-mediated adjuvanticity. Antigen-presenting cells (APCs) can sense antigens (contained within adjuvants, such as alum) through the inﬂammasome. Inﬂammasome activation leads to the release of caspase-1-dependent cytokines including IL-1β, IL-18, and IL-33, which may in an autocrine manner trigger their respective receptors to promote antigen presentation, upregulation of costimulatory molecules, and the release of cytokines, which ultimately result in the stimulation of antigen-speciﬁc T and B cells (1). Secreted IL-1β, IL-18, and possibly IL-33 may also directly contribute to the activation of T and B cells (2). The question marks illustrate the possibility that the inﬂammasome may activate an as yet unidentiﬁed factor that would signal in a MyD88-independent manner. This could explain the observation that inﬂammasome-mediated adjuvanticity is MyD88-independent. Yet to be identiﬁed substrates and signaling pathways linked to the inﬂammasome may also directly enhance costimulatory signals (3).

adaptive immunity. A critical question that remains to be addressed is how the inﬂammasome initiates lymphocyte activation and how it favors Th2 immunity (Figure 6). The three cytokines that we know to depend for activity on the NALP3 inﬂammasome are IL-1β, IL-18, and possibly IL-33, which are all known to trigger various aspects of Th2 immune responses. For example, IL-1 is crucial in regulating Th2 responses during gastrointestinal nematode infection (232) and was suggested more than 20 years ago as mediating Th2 responses upon immunization with an antigen in alum (133). IL-33 induces the expression of IL-4, IL-5, and IL-13 and mediates Th2 polarization by engaging the ST2 receptor (233). IL-18, which is commonly considered to promote Th1 immunity, can amplify Th2 responses and promote Th2-biased pathologies, such as asthma, 252

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by triggering the production of IgE antibodies (234). These observations strongly suggest that the inﬂammasome-generated cytokines play a role in the adjuvant properties of alum and MSU. Surprisingly, no defect in alum adjuvanticity was observed in MyD88-deﬁcient mice (113, 227). MyD88 is a crucial signaling molecule downstream of IL-1 and IL-18. Moreover, MyD88 is downstream of IL-33, suggesting that all three cytokines may not be directly involved in regulating the adjuvant properties of alum, or that MyD88independent signals, triggered by ST2 or other IL-1R family members, are involved in this process. New inﬂammasome and caspase-1 substrates have recently been identiﬁed, but the role of these proteins in regulating immune responses remains to be investigated in the context of antigen immunization (171, 211–213).

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The role of inﬂammasomes in regulating the adaptive immune response is supported by the ﬁndings showing that polymorphisms in NALP1 are associated with vitiligo-associated multiple autoimmune disease (235), a disorder in which the patients, in addition to generalized vitiligo characterized by loss of skin pigments, have increased frequency of several other autoimmune diseases, particularly autoimmune thyroid disease, latent autoimmune diabetes, rheumatoid arthritis, psoriasis, pernicious anemia, systemic lupus erythematosus, and Addison’s disease. Ironically, although the role of the inﬂammasome in promoting the adaptive immune response and in autoimmunity is now widely accepted, most of the diseases with established inﬂammasome overactivation lead to autoinﬂammatory syndromes that, by deﬁnition, are devoid of autoreactive T cells and autoreactive antibodies. A better understanding of the role of the various inﬂammasome mediators in the regulation of the inﬂammatory (innate) response and the adaptive response may help to solve this paradox.

Inflammasomes and Pyroptosis Under certain conditions, activation of inﬂammasomes, and thus inﬂammatory caspases, leads to cell death. The word pyroptosis— which is derived from the Greek “pyro” (ﬁre), to denote the release of proinﬂammatory mediators, and “ptosis,” which in Greek means falling, a term commonly used to describe cell death—was introduced for this particular type of cell death (236). Pyroptosis is dependent on the activation of caspase-1, is often associated with a high inﬂammatory state in contrast to the silent apoptotic death, and frequently occurs upon infection with intracellular pathogens. It has been best studied in the context of Shigella-infected macrophages. S. ﬂexneri is a human intestinal pathogen, causing dysentery by invading the epithelium of the colon. This facultative intracellular pathogen evades the phagosome to enter the cytosol where it can trigger cell death (237). Shigella-induced

macrophage death requires the inﬂammatory caspase-1, but not the apoptotic caspase-3 (238, 239). Similarly, caspase-1-dependent cell death has been reported in macrophages infected with Salmonella typhimurium (240), Pseudomonas aeruginosa (186), Francisella tularensis (241), Legionella pneumophila (180), and Listeria monocytogenes (242). Surprisingly, inﬂammasome requirements differ between caspase-1-dependent IL-1β and IL-18 secretion and pyroptosis. Both IPAF and ASC are required for cytokine production by Salmonella-infected macrophages; however, only IPAF-deﬁcient macrophages are completely resistant to Salmonella-induced pyroptosis, whereas ASC-deﬁcient macrophages are only partially protected (77). Similarly, S. ﬂexneri and P. aeruginosa require both IPAF and ASC to induce IL-1β secretion, whereas ASC is dispensable for triggering pyroptosis (186–188). In the case of Bacillus anthracis, the causative agent of anthrax, pyroptosis depends on NALP1 (176, 243). These ﬁndings are in contradiction with an in vitro study proposing that pyroptosis is triggered by an ASCdependent, but NALP-independent, complex (termed pyroptosome) (198, 244). Overall, these studies support a model whereby ASC as well as NALP and IPAF inﬂammasomes form distinct complexes, possibly with different cellular localization or different substrate speciﬁcity. Detailed biochemical studies are required to shed some light on the various types of inﬂammasomes formed upon infection of macrophages with live pathogens. It is noteworthy that ASC-dependent inﬂammasome activation leads to a very rapid secretion of inﬂammatory caspases including caspase-1 and caspase-5 (70). It is therefore tempting to postulate that the rapid secretion of the inﬂammatory caspases may be part of a regulatory mechanism aimed at reducing cell death associated with ASC and NALP inﬂammasomes.

Pyroptosis: a form of cell death associated with antimicrobial responses during inﬂammation and dependent on the activation of an inﬂammasome and inﬂammatory caspases such as caspase-1

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inﬂammatory mediators in pathways and pathologies that may involve inﬂammasome activation, including neurodegenerative disorders, cancer, and fertility-associated conditions (245–247). IL-1 is a key mediator of experimentally induced neurodegeneration, and its inhibition is neuroprotective in vitro and in vivo (246, 248). Interestingly, NAIP (a NLR associated with the IPAF inﬂammasome) is partially deleted in individuals with the neurodegenerative disease spinal muscular atrophy (249). The ﬁbrillar peptide amyloid-β has a key function in the pathogenesis of Alzheimer’s disease (250). Similar to uric acid crystals, the phagocytosis of amyloid-β was found to activate the NALP3 inﬂammasome and to be critical for the recruitment of microglia to exogenous amyloid-β in the brain. Thus, the activation of the NALP3 inﬂammasome may be important for inﬂammation and tissue damage in Alzheimer’s disease. IL-1β is known to play a role in both ovulation and oocyte maturation (251). In the mare, intrafollicular injection of IL-1β leads to increased ovulation, but also to a very low rate of embryo development, most likely owing to a defect in oocyte maturation (252). Similarly, IL-1β perfusion in the rabbit ovary blocks embryo development at the four-cell stage (253). It is therefore possible that inﬂammasomes may link some aspects of innate immunity to reproductive biology. Indeed, the expression proﬁles of NALP4, NALP5, NALP8, and NALP9 in gametes and preimplantation embryos, together with genetic studies, suggest a possible function for these proteins in the biology of reproduction (33). NALP5 (Mater)-deﬁcient female mice are sterile owing to an arrest at the two-cell stage in the development of the embryos (254). Furthermore, mutations in NALP7 cause recurrent hydatidiform moles, an abnormal human pregnancy with no embryo and cystic degeneration of placental villi in humans (255–257). Although it is known that inﬂammation and bacterial infection cause infertility, ectopic pregnancy, and abortion, the role of NALP7 in this disease is unknown. Similarly, the ability of NALP7, as well as many

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other NALPs, to form inﬂammasomes similar to the NALP3 inﬂammasome has not being investigated.

CONCLUDING REMARKS In the past decade, our understanding of the cellular and molecular mechanisms by which the innate immune system molecules sense speciﬁc molecular patterns from components of invading organisms of both bacterial and viral origin has increased tremendously. The NLRs, together with the TLRs, are now appreciated to be part of this important sensing system that allows the host to mount an effective immune response for elimination of the microbe and for establishment of an effective adaptive immune response for long-lasting immunity. What is fascinating is the realization that at least one NLR member, NALP3, a receptor that was postulated more than 10 years ago, also detects various endogenous, sterile danger signals in the absence of microbial infections. Danger signals include several particles such as uric acid crystals, asbestos, or aluminum, which cause the assembly of the NALP3 inﬂammasome and the generation of the proinﬂammatory cytokine IL-1. The remarkable progress in this ﬁeld offers new hope for many patients. We can anticipate a new generation of IL-1 antagonists in the near future. A new IL-1β antibody is currently in Phase II trials for rheumatoid arthritis, and various inﬂammatory diseases, such as gout, are now successfully treated with an IL-1 inhibitor. Moreover, the efﬁciency of aluminum as an adjuvant can now be explained at the molecular level and may help to design effective, but safe, adjuvants in the future. Yet despite our increased knowledge, many questions remain. The precise roles and needs for molecules such as IPAF and NAIP remain ill deﬁned. The ligands of many other NLRs are unknown and their functions elusive. Although mutations in NOD2 and NALP3 genes provide a basis for susceptibility to Crohn’s disease and inﬂammatory diseases, why is a mutation in NALP1 associated with vitiligo? What is the signiﬁcance of proteins other than IL-1 and

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IL-18 cleaved by the inﬂammasome-activated caspase-1, such as caspase-7 or enzymes of the glycolytic pathway (258)? From what we have learned about the function of NLRs in the short time since their discovery, it is obvious that a future better un-

derstanding of the biology of NLRs will not only help to elucidate their role in host responses to infectious agents and to danger signals, but will certainly contribute to the development of desperately needed novel types of anti-inﬂammatory drugs.

DISCLOSURE STATEMENT

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The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

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70. Describes the inflammasome for the first time.

77. Identifies ASC and IPAF as essential inflammasome components in mice.

78. Describes the NALP3 inflammasome and its overactivation in MWS patients for the first time.

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94. Kahlenberg JM, Dubyak GR. 2004. Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release. Am. J. Physiol. Cell Physiol. 286:C1100–8 ˜ G. 2007. Differential requirement of P2X7 receptor and 95. Franchi L, Kanneganti T, Dubyak GR, Nu´ nez intracellular K+ for caspase-1 activation induced by intracellular and extracellular bacteria. J. Biol. Chem. 282:18810–18 96. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, et al. 2006. Cryopyrin activates the inﬂammasome in response to toxins and ATP. Nature 440:228–32 97. Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, et al. 2006. Critical role for NALP3/CIAS1/cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24:317–27 ¨ 98. Kanneganti TD, Ozoren N, Body-Malapel M, Amer A, Park JH, et al. 2006. Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 440:233–36 ¨ 99. Haag F, Adriouch S, Braß A, Jung C, Moller S, et al. 2007. Extracellular NAD and ATP: partners in immune cell modulation. Purinergic Signal 3:71–81 ¨ 100. Eckle T, Fullbier L, Wehrmann M, Khoury J, Mittelbronn M, et al. 2007. Identiﬁcation of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury. J. Immunol. 178:8127–37 101. Communi D, Janssens R, Suarez-Huerta N, Robaye B, Boeynaems JM. 2000. Advances in signalling by extracellular nucleotides: the role and transduction mechanisms of P2Y receptors. Cell Signal. 12:351–60 102. Novak I. 2003. ATP as a signaling molecule: the exocrine focus. News Physiol. Sci. 18:12–17 103. MacDonald PE, Braun M, Galvanovskis J, Rorsman P. 2006. Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells. Cell Metab. 4:283–90 104. Koshlukova SE, Lloyd TL, Araujo MW, Edgerton M. 1999. Salivary histatin 5 induces nonlytic release of ATP from Candida albicans leading to cell death. J. Biol. Chem. 274:18872–79 105. Laliberte RE, Eggler J, Gabel CA. 1999. ATP treatment of human monocytes promotes caspase-1 maturation and externalization. J. Biol. Chem. 274:36944–51 106. Kanneganti TD, Lamkanﬁ M, Kim YG, Chen G, Park JH, et al. 2007. Pannexin-1-mediated recognition of bacterial molecules activates the cryopyrin inﬂammasome independent of Toll-like receptor signaling. Immunity 26:433–43 107. Locovei S, Bao L, Dahl GP. 2006. Pannexin 1 in erythrocytes: function without a gap. Proc. Natl. Acad. Sci. USA 103:7655–59 108. Piccini A, Carta S, Tassi S, Lasigli´e D, Fossati G, Rubartelli A. 2008. ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1β and IL-18 secretion in an autocrine way. Proc. Natl. Acad. Sci. USA 105:8067–72 109. Yamamoto M, Yaginuma K, Tsutsui H, Sagara J, Guan X, et al. 2004. ASC is essential for LPS-induced activation of procaspase-1 independently of TLR-associated signal adaptor molecules. Genes Cells 9:1055– 67 110. Idzko M, Hammad H, van Nimwegen M, Kool M, Willart MA, et al. 2007. Extracellular ATP triggers and maintains asthmatic airway inﬂammation by activating dendritic cells. Nat. Med. 13:913–19 111. Shi Y, Evans JE, Rock KL. 2003. Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 425:516–21 112. Chen CJ, Shi Y, Hearn A, Fitzgerald K, Golenbock D, et al. 2006. MyD88-dependent IL-1 receptor signaling is essential for gouty inﬂammation stimulated by monosodium urate crystals. J. Clin. Invest. 116:2262–71 113. Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA. 2008. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453:1122–6 114. Shi Y, Galusha SA, Rock KL. 2006. Cutting edge: elimination of an endogenous adjuvant reduces the activation of CD8 T lymphocytes to transplanted cells and in an autoimmune diabetes model. J. Immunol. 176:3905–8 115. Orengo J, Evans J, Bettiol E, Leliwa-Sytek A, Day K, et al. 2008. Plasmodium-induced inﬂammation by uric acid. PLoS Pathogens 4:e1000013 116. Kool M, Soulli´e T, Van Nimwegen M, Willart MA, Muskens F, et al. 2008. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inﬂammatory dendritic cells. J. Exp. Med. 205:869–82 www.annualreviews.org • The Inflammasomes

97. Demonstrates for the first time that the NALP3 inflammasome is implicated in Type IV hypersensitivity.

111. Identifies uric acid as an endogenous adjuvant released by damaged cells.

113. Describes, as does 117, 118, 119, and 129, the activation of the NALP3 inflammasome by alum adjuvants.

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167. Kumagai Y, Takeuchi O, Akira S. 2008. TLR9 as a key receptor for the recognition of DNA. Adv. Drug Deliv. Rev. 60:795–804 168. Muruve DA, P´etrilli V, Zaiss AK, White LR, Clark SA, et al. 2008. The inﬂammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response. Nature 452:103–7 169. Kanneganti TD, Body-Malapel M, Amer A, Park JH, Whitﬁeld J, et al. 2006. Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA. J. Biol. Chem. 281:36560–68 170. Gonzalez MR, Bischofberger M, Pernot L, Van Der Goot FG, Frˆeche B. 2008. Bacterial pore-forming toxins: the (w)hole story? Cell Mol. Life Sci. 65:493–507 171. Gurcel L, Abrami L, Girardin S, Tschopp J, Van Der Goot FG. 2006. Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell 126:1135– 45 ¨ 172. Ozoren N, Masumoto J, Franchi L, Kanneganti TD, Body-Malapel M, et al. 2006. Distinct roles of TLR2 and the adaptor ASC in IL-1β/IL-18 secretion in response to Listeria monocytogenes. J. Immunol. 176:4337–42 173. Hara H, Tsuchiya K, Nomura T, Kawamura I, Shoma S, Mitsuyama M. 2008. Dependency of caspase-1 activation induced in macrophages by Listeria monocytogenes on cytolysin, listeriolysin O, after evasion from phagosome into the cytoplasm. J. Immunol. 180:7859–68 174. Warren SE, Mao DP, Rodriguez AE, Miao EA, Aderem A. 2008. Multiple Nod-like receptors activate caspase 1 during Listeria monocytogenes infection. J. Immunol. 180:7558–64 175. Boyden E, Dietrich W. 2006. Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin. Nat. Genet. 38:240–44 176. Fink SL, Bergsbaken T, Cookson BT. 2008. Anthrax lethal toxin and Salmonella elicit the common cell death pathway of caspase-1-dependent pyroptosis via distinct mechanisms. Proc. Natl. Acad. Sci. USA 105:4312–17 177. Wickliffe KE, Leppla SH, Moayeri M. 2008. Anthrax lethal toxin-induced inﬂammasome formation and caspase-1 activation are late events dependent on ion ﬂuxes and the proteasome. Cell Microbiol. 10:332–43 178. Squires RC, Muehlbauer SM, Brojatsch J. 2007. Proteasomes control caspase-1 activation in anthrax lethal toxin-mediated cell killing. J. Biol. Chem. 282:34260–67 179. Lara-Tejero M, Sutterwala FS, Ogura Y, Grant EP, Bertin J, et al. 2006. Role of the caspase-1 inﬂammasome in Salmonella typhimurium pathogenesis. J. Exp. Med. 203:1407–12 180. Molofsky AB, Byrne BG, Whitﬁeld NN, Madigan CA, Fuse ET, et al. 2006. Cytosolic recognition of ﬂagellin by mouse macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203:1093–104 181. Ren T, Zamboni DS, Roy CR, Dietrich W, Vance RE. 2006. Flagellin-deﬁcient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity. PLoS Pathog. 2:e18 182. Zamboni DS, Kobayashi KS, Kohlsdorf T, Ogura Y, Long EM, et al. 2006. The Birc1e cytosolic patternrecognition receptor contributes to the detection and control of Legionella pneumophila infection. Nat. Immunol. 7:318–25 ˜ 183. Lamkanﬁ M, Amer A, Kanneganti TD, Munoz-Planillo R, Chen G, et al. 2007. The Nod-like receptor family member Naip5/Birc1e restricts Legionella pneumophila growth independently of caspase-1 activation. J. Immunol. 178:8022–27 184. Pedra JH, Sutterwala FS, Sukumaran B, Ogura Y, Qian F, et al. 2007. ASC/PYCARD and caspase-1 regulate the IL-18/IFN-γ axis during Anaplasma phagocytophilum infection. J. Immunol. 179:4783–91 185. Miao EA, Ernst RK, Dors M, Mao DP, Aderem A. 2008. Pseudomonas aeruginosa activates caspase 1 through Ipaf. Proc. Natl. Acad. Sci. USA 105:2562–67 186. Sutterwala FS, Mijares LA, Li L, Ogura Y, Kazmierczak BI, Flavell RA. 2007. Immune recognition of Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inﬂammasome. J. Exp. Med. 204:3235–45 ˜ G. 2007. Critical role for Ipaf in 187. Franchi L, Stoolman J, Kanneganti TD, Verma A, Ramphal R, Nu´ nez Pseudomonas aeruginosa-induced caspase-1 activation. Eur. J. Immunol. 37:3030–39 188. Suzuki T, Franchi L, Toma C, Ashida H, Ogawa M, et al. 2007. Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages. PLoS Pathog. 3:e111

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189. Henry T, Brotcke A, Weiss DS, Thompson LJ, Monack DM. 2007. Type I interferon signaling is required for activation of the inﬂammasome during Francisella infection. J. Exp. Med. 204:987–94 190. Greten FR, Arkan MC, Bollrath J, Hsu LC, Goode J, et al. 2007. NF-κB is a negative regulator of IL-1β secretion as revealed by genetic and pharmacological inhibition of IKKβ. Cell 130:918–31 191. Stehlik C, Dorﬂeutner A. 2007. COPs and POPs: Modulators of inﬂammasome activity. J. Immunol. 179:7993–98 192. Dorﬂeutner A, Talbott SJ, Bryan NB, Funya KN, Rellick SL, et al. 2007. A Shope Fibroma virus PYRINonly protein modulates the host immune response. Virus Genes 35:685–94 193. Benedict CA, Ware CF. 2005. Poxviruses aren’t stuPYD. Immunity 23:553–55 194. Johnston JB, Barrett JW, Nazarian SH, Goodwin M, Ricciuto D, et al. 2005. A poxvirus-encoded pyrin domain protein interacts with ASC-1 to inhibit host inﬂammatory and apoptotic responses to infection. Immunity 23:587–98 195. Bedoya F, Sandler LL, Harton JA. 2007. Pyrin-only protein 2 modulates NF-κB and disrupts ASC:CLR interactions. J. Immunol. 178:3837–45 196. The French FMF Consortium. 1997. A candidate gene for familial Mediterranean fever. Nat. Genet. 17:25–31 197. Chae JJ, Komarow HD, Cheng J, Wood G, Raben N, et al. 2003. Targeted disruption of pyrin, the FMF protein, causes heightened sensitivity to endotoxin and a defect in macrophage apoptosis. Mol. Cell 11:591–604 198. Fernandes-Alnemri T, Wu J, Yu JW, Datta P, Miller B, et al. 2007. The pyroptosome: a supramolecular assembly of ASC dimers mediating inﬂammatory cell death via caspase-1 activation. Cell Death Differ. 14:1590–604 199. McDermott MF. 2004. A common pathway in periodic fever syndromes. Trends Immunol. 25:457–60 200. Shoham NG, Centola M, Mansﬁeld E, Hull KM, Wood G, et al. 2003. Pyrin binds the PSTPIP1/ CD2BP1 protein, deﬁning familial Mediterranean fever and PAPA syndrome as disorders in the same pathway. Proc. Natl. Acad. Sci. USA 100:13501–6 201. Grosse J, Chitu V, Marquardt A, Hanke P, Schmittwolf C, et al. 2006. Mutation of mouse Mayp/Pstpip2 causes a macrophage autoinﬂammatory disease. Blood 107:3350–58 202. Ferguson PJ, Bing X, Vasef MA, Ochoa LA, Mahgoub A, et al. 2006. A missense mutation in pstpip2 is associated with the murine autoinﬂammatory disorder chronic multifocal osteomyelitis. Bone 38:41–47 203. da Cunha JP, Galante PA, de Souza SJ. 2008. Different evolutionary strategies for the origin of caspase-1 inhibitors. J. Mol. Evol. 66:591–97 204. Kersse K, Vanden Berghe T, Lamkanﬁ M, Vandenabeele P. 2007. A phylogenetic and functional overview of inﬂammatory caspases and caspase-1-related CARD-only proteins. Biochem. Soc. Trans. 35:1508–11 205. Saleh M, Vaillancourt JP, Graham RK, Huyck M, Srinivasula SM, et al. 2004. Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms. Nature 429:75–79 206. Kachapati K, O’Brien TR, Bergeron J, Zhang M, Dean M. 2006. Population distribution of the functional caspase-12 allele. Hum. Mutat. 27:975 207. Wang X, Grus W, Zhang J. 2006. Gene losses during human origins. PLoS Biol. 4:e52 208. Xue Y, Daly A, Yngvadottir B, Liu M, Coop G, et al. 2006. Spread of an inactive form of caspase-12 in humans is due to recent positive selection. Am. J. Hum. Genet. 78:659–70 209. Saleh M, Mathison JC, Wolinski MK, Bensinger SJ, Fitzgerald P, et al. 2006. Enhanced bacterial clearance and sepsis resistance in caspase-12-deﬁcient mice. Nature 440:1064–68 210. Roy S, Sharom JR, Houde C, Loisel TP, Vaillancourt JP, et al. 2008. Conﬁnement of caspase-12 proteolytic activity to autoprocessing. Proc. Natl. Acad. Sci. USA 105:4133–38 211. Miggin SM, P˚alsson-McDermott E, Dunne A, Jefferies C, Pinteaux E, et al. 2007. NF-κB activation by the Toll-IL-1 receptor domain protein MyD88 adapter-like is regulated by caspase-1. Proc. Natl. Acad. Sci. USA 104:3372–77 ¨ 212. Keller M, Ruegg A, Werner S, Beer HD. 2008. Active caspase-1 is a regulator of unconventional protein secretion. Cell 132:818–31 213. Chae JJ, Wood G, Richard K, Jaffe H, Colburn NT, et al. 2008. The familial Mediterranean fever protein, pyrin, is cleaved by caspase-1 and activates NF-κB through its N-terminal fragment. Blood 112:1794–803 www.annualreviews.org • The Inflammasomes

205. Demonstrates, as does 209, the role of caspase-12 in regulating endotoxin responsiveness.

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217. Discusses the immunological disease continuum ranging from autoinflammatory diseases to autoimmune diseases.

224. Demonstrates the spectacular effect of IL-1 signaling blockade in diseases characterized by inflammasome overactivation for the first time.

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237. Zychlinsky A, Prevost MC, Sansonetti PJ. 1992. Shigella ﬂexneri induces apoptosis in infected macrophages. Nature 358:167–69 238. Chen Y, Smith MR, Thirumalai K, Zychlinsky A. 1996. A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J. 15:3853–60 239. Hilbi H, Moss JE, Hersh D, Chen Y, Arondel J, et al. 1998. Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB. J. Biol. Chem. 273:32895–900 240. Hersh D, Monack DM, Smith MR, Ghori N, Falkow S, Zychlinsky A. 1999. The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc. Natl. Acad. Sci. USA 96:2396–401 241. Mariathasan S, Weiss DS, Dixit VM, Monack DM. 2005. Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis. J. Exp. Med. 202:1043–49 242. Cervantes J, Nagata T, Uchijima M, Shibata K, Koide Y. 2008. Intracytosolic Listeria monocytogenes induces cell death through caspase-1 activation in murine macrophages. Cell Microbiol. 10:41–52 243. Reig N, Jiang A, Couture R, Sutterwala FS, Ogura Y, et al. 2008. Maturation modulates caspase-1independent responses of dendritic cells to Anthrax lethal toxin. Cell Microbiol. 10:1190–207 244. Yu JW, Fernandes-Alnemri T, Datta P, Wu J, Juliana C, et al. 2007. Pyrin activates the ASC pyroptosome in response to engagement by autoinﬂammatory PSTPIP1 mutants. Mol. Cell 28:214–27 245. Scott AM, Saleh M. 2007. The inﬂammatory caspases: guardians against infections and sepsis. Cell Death Differ. 14:23–31 246. Simi A, Tsakiri N, Wang P, Rothwell NJ. 2007. Interleukin-1 and inﬂammatory neurodegeneration. Biochem. Soc. Trans. 35:1122–26 247. McConnell B, Vertino P. 2004. TMS1/ASC: the cancer connection. Apoptosis 9:5–18 248. Trendelenburg G. 2008. Acute neurodegeneration and the inﬂammasome: central processor for danger signals and the inﬂammatory response? J. Cereb. Blood Flow Metab. 28:867–81 249. Roy N, Mahadevan MS, McLean M, Shutler G, Yaraghi Z, et al. 1995. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell 80:167–78 250. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, et al. 2008. The NALP3 inﬂammasome is involved in the innate immune response to amyloid-β. Nat. Immunol. 9:857–65 251. G´erard N, Caillaud M, Martoriati A, Goudet G, Lalmanach AC. 2004. The interleukin-1 system and female reproduction. J. Endocrinol. 180:203–12 252. Caillaud M, Duchamp G, G´erard N. 2005. In vivo effect of interleukin-1β and interleukin-1RA on oocyte cytoplasmic maturation, ovulation, and early embryonic development in the mare. Reprod. Biol. Endocrinol. 3:26 253. Takehara Y, Dharmarajan AM, Kaufman G, Wallach EE. 1994. Effect of interleukin-1 β on ovulation in the in vitro perfused rabbit ovary. Endocrinology 134:1788–93 254. Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, et al. 2000. Mater, a maternal effect gene required for early embryonic development in mice. Nat. Genet. 26:267–68 255. Qian J, Deveault C, Bagga R, Xie X, Slim R. 2007. Women heterozygous for NALP7/NLRP7 mutations are at risk for reproductive wastage: report of two novel mutations. Hum. Mutat. 28:741 256. Slim R, Mehio A. 2007. The genetics of hydatidiform moles: new lights on an ancient disease. Clin. Genet. 71:25–34 257. Murdoch S, Djuric U, Mazhar B, Seoud M, Khan R, et al. 2006. Mutations in NALP7 cause recurrent hydatidiform moles and reproductive wastage in humans. Nat. Genet. 38:300–2 258. Shao W, Yeretssian G, Doiron K, Hussain SN, Saleh M. 2007. The caspase-1 digestome identiﬁes the glycolysis pathway as a target during infection and septic shock. J. Biol. Chem. 282:36321–29 259. Li J, Yin HL, Yuan J. 2008. Flightless-I regulates proinﬂammatory caspases by selectively modulating intracellular localization and caspase activity. J. Cell Biol. 181:321–33 ¨ 260. Young JL, Sukhova GK, Foster D, Kisiel W, Libby P, Schonbeck U. 2000. The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1β-converting enzyme (caspase-1) activity in human vascular smooth muscle cells. J. Exp. Med. 191:1535–44 261. Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, et al. 1992. Viral inhibition of inﬂammation: cowpox virus encodes an inhibitor of the interleukin-1 β converting enzyme. Cell 69:597–604

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Human Marginal Zone B Cells Jean-Claude Weill, Sandra Weller, and Claude-Agn`es Reynaud INSERM U783, D´eveloppement du Syst`eme Immunitaire, Universit´e Paris Descartes, Facult´e de M´edecine, Site Necker-Enfants Malades, 75730 Paris Cedex 15 France; email: [email protected], [email protected]

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Key Words

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

IgM memory, somatic hypermutation, hyper-IgM syndrome, antipolysaccharide response

This article’s doi: 10.1146/annurev.immunol.021908.132607 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0267$20.00

Abstract Human marginal zone (MZ) B cells are, in a sense, a new entity. Although they share many properties with their mouse counterpart, they also display striking differences, such as the capacity to recirculate and the presence of somatic mutations in their B cell receptor. These differences are the reason they are often not considered a separate, rodent-like B cell lineage, but rather are considered IgM memory B cells. We review here our present knowledge concerning this subset and the arguments in favor of the proposition that humans have evolved for their MZ B cell compartment a separate B cell population that develops and diversiﬁes its Ig receptor during ontogeny outside T-dependent or T-independent immune responses.

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INTRODUCTION Splenic marginal zone: a ring structure around B cell follicles in the spleen, composed mainly of B lymphocytes, together with speciﬁc resident macrophages and stromal/reticular cells

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MZ: marginal zone B cell receptor (BCR): a term that refers to the surface immunoglobulin molecule irrespective of its heavy chain isotype Germinal center (GC): a specialized structure in secondary lymphoid organs involved in the afﬁnity maturation of T-dependent responses through proliferation, hypermutation, isotype switch, and selection of antigenresponsive B cells

Most of the actual knowledge on splenic marginal zone (MZ) B cells, deﬁned 25 years ago as a distinct lymphocyte lineage, comes from studies performed in rodents (1–3). These cells in mice and humans share many common properties, but, as developed in this review, there are also striking differences concerning their anatomical localization and their physiological and diversiﬁcation properties. Concerning the latter point, the presence of somatic mutations in the IgM receptor of human MZ B cells has caused confusion about their deﬁnition, leading these cells to be called IgM memory B cells by most authors, a term never used for rodent MZ B cells, which mostly bear an unmutated B cell receptor (BCR) (4, 5). This confusion was reenforced by the fact that the MZ is a complex niche in which true memory B cells generated by T cell–dependent responses can transit, as observed in several experimental settings in rodents (6, 7). Are these mutated IgM+ B cells, which constitute most of the splenic human MZ, bona ﬁde memory B cells derived at an early stage of the germinal center (GC) reaction induced by T-dependent responses? Are they products of T-independent immune responses that produce B cells with fewer somatic mutations (8)? Or are they, along with their circulating counterpart, a separate B cell lineage as in rodents? We try to develop the arguments that have fostered this controversy and present the data that, in our opinion, support the notion that human MZ B cells are a true separate lineage with unique, species-speciﬁc properties.

ANATOMICAL LOCALIZATION The Spleen Rodents. Blood enters the spleen through the splenic artery and then exits the circulation in the marginal sinus. Blood ﬂows through the MZ to the white and red pulp before leaving the spleen through the venous circulation. MZ B cells express the S1P1 and S1P3 lysophospholipid receptors that permit, along with the integrins αL β2 and α4 β1 , the B cells to be retained 268

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in the MZ (9, 10). Because recirculating naive lymphocytes do not express these molecules, they may cross the MZ freely. While passing through the marginal sinus, blood circulation slows down, thus favoring the encounter between blood-borne antigens and MZ B cells. Although they are considered a sessile B cell subset, MZ B cells do not always stay in the MZ because they can shuttle to the follicles and transport antigens to follicular dendritic cells through their complement receptor (11). They can also, after activation through their BCR, move to the T cell zone and either activate T cells or differentiate into plasmablasts. The rodent MZ contains, in addition to B cells, MZ macrophages harboring a unique set of pattern-recognition receptors, the C-type lectin SIGNR1, and the type I scavenger receptor MARCO. It also contains metallophilic macrophages located at the white pulp border that harbor the sialoadhesin SIGLEC-1 and a set of nonresident dendritic cells (10, 12). Humans. In humans, B cell follicles are not separated from the MZ by marginal sinuses. There is nevertheless a clear MZ (which, as in rodents, is more clearly identiﬁed when there is a GC) separated into an outer and inner part by a MAdCAM-1-positive ﬁbroblast layer. Surrounding the outer MZ is a large perifollicular zone containing sialoadhesin-positive macrophages. Because terminal vessels directly open into the perifollicular zone, lymphocytes may exit the circulation in this structure and then cross the MZ in their route back to the white pulp. Thus, despite anatomical differences that also include differences in the cellular content of this region, as for example the absence of metallophilic MZ macrophages, human MZ B cells are also constantly brought in contact with blood-borne pathogens (9, 13, 14). As we discuss below, the human splenic MZ has a unique role because it is a place where MZ B cells both reside and respond to T-independent antigens and a place they transit through on their way to the general circulation. Resident stromal cells may also contribute to the unique characteristics of the human MZ (15).

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MZ-like B cells are present in the inner wall of the subcapsular sinus in lymph nodes, in the crypt epithelium of tonsils, and under the dome epithelium of Peyer’s patches in gut-associated lymphoid tissues (GALT) (4, 16–19). The central role of the spleen in the defense against encapsulated bacteria tends to indicate that these accessory MZ-like regions may not have an immune protective capacity similar to the spleen (20–22). Nevertheless, they could play a protective role when the spleen is absent and also during ontogeny as sites where MZ B cells could possibly circulate, develop, and diversify their Ig receptor (see below).

MARGINAL ZONE B CELLS ARE PRESENT IN BLOOD AND IN SPLEEN Maurer et al. (23) originally reported the presence of a large CD27+ B cell subset in the adult peripheral blood in humans, a subset further divided into IgD+ and IgD− B cells (24). Moreover, unlike CD27− B cells, CD27+ B

cells were deﬁned as large cells with abundant cytoplasm that could secrete Ig in the presence of SAC (Staphylococcus aureus Conwan strain) and IL-2. Although IgM+ B cells carrying a mutated BCR had been described in human blood and bone marrow (25–27), a direct link between the presence of the CD27 marker and somatic mutations in the BCR was established by Klein and collaborators (28), who concluded that 40% of the blood B cells were CD27+ , comprising IgM+ IgD+ , IgM-only, and switched IgM− IgD− B cells in roughly equal proportions, all with mutated Ig genes. Moreover, these authors conﬁrmed that they were large cells prone to secrete Ig, and they therefore proposed that blood CD27+ B cells were, most likely, antigen-primed, post-GC memory B cells. Today, most investigators in the ﬁeld agree that there are two main B cell compartments in blood, a naive CD27− subset accounting for 60–70% of total B cells and a so-called memory CD27+ subset composed of IgM+ IgD+ and IgM− IgD− B cell fractions, representing each around 15–20% of total B cells (with large individual variations in the percentages) (Figure 1a). The CD27+ IgM-only

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subset accounts for only a small percentage of total B cells in normal individuals, but it can be expanded in some speciﬁc pathological cases (autoimmunity or genetic deﬁciency; see below) (29–31). The CD27 marker does not appear as an unequivocal marker of memory B cells, however. Effectively, isotype-switched CD27− B cells expressing a mutated receptor may represent in blood up to one-third of the total IgG+ or IgA+ B cells (32–34). Naive B cells can be discriminated from these bona ﬁde CD27− memory B cells by their expression of the ATP-binding cassette B1 transporter (32). Conversely, surface CD27 expression was also reported for nonmemory B cells, notably for CD19+ Ig-negative B cell precursors in bone marrow (35–37). The same three major B cell subsets can be observed in the human adult spleen, with the addition of two populations in young children: transitional B cells (also detectable in blood) (38–40) and GC B cells that constitute the major 104 10

fraction of the IgD− CD27+ subset in individuals undergoing classical childhood vaccinations (Figure 2). IgM+ IgD+ CD27+ B cells in blood and spleen display a similar phenotype to one another, IgMhigh IgDlow CD23− CD21+ CD1c+ and mainly CD5− (30, 41, 42). This unique combination of markers is reminiscent of the markers that characterize MZ B cells in rodents, which are described as CD23− CD21+ CD1d+ (2). CD1 molecules are nonpolymorphic MHC class I–like molecules containing an antigenbinding hydrophobic pocket in which lipid antigens can be anchored and presented to lipid-speciﬁc T cells. Mice only express the CD1d molecule, which can present lipid antigens to a set of natural killer T cells with a restricted T cell receptor repertoire (43). Humans express a family of CD1 isoforms (CD1a to CD1e) that present lipids to clonally diverse T cells (44). The function of the CD1c receptor in human MZ B cells remains unknown. It is tempting to speculate that speciﬁc

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CD38 Figure 2 Transitional and germinal center (GC) B cells decline with age in the human spleen. Flow cytometry analysis of CD19-gated B cells after CD19, CD24, and CD38 labeling allows the identiﬁcation of transitional/ immature (CD38high CD24high ) and GC (CD38high CD24− ) B cell subsets from splenic samples at different ages (patients described in Reference 60, with one additional case). Splenic transitional B cells have a CD27−/low phenotype, whereas GC B cells are CD27low/+ (not shown). 270

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CD1c-restricted T cells may contribute to the development or to the immune response of these B cells through recognition of some particular self or exogenous lipid molecules they would present (45–47).

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HISTOLOGICAL CHARACTERIZATION OF SPLENIC MARGINAL ZONE B CELLS Splenic MZ B cells were deﬁned primarily on the basis of their anatomical localization and their characteristic morphology: slightly larger than mantle zone B cells and with paler, more irregular nuclei in conventional histology (4, 48). The human splenic MZ can show great interindividual variability, but immunohistological studies have shown that in adults, most of the B cells localized in the MZ are characterized by a sIgMhigh sIgDlow CD27+ phenotype, whereas mantle zone B cells are sIgM+ sIgDhigh CD27− (14, 15, 30, 49, 50). MZ B cells also express more CD21 and CD1c than do mantle zone B cells, but they are negative for CD5, CD9, CD10, and CD23 (30, 48, 51, 52). However, as already mentioned, the MZ is a complex niche that can harbor a few IgDhigh CD27− cells, usually scattered over the entire MZ but sometimes also occurring as a ring structure in the outer MZ and most probably corresponding to recirculating follicular B cells. Few surface IgApositive B cells can be observed among CD27+ B cells in the MZ, whereas IgM− CD27+ , which most likely represent switched bona ﬁde memory cells, can be seen in the outer MZ (53). Ettinger et al. (41) described recently by ﬂow cytometry the presence of an IgG+ B cell subset among splenic CD21high CD23− CD27+ B cells, but their localization in the MZ, as proposed in this study, remains to be established.

microdissection studies performed on histological sections of spleen and lymph nodes, which showed that rearranged VH genes ampliﬁed from such isolated cells bore somatic mutations (54, 55). Fewer proliferating Ki67+ cells could be seen in the MZ compared with the adjacent GC, but, whereas clonal expansions could be found among MZ B cells, no sharing of clones was observed between these two regions. The authors logically concluded from these data that “the marginal zone of human spleen is a reservoir of memory B cells” (54). It should be stressed, however, that this work, which is widely cited to support the notion that authentic memory B cells may reside in the MZ, was in fact concluding that the main MZ subset, the IgM+ IgD+ CD27+ B cells, harbored mutated Ig genes. Since the report of Klein et al. (28) describing the presence of somatic mutations in CD27+ peripheral B cells, several publications have documented the mutation frequency of the different CD27+ subsets in both blood and spleen. These estimates were performed either on nonselected regions of the Ig locus (notably the JH intronic sequence downstream of rearranged VH genes) or on functional VH sequences, either at the single-cell level or by restricting the analysis to speciﬁc VH genes (e.g., V3-23) (28, 30, 31). All these data point to an approximately twofold lower mutation frequency in the IgM+ IgD+ CD27+ subset compared with isotype-switched cells (Figure 1b). Most importantly, somatic mutations are detected in at least 90% of the sequences analyzed at the adult stage, indicating that the somatic mutation diversiﬁcation process concerns the IgM+ IgD+ CD27+ population as a whole, and not a restricted subset of these cells.

MUTATIONAL STATUS OF MARGINAL ZONE B CELLS

BLOOD IgM+ IgD+ CD27+ B CELLS WITH MUTATED IMMUNOGLOBULIN GENES ARE PRESENT IN HYPER-IgM PATIENTS

The ﬁrst results reporting the mutational status of splenic human MZ B cells came from

In patients with a hyper-IgM syndrome owing to a CD40 or CD40L genetic deﬁciency, the www.annualreviews.org • Human MZ B Cells

Mantle zone (or corona): histological characterization of naive B cells surrounding germinal centers in secondary lymphoid tissues Hyper-IgM syndrome: a genetic disease corresponding to a failure to perform isotype switching owing either to impaired T-B collaboration or to intrinsic B cell defects in the isotype switch mechanism

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cognate T-B collaboration and the formation of GCs cannot take place. These patients display a mutated IgM+ IgD+ CD27+ B cell subset in the total absence of CD27+ IgM-only and switched B cells in blood. This B cell subset has a low to normal frequency of mutations on its BCR, but in most cases it represents quantitatively about one-third of the value found in age-matched controls (Figure 3) (30, 56–58). These results formed the basis of the original proposition that IgM+ IgD+ CD27+ B cells may develop and mutate along a GC-independent pathway and could be involved in T-independent responses (29, 58). In contrast, in patients with a hyperIgM syndrome caused by a genetic defect in the activation-induced cytidine deaminase (AID) gene, the GC reaction develops but class switch recombination (CSR) and somatic hypermutation (SHM) cannot take place. Interestingly, these patients possess an IgM+ IgD+ CD27+ blood subset and an elevated IgM-only CD27+ subset (both with an unmutated BCR) (30). This supports the notion that IgM-only memory B cells are GC derived, being the direct precursors of switched B cells and therefore accumulating when CSR is compromised (Figure 3).

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DEVELOPMENT AND DIVERSIFICATION OF IgM+ IgD+ CD27+ B CELLS IN YOUNG CHILDREN Children younger than age two fail to respond to T-independent antigens and, notably, to plain polysaccharidic vaccines. This observation led to the propositions that the splenic MZ might be nonfunctional, probably not fully developed (59), or that MZ B cells might be absent at these young ages (29). This is not the case, however, because, starting with a very low level of 0.5–1% of total B cells in cord blood, the number of IgM+ IgD+ CD27+ B cells progressively increases in the blood and spleen of infants, displaying values comparable to those observed for switched B cells at the same age (58, 60) (Figure 4). In parallel with this increase in cell numbers, the V gene mutation frequency gradually increases as the blood IgM+ IgD+ CD27+ B cell subset matures, reaching adult values by two to three years of age (30). Lower mutation frequencies have been observed in spleen samples of the same age (60), but a detailed description of mutation accumulation during the ﬁrst years of life remains to be established

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sections of an 8-month spleen sample, however (60) (Figure 5). A likely explanation for these discrepancies may be the origin of the tissue sample analyzed; surgical samples are probably better protected from autolysis of some surface markers than the ones obtained from autopsies. A more exhaustive analysis of surface marker expression nevertheless remains to be performed to determine how much splenic MZ B cells in infants may differ phenotypically from their adult equivalents.

Figure 4 Parallel emergence of IgM+ IgD+ CD27+ and switched B cells in the blood of children less than two years old. Taken from Reference 60 supplemental data.

for the splenic IgM+ IgD+ CD27+ B cell compartment. It has been reported that the splenic MZ is populated by naive B cells in infants (50). This observation seems to contradict data obtained by ﬂow cytometry. A clear MZ compartment with low CD27, high IgD, and CD1c staining can clearly be distinguished in histological

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REPERTOIRE DIVERSIFICATION OF IgM+ IgD+ CD27+ B CELLS OCCURS OUTSIDE T-DEPENDENT AND T-INDEPENDENT RESPONSES IN INFANTS Switched and IgM+ IgD+ CD27+ B cells share many properties in addition to their mutational status: their CD21+ CD23− phenotype, their large cytoplasm, their propensity to secrete Ig under stimulation, and their parallel emergence during ontogeny. These common

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Figure 5 Presence of IgD+ CD27+ CD1c+ B cells in the splenic marginal zone (MZ) of an 8-month old child. Analysis by confocal microscopy of serial sections of an 8-month splenic sample, double labeled with anti-IgD and anti-CD27 antibodies (a–d ) or with anti-IgD and anti-CD1c antibodies (e–h). CD27low cells were present in the MZ, corresponding to the outer part of the IgD-positive ring surrounding the germinal center (GC). Intense CD27 staining corresponds to T cells. Co-expression of CD1c and IgD at similar intensities was observed in the MZ, resulting in a yellow appearance in the merged panels ( g, h). The region boxed with dotted lines in panels a and b is magniﬁed in panels c and d, and the one marked in panel g is magniﬁed in h. “Co” indicates corona (or mantle zone). Original magniﬁcation: 20x for panels a and b, 40x for panels c, d, and h, 10x for panels e, f, and g. Taken from Reference 60. www.annualreviews.org • Human MZ B Cells

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CDR3: third complementaritydetermining region, constituting a clonal B cell marker for heavy chain V-D-J junctions CDR3 spectratyping: analysis by capillary or standard gel electrophoresis of the Ig heavy chain CDR3 size distribution; discrete electrophoretic peaks corresponding to functional sequences are resolved with a 3-nucleotide spacing TLRs: Toll-like receptors

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features could raise doubts as to whether MZ B cells are really a separate lineage and could be taken as arguments in favor of the contrary proposition, namely that these B cells are memory B cells that have undergone hypermutation in the GC during a T-dependent response but exited the GC at an early IgM-expressing stage before undergoing isotype switch (61, 62). In a recent study, we used the dissociation between T-dependent and T-independent responses that is observed in toddlers, together with the numerous antigenic challenges provided by children’s vaccinations, to determine whether the IgM+ IgD+ CD27+ and switched subsets may be functionally related. We evaluated the clonal diversity of their repertoire by analyzing the distribution of CDR3 lengths of speciﬁc rearranged VH genes (CDR3 spectratyping) and the number of nonredundant sequences corresponding to a deﬁned CDR3 size. In all children analyzed between ages 8 and 24 months, the IgM+ IgD+ CD27+ subset, whether in blood or in spleen, showed a high clonal diversity comparable to naive B cells, while harboring somatic mutations. Switched B cells, in contrast, showed a much more restricted repertoire, which reﬂected the clonal expansions that occurred during antigen-driven responses (Figure 6). Moreover, the abundant GCs present in spleen samples at these young ages allowed the speciﬁc analysis of both mu and gamma transcripts from GC B cells. Here again, GC B cells showed a restricted repertoire, whether analyzed at the mu or gamma transcript level, in marked contrast with IgM+ IgD+ CD27+ splenic B cells, an observation that seems a priori to preclude a differentiation scheme in which IgM-positive GC B cells would be the precursors of the mutated MZ subset (60). Together, the presence of somatic mutations in Ig genes in the absence of any antigen-driven clonal expansion in IgM+ IgD+ CD27+ B cells from young children supports the proposition that this B cell subset may develop and mutate during the ﬁrst years of life without being engaged in T-dependent or T-independent immune responses. Weill

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A ROLE FOR TOLL-LIKE RECEPTORS IN MZ B CELL DEVELOPMENT? Human blood naive CD27− B cells do not express Toll-like receptors (TLRs) and require stimulation through their BCR to express TLR9 and become responsive to CpG stimulation (63). In contrast, IgM+ IgD+ CD27+ B cells and switched B cells express TLR2, 6, 7, 8, 9, and 10 constitutively and proliferate and secrete IgM and IgG or IgA, respectively, upon CpG stimulation (64–66). This observation led Lanzavecchia and colleagues (67) to propose that naive B cells need cognate stimulation to be activated by CpG, whereas memory B cells can be activated by CpG (or by T cell help) in a noncognate mode, with this bystander stimulation contributing to the continual renewal of the plasma cell pool and the serum antibody level. Is there a role for TLRs in the development of MZ B cells and in the responses to T-independent antigens such as encapsulated bacteria? Carsetti and colleagues (68) have reported recently that transitional B cells, characterized as CD24high CD38high and representing 1–2% of peripheral B cells, can, unlike naive B cells, differentiate upon CpG activation into three different subsets, IgM+ IgD+ CD27+ B cells, antibody-secreting cells that produce IgM and switched Ig isotypes, and naive B cells. These authors thus proposed that IgM+ IgD+ CD27+ B cells could be derived from transitional B cells through TLR9 stimulation (68). Our preliminary analysis, performed in collaboration with Casanova and colleagues, of blood samples from children and young adults bearing various genetic deﬁciencies in molecules involved in the TLR signaling pathways, are somehow in contradiction with these results. We have indeed observed in some patients a speciﬁc deﬁciency in the IgM+ IgD+ CD27+ B cell subset, along with a normal naive and switched memory B cell compartment, but the defect harbored by these patients did not seem to involve the TLR9 pathway (S. Weller, H. Delagreverie, M. Bonnet,

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Figure 6 Absence of antigen-induced clonal expansion in blood or splenic MZ B cell subsets of young children. Repertoire diversity was estimated by analyzing the distribution of V3-15 heavy chain CDR3 lengths (CDR3-spectratyping) and by sequencing selected peaks. The regular, quasi-gaussian distribution of CDR3 sizes from IgM+ IgD+ CD27+ B cells contrasts with the irregular CDR3 proﬁle of germinal center or switched B cells. A shift toward smaller CDR3 sizes is observed in the IgM+ IgD+ CD27+ subset compared with naive B cells. Adapted from Reference 60.

A. Puel, C. Picard, E. Meffre, C-A. Reynaud, JC. Weill, J-L. Casanova, manuscript in preparation). The relative contribution of TLR signaling to the development, maintenance, and/or activation of MZ B cells nevertheless remains to be established.

IN VITRO ACTIVATION OF IgM+ IgD+ CD27+ B CELLS Several groups have studied the behavior of IgM+ IgD+ CD27+ B cells after various types of stimulations in vitro and compared them

with the other B cell subsets. Both IgM+ and switched CD27+ B cells are large and granular compared with naive B cells and are more prone to proliferation and Ig secretion under various stimuli (23, 24, 69, 70). IL-21, which is produced by T helper follicular cells, is a potent differentiation factor for human B cells (71–73). In the presence of CD40L, IL-21 induces the differentiation of a small proportion of naive B cells into IgM-secreting cells, with a few of them producing IgG and IgA (73). A larger proportion of CD27+ B cells differentiate into Ig-secreting cells under the same www.annualreviews.org • Human MZ B Cells

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stimulation. IgM+ IgD+ CD27+ B cells produce IgM, but also some IgG and IgA, whereas switched memory B cells produce IgG and IgA. These data conﬁrm that signaling through CD40 in the presence of IL-21 has the capacity to enhance the differentiation of blood B cells into plasmocytes and to induce their switching to other isotypes. Switching of these B cells in vivo upon T-independent antigenic stimulation may thus require speciﬁc interactions such as CD40-CD40L as well as TLR signals that may be provided by accessory cells or T cells in a noncognate fashion. This observation can explain why hyper-IgM patients who have a CD40-CD40L signaling defect and who display a mutated IgM+ IgD+ CD27+ B cell population are nevertheless unable to produce protective IgG or IgA antibodies against encapsulated bacteria.

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THE PARADOX OF THE ANTI-POLYSACCHARIDE RESPONSE IN HUMANS The human antibody response to nonconjugated vaccines against encapsulated bacteria (Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus inﬂuenzae) is essentially composed of mutated antibodies, with a recurrent usage of a few VH genes (V3-15, V3-23, V330). Moreover, shared modiﬁcations between different isotypes indicate that intraclonal class switching arises during the maturation of the response (e.g., IgG to IgA) and that, depending on the serotypes analyzed that differ essentially by their sugar contents, the response can be more or less oligoclonal (74, 75). It remains striking that humans, as opposed to rodents, have selected mutated antibodies for their protection against encapsulated bacteria (76). Effectively, unmutated natural polyreactive IgM antibodies that bind all pneumoccocal polysaccharide capsule serotypes are present in healthy individuals but display a weak protective capacity against pneumoccocal infections when injected into mice, with the animal’s death delayed by only a maximum of 24 h after a lethal challenge of Streptococcus pneumoniae (77). Strik276

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ingly, in the same test but after a pneumococcal or a Haemophilus inﬂuenza vaccination, the mutated antibodies raised could confer almost complete protection (78, 79). The sequence of events taking place during a T-independent immune response has been followed in a case of splenectomy performed 8 days after immunization with nonconjugated pneumococcal and meningococcal vaccines. Blood was analyzed before vaccination, 8 days after vaccination at the time of splenectomy, and 5 weeks after vaccination, together with the splenic sample. A speciﬁc B cell clone (with a V3-15 rearrangement known to be mobilized in such vaccinations) was expanded 8 days after vaccination in the splenic and blood IgM+ IgD+ CD27+ B cell subsets, while present at a low frequency in the blood before vaccination and already mutated at this stage. Strikingly, the same clone was also expanded in mu- and gamma-expressing plasma cells isolated from the spleen after vaccination (S. Weller, C-A. Reynaud, J-C. Weill, unpublished observations), and was still present, albeit at a somewhat reduced frequency, in blood IgM+ IgD+ CD27+ B cells four weeks later (30). The mobilization of B cells with mutated Ig genes in antipolysaccharidic responses has constituted a long-standing paradox (80). The fast timing of their emergence appeared incompatible with a de novo–triggered mutagenesis of naive B cells, suggesting that they are recruited from a pool of preexisting memory B cells generated during classical T-dependent responses. If this were the case, it would remain difﬁcult to explain why switched memory B cells that are generated by T-dependent responses soon after birth would only respond to encapsulated bacteria after the age of two years. The ontogenic diversiﬁcation and maturation of MZ B cells in humans, together with their preactivated phenotype, appears as a straightforward explanation for these puzzling observations: Prediversiﬁed human splenic MZ B cells will be mobilized very rapidly after injection of a polysaccharidic vaccine and, once triggered, will proliferate, switch isotype, and differentiate into IgM-, IgG-, and IgA-secreting

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plasmocytes without generating any memory (29, 30, 80).

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MAINTENANCE AND DIVERSIFICATION OF THE HUMAN MZ B CELL POPULATION It has been proposed that mouse splenic MZ B cells are derived from T2 transitional B cells (81, 82) and that the basis of this lineage choice is under the control of the Notch2 signaling pathway (83). Moreover, data indicate that, in the absence of a continual export of newly formed B cells from bone marrow, mouse MZ B cells are able to maintain themselves, unlike naive B cells that show progressive decay, implying that MZ B cells can self-renew in these speciﬁc conditions (84, 85). Nevertheless, one cannot infer from these data that the mouse MZ B cell population self-renews constantly in the physiological context. It is also not possible to decide whether there is one central wave of MZ B cell production during ontogeny or whether these cells are, like naive B cells, constantly generated from bone marrow precursors. We know even less concerning human MZ B cells. In humans, mutations accumulate with time in the MZ B cell subset of very young children, their frequency reaching adult values by two to four years of age. Moreover, human MZ B cells do not display a V gene repertoire that differs much from the one displayed by naive B cells (31). It seems reasonable, therefore, to propose that there is in humans one wave of development and diversiﬁcation of the MZ B cell subpopulation during the ﬁrst years of life and that, as in rodents, these cells may diverge from naive B cells at the transitional stage in the spleen. The MZ subset can be replenished in adult life after bone marrow grafting or after anti-CD20 B cell depletion, the slow recovery of this subset in fact following a normal ontogenic time scale (86–88). One can therefore envision that a small renewal of the MZ population from bone marrow precursors may occur throughout life in speciﬁc lymphopenic situations.

To understand human MZ B cell ontogeny, one obviously needs to know in which sites these cells diversify their BCRs (89). No AID expression was detected in splenic MZ B cells (61), but a low expression level was observed in splenic IgM+ IgD+ CD27+ B cells of young children (under two years), implying that this could be a site where these cells would slowly and progressively diversify (60). The importance of the GC compartment at these ages nevertheless makes any ﬁrm conclusion difﬁcult. By analogy with other species using the same type of strategies to generate their B cell preimmune repertoire, SHM could take place in MZ-like regions in GALT during the ﬁrst years of life, with B cell proliferation driven by speciﬁc bacterial superantigens, as recently demonstrated for the development of the antibody repertoire in the rabbit model (90–92).

Congenital asplenia: a genetic deﬁciency of unknown origin in humans, resulting in the absence of splenic development, frequently associated with cardiovascular malformations

THE MZ B CELL SUBSET CAN BE FORMED IN THE ABSENCE OF THE SPLEEN BUT DECLINES WITH AGE If there is one central wave of MZ B cell formation during ontogeny, these cells have to maintain themselves throughout life. In patients having undergone splenectomy, there is a slow decrease of their blood MZ B cells, as if the splenic MZ was the place in which these cells would not only respond to blood-borne antigens but also receive signals allowing them to survive and to recirculate (Reference 93 and Figure 7a). Strikingly, and in opposition with results published by Carsetti and colleagues (29), a normal blood MZ compartment was observed in young patients with congenital asplenia, implying that the spleen was not strictly required for this population to develop and diversify during ontogeny (30) (Figure 7b). As observed for splenectomized patients, this circulating MZ subset failed to expand in older asplenic individuals (29, 30) (Figure 7b): The milder impact observed in the case of asplenia could correspond to the fact that MZ precursors might ﬁnd alternative sites to develop, albeit with a lower efﬁciency, whereas splenectomy www.annualreviews.org • Human MZ B Cells

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Figure 7 Impact of splenectomy and asplenia on the peripheral MZ subset. (upper part) IgM+ IgD+ CD27+ and switched B cell subsets are analyzed in patients who had undergone splenectomy 7–50 years previously (SPL, splenectomized patients; HC, healthy controls). (lower part) Patients with congenital asplenia (ASP) are analyzed, classiﬁed in two categories, under 4 years old and over 4 years old. IgM+ IgD+ CD27+ B cells declined markedly in splenectomized patients, whereas both IgD+ CD27+ and switched subsets were affected, albeit slightly differently, in asplenic adults. Data are taken from Reference 30 for asplenic patients; splenectomized patients are described in Reference 105, with additional unpublished cases for both categories.

would result in a massive depletion of the mature MZ pool that would fail to properly self-renew outside the spleen. Nevertheless, the higher susceptibility to encapsulated bacterial infections displayed by asplenic and splenectomized patients indicates that a MZ B cell subset and a functional spleen are both necessary for an efﬁcient antibacterial defense.

POSITIVE AND NEGATIVE SELECTION IN THE MZ SUBSET Rosner et al. (94) reported that human blood IgM+ IgD+ CD27+ B cells harbor shorter VH CDR3 lengths compared with naive B cells, 278

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an observation previously made for rodent MZ B cells (5, 95, 96). This repertoire bias was also observed for either blood or spleen IgM+ IgD+ CD27+ B cells in infants, indicating that such a selection takes place during their emergence in the absence of antigenic responses (60). For mouse MZ B cells, positive selection may occur according to a signal strength model (97), and, similarly, weak signaling through self or commensal antigens could select for speciﬁc CDR3 lengths. Alternatively, a shorter CDR3 may impinge on the tonic signal delivered by the BCR and direct these cells preferentially toward the MZ lineage.

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Figure 8 A differentiation scheme for MZ B cells. Formation of MZ B cells is schematized, by analogy with the mouse B cell system, as a branching point from immature/transitional B cells (branch A). Alternative views are represented (dotted lines) as either a differentiation branching from naive B cells (branch B), or as an early product of the GC reaction (branch C). The three checkpoints of autoreactivity described by M. Nussenzweig and colleagues (marked 1, 2, and 3) (98, 99) are represented in the context of the ﬁrst differentiation scheme.

Negative selection also operates at distinct steps during the differentiation of human B cells by purging these cells from autoreactivity at two checkpoints in bone marrow: (a) at the pre-B to immature B transition and (b) at the periphery, between transitional/immature and naive B cells (98). MZ B cells go through a speciﬁc self-reactivity checkpoint before the appearance of somatic mutations (99). Depending on the differentiation scheme proposed for these cells, such a selection step would constitute either a third checkpoint in the emergence of the MZ B cell subset or a second one, symmetrical to the one operating on naive B cells (Figure 8). Moreover, switched memory B cells show a rather high level of autoreactivity compared with naive B cells, an autoreactiv-

ity most probably produced by somatic mutations (100). Such increase in autoreactivity was not observed in mutated IgM+ IgD+ CD27+ B cells, suggesting the existence of an additional control as these cells diversify their Ig receptors.

CONCLUSION Human MZ B cells are rarely named as such in the scientiﬁc literature because of their mutated BCRs that cause them to be considered classical memory B cells. We and other authors have shown that birds, sheep, cattle, and rabbits diversify their B cell antibody preimmune repertoire in GALT by postrearrangement processes such as gene conversion and/or SHM www.annualreviews.org • Human MZ B Cells

Autoreactivity: capacity of lymphocytes to recognize and to be activated by self-antigens

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(101, 102). The description of a B cell subset that diversiﬁes its Ig genes outside T-dependent or T-independent responses suggests that humans may have conserved for MZ B cells the strategies used by many animal species to generate their B cell preimmune repertoire (103,

104). This prediversiﬁcation by hypermutation may provide the MZ B cell repertoire with the avidity and afﬁnity required for a fast protective response against highly pathogenic encapsulated bacteria that do not trigger classical T-dependent responses.

FUTURE ISSUES 1. Where and when do MZ B cells diversify their BCR during development? Annu. Rev. Immunol. 2009.27. Downloaded from arjournals.annualreviews.org by University of Bergen UNIVERSITETSBIBLIOTEKET on 01/13/09. For personal use only.

2. Which ligands/signaling pathways are necessary to drive their development? 3. Is there a single wave of MZ B cell formation during ontogeny? 4. What causes the unresponsiveness to T-independent antigens in infants and elderly people? Is it possible to design adjuvants that could promote long-lasting T-independent responses? 5. Could one develop a differentiation system in humanized mice to study and manipulate MZ B cell ontogeny? 6. Could one identify speciﬁc differentiation/activation stages of MZ B cells that would represent the normal counterparts of marginal zone lymphomas?

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

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12. Balazs M, Martin F, Zhou T, Kearney J. 2002. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 17:341–52 13. Timens W, Poppema S. 1985. Lymphocyte compartments in human spleen. An immunohistologic study in normal spleens and uninvolved spleens in Hodgkin’s disease. Am. J. Pathol. 120:443–54 14. Steiniger B, Timphus EM, Barth PJ. 2006. The splenic marginal zone in humans and rodents: an enigmatic compartment and its inhabitants. Histochem. Cell Biol. 126:641–48 15. Steiniger B, Barth P, Hellinger A. 2001. The perifollicular and marginal zones of the human splenic white pulp: Do ﬁbroblasts guide lymphocyte immigration? Am. J. Pathol. 159:501–12 16. Stein H, Bonk A, Tolksdorf G, Lennert K, Rodt H, Gerdes J. 1980. Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin’s lymphomas. J. Histochem. Cytochem. 28:746– 60 17. Spencer J, Finn T, Pulford KA, Mason DY, Isaacson PG. 1985. The human gut contains a novel population of B lymphocytes which resemble marginal zone cells. Clin. Exp. Immunol. 62:607–12 18. Liu YJ, Barthelemy C, de Bouteiller O, Arpin C, Durand I, Banchereau J. 1995. Memory B cells from human tonsils colonize mucosal epithelium and directly present antigen to T cells by rapid up-regulation of B7-1 and B7-2. Immunity 2:239–48 19. Dono M, Zupo S, Leanza N, Melioli G, Fogli M, et al. 2000. Heterogeneity of tonsillar subepithelial B lymphocytes, the splenic marginal zone equivalents. J. Immunol. 164:5596–604 20. Schutze GE, Mason EO Jr, Barson WJ, Kim KS, Wald ER, et al. 2002. Invasive pneumococcal infections in children with asplenia. Pediatr. Infect. Dis. J. 21:278–82 21. Zandvoort A, Timens W. 2002. The dual function of the splenic marginal zone: essential for initiation of anti-TI-2 responses but also vital in the general ﬁrst-line defense against blood-borne antigens. Clin. Exp. Immunol. 130:4–11 22. Brendolan A, Rosado MM, Carsetti R, Selleri L, Dear TN. 2007. Development and function of the mammalian spleen. Bioessays 29:166–77 23. Maurer D, Fischer GF, Fae I, Majdic O, Stuhlmeier K, et al. 1992. IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset. J. Immunol. 148:3700–5 24. Agematsu K, Nagumo H, Yang FC, Nakazawa T, Fukushima K, et al. 1997. B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production. Eur. J. Immunol. 27:2073–79 25. van Es JH, Meyling FH, Logtenberg T. 1992. High frequency of somatically mutated IgM molecules in the human adult blood B cell repertoire. Eur. J. Immunol. 22:2761–64 26. Klein U, Kuppers R, Rajewsky K. 1997. Evidence for a large compartment of IgM-expressing memory B cells in humans. Blood 89:1288–98 27. Paramithiotis E, Cooper MD. 1997. Memory B lymphocytes migrate to bone marrow in humans. Proc. Natl. Acad. Sci. USA 94:208–12 28. Klein U, Rajewsky K, Kuppers R. 1998. Human immunoglobulin (Ig)M+ IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J. Exp. Med. 188:1679–89 29. Kruetzmann S, Rosado MM, Weber H, Germing U, Tournilhac O, et al. 2003. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. J. Exp. Med. 197:939–45 30. Weller S, Braun MC, Tan BK, Rosenwald A, Cordier C, et al. 2004. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversiﬁed immunoglobulin repertoire. Blood 104:3647–54 31. Tian C, Luskin GK, Dischert KM, Higginbotham JN, Shepherd BE, Crowe JE Jr. 2007. Evidence for preferential Ig gene usage and differential TdT and exonuclease activities in human naive and memory B cells. Mol. Immunol. 44:2173–83 32. Wirths S, Lanzavecchia A. 2005. ABCB1 transporter discriminates human resting naive B cells from cycling transitional and memory B cells. Eur. J. Immunol. 35:3433–41 33. Fecteau JF, Cote G, Neron S. 2006. A new memory CD27− IgG+ B cell population in peripheral blood expressing VH genes with low frequency of somatic mutation. J. Immunol. 177:3728–36 www.annualreviews.org • Human MZ B Cells

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77. Baxendale HE, Johnson M, Stephens RC, Yuste J, Klein N, et al. 2008. Natural human antibodies to pneumococcus have distinctive molecular characteristics and protect against pneumococcal disease. Clin. Exp. Immunol. 151:51–60 78. Lucas AH, Larrick JW, Reason DC. 1994. Variable region sequences of a protective human monoclonal antibody speciﬁc for the Haemophilus inﬂuenzae type B capsular polysaccharide. Infect. Immun. 62:3873–80 79. Zhong Z, Burns T, Chang Q, Carroll M, Pirofski L. 1999. Molecular and functional characteristics of a protective human monoclonal antibody to serotype 8 Streptococcus pneumoniae capsular polysaccharide. Infect. Immun. 67:4119–27 80. Weller S, Reynaud CA, Weill JC. 2005. Vaccination against encapsulated bacteria in humans: Paradoxes. Trends Immunol. 26:85–89 81. Allman D, Srivastava B, Lindsley RC. 2004. Alternative routes to maturity: branch points and pathways for generating follicular and marginal zone B cells. Immunol. Rev. 197:147–60 82. Su TT, Guo B, Wei B, Braun J, Rawlings DJ. 2004. Signaling in transitional type 2 B cells is critical for peripheral B-cell development. Immunol. Rev. 197:161–78 83. Saito T, Chiba S, Ichikawa M, Kunisato A, Asai T, et al. 2003. Notch2 is preferentially expressed in mature B cells and indispensable for marginal zone B lineage development. Immunity 18:675–85 84. Carvalho TL, Mota-Santos T, Cumano A, Demengeot J, Vieira P. 2001. Arrested B lymphopoiesis and persistence of activated B cells in adult interleukin 7−/− mice. J. Exp. Med. 194:1141–50 85. Hao Z, Rajewsky K. 2001. Homeostasis of peripheral B cells in the absence of B cell inﬂux from the bone marrow. J. Exp. Med. 194:1151–64 86. Avanzini MA, Locatelli F, Dos Santos C, Maccario R, Lenta E, et al. 2005. B lymphocyte reconstitution after hematopoietic stem cell transplantation: functional immaturity and slow recovery of memory CD27+ B cells. Exp. Hematol. 33:480–86 87. Roll P, Palanichamy A, Kneitz C, Dorner T, Tony HP. 2006. Regeneration of B cell subsets after transient B cell depletion using anti-CD20 antibodies in rheumatoid arthritis. Arthritis. Rheum. 54:2377–86 88. Anolik JH, Friedberg JW, Zheng B, Barnard J, Owen T, et al. 2007. B cell reconstitution after rituximab treatment of lymphoma recapitulates B cell ontogeny. Clin. Immunol. 122:139–45 89. Weller S, Reynaud CA, Weill JC. 2005. Splenic marginal zone B cells in humans: Where do they mutate their Ig receptor? Eur. J. Immunol. 35:2789–92 90. Rhee KJ, Sethupathi P, Driks A, Lanning DK, Knight KL. 2004. Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire. J. Immunol. 172:1118–24 91. Rhee KJ, Jasper PJ, Sethupathi P, Shanmugam M, Lanning D, Knight KL. 2005. Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues. J. Exp. Med. 201:55–62 92. Weill JC, Reynaud CA. 2005. Do developing B cells need antigen? J. Exp. Med. 201:7–9 93. Di Sabatino A, Rosado MM, Ciccocioppo R, Cazzola P, Morera R, et al. 2005. Depletion of immunoglobulin M memory B cells is associated with splenic hypofunction in inﬂammatory bowel disease. Am. J. Gastroenterol. 100:1788–95 94. Rosner K, Winter DB, Tarone RE, Skovgaard GL, Bohr VA, Gearhart PJ. 2001. Third complementaritydetermining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than nonmutated genes. Immunology 103:179–87 95. Schelonka RL, Tanner J, Zhuang Y, Gartland GL, Zemlin M, Schroeder HW Jr. 2007. Categorical selection of the antibody repertoire in splenic B cells. Eur. J. Immunol. 37:1010–21 96. Martin F, Kearney JF. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2:323–35 97. Pillai S, Cariappa A, Moran ST. 2004. Positive selection and lineage commitment during peripheral B-lymphocyte development. Immunol. Rev. 197:206–18 98. Wardemann H, Yurasov S, Schaefer A, Young JW, Meffre E, Nussenzweig MC. 2003. Predominant autoantibody production by early human B cell precursors. Science 301:1374–77 99. Tsuiji M, Yurasov S, Velinzon K, Thomas S, Nussenzweig MC, Wardemann H. 2006. A checkpoint for autoreactivity in human IgM+ memory B cell development. J. Exp. Med. 203:393–400 100. Tiller T, Tsuiji M, Yurasov S, Velinzon K, Nussenzweig MC, Wardemann H. 2007. Autoreactivity in human IgG+ memory B cells. Immunity 26:205–13 101. Weill JC, Reynaud CA. 1998. Galt versus bone marrow models of B cell ontogeny. Dev. Comp. Immunol. 22:379–85

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102. Lanning D, Osborne BA, Knight KL. 2004. Immunoglobulin genes and generation of antibody repertoires in higher vertebrates: A key role for GALT. In Molecular Biology of B Cells, ed. FW Alt, T Honjo, MS Neuberger, pp. 433–48. London: Elsevier 103. Tarlinton D. 2008. Sheepish B cells: evidence for antigen-independent antibody diversiﬁcation in humans and mice. J. Exp. Med. 205:1251–54 104. Weill JC, Weller S, Reynaud CA. 2004. A bird’s eye view on human B cells. Semin. Immunol. 16:277–81 105. Mamani-Matsuda M, Cosma A, Weller S, Faili A, Staib C, et al. 2008. The human spleen is a major reservoir for long-lived vaccinia virus-speciﬁc memory B cells. Blood 111:4653–59

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Diane Mathis and Christophe Benoist Section on Immunology and Immunogenetics, Joslin Diabetes Center; Department of Medicine, Brigham and Women’s Hospital; Harvard Medical School; and the Harvard Stem Cell Institute, Boston, Massachusetts 02215; email: [email protected]

Annu. Rev. Immunol. 2009. 27:287–312

Key Words

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

T cell tolerance, autoimmunity, thymus, transcription factor

This article’s doi: 10.1146/annurev.immunol.25.022106.141532

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0287$20.00

Mutations in the transcriptional regulator, Aire, cause APECED, a polyglandular autoimmune disease with monogenic transmission. Animal models of APECED have revealed that Aire plays an important role in T cell tolerance induction in the thymus, mainly by promoting ectopic expression of a large repertoire of transcripts encoding proteins normally restricted to differentiated organs residing in the periphery. The absence of Aire results in impaired clonal deletion of self-reactive thymocytes, which escape into the periphery and attack a variety of organs. In addition, Aire is a proapoptotic factor, expressed at the ﬁnal maturation stage of thymic medullary epithelial cells, a function that may promote cross-presentation of the antigens encoded by Aireinduced transcripts in these cells. Transcriptional regulation by Aire is unusual in being very broad, context-dependent, probabilistic, and noisy. Structure/function analyses and identiﬁcation of its interaction partners suggest that Aire may impact transcription at several levels, including nucleosome displacement during elongation and transcript splicing or other aspects of maturation.

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For decades, immunological tolerance has been one of the favorite enigmas of immunologists. How does the immune system exercise its primary function of dispensing with microbial invaders while remaining inert to the body’s own constituents? The short answer is that the immune system—more speciﬁcally, the lymphocyte component of the adaptive immune system—is rendered tolerant to the latter but not to the former. Over the years, it has become apparent that an intricate network of mechanisms establishes and maintains lymphocyte tolerance (reviewed in 1–3). These mechanisms have classically been grouped into two broad categories: central and peripheral. Central tolerance relates to immature lymphocytes as they differentiate in the primary lymphoid organs, the thymus for T cells and the fetal liver or bone marrow for B cells. Hence, the relevant antigens would be those synthesized speciﬁcally by stromal cells in these organs, speciﬁcally by hematopoietic cells that circulate through them or, ubiquitously, by all cells. The major mechanisms in operation in central tolerance are clonal deletion, inactivation, or diversion of self-reactive lymphocytes. Peripheral tolerance, on the other hand, concerns mature lymphocytes after they have exited the primary lymphoid organs and are circulating through the blood, lymph, and secondary lymphoid organs or are percolating through parenchymal tissues. Relevant antigens would be tissular substances not encountered previously, during lymphocyte differentiation. Clonal deletion, anergy, and diversion are operative in peripheral tolerance as well, but a variety of other mechanisms also come into play, including clonal ignorance and suppression of self-reactive lymphocytes. Thus, it seems that evolution has furnished organisms with a comprehensive net of protection against potentially destructive selfdirected—or autoimmune—responses. However, this impression has derived largely from results on genetically engineered mouse models. One is forced to ask how these different mechanisms integrate to enforce tolerance in unmanipulated mice and humans: Which of the

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diverse tolerization modes dominate? Which are experimental anomalies? To what extent are they interdependent or redundant? How do their roles vary with genetics, environment, or developmental stage? One approach to addressing such questions concerning how a tolerant state is achieved or maintained is to explore how it is lost, i.e., in contexts of autoimmunity. In this regard, the multiorgan autoimmune disease autoimmune polyendocrinopathy-candidiasesectodermal dystrophy (APECED) has yielded important insights of late (reviewed in 4).

APECED REVEALS AIRE APECED is a rare but devastating primary immunodeﬁciency disease. Classiﬁed as a “Disease of Immune Dysregulation” (Type IV PID) (5), APECED is characterized by a set of three abnormal features—chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufﬁciency (6)—a triad ﬁrst reported in 1946 (7). Classically, an individual must present with at least two of these three clinical abnormalities to be diagnosed with APECED, which usually occurs before the age of ten. However, more recently, identiﬁcation of the causal genetic lesion has permitted extension of the diagnosis to patients with atypical presentations—for example, with one of the hallmark features in isolation (8–12). Most patients also routinely exhibit a variable number of other autoimmune manifestations, including thyroiditis, type 1 diabetes (T1D), ovarian failure, alopecia, or hepatitis. These secondary features differ widely from individual to individual, even between siblings with exactly the same genetic lesion and similar environmental exposures. APECED has unusually simple genetics for an autoimmune disease (13). Almost always exhibiting an autosomal recessive mode of inheritance, it is generally considered to be a monogenic disorder, in striking contrast to most other autoimmune diseases, with their often knotty genetic inﬂuences. However, more recent analyses have revealed effects of additional genetic loci, in particular the human leukocyte antigen (HLA) complex, on certain

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disease manifestations (e.g., 14, 15). APECED is a rare disorder, although there are pockets of elevated frequency in Finland, Sardinia, and Iran, where it can afﬂict as many as 1 in 10,000 people, probably reﬂecting particular founder mutations (16). Family studies permitted localization of the underlying gene to the q22 region of chromosome 21 (17). A major leap forward in our understanding of APECED came in 1997 when two groups used heroic positional strategies to clone the responsible gene (18, 19). The protein encoded at this locus was termed Aire for autoimmune regulator. As detailed below (see section entitled Molecular Mechanisms of Aire), this 545 amino acid protein has several structural domains, reminiscent of those found in known transcription factors, prompting the speculation that it is some sort of transcriptional regulator. Over 60 mutations have by now been localized in the AIRE genes of different APECED patients. Given their scatter throughout the protein sequence, they have not so far provided many insights into its function; in addition, save for one possible exception (8, 20), the different mutations have not to date been convincingly associated with particular disease manifestations. Just after Aire was identiﬁed, there was signiﬁcant controversy over its pattern of expression, with some groups claiming a very broad organ distribution (18) and others a quite restricted localization to lymphoid organs, in particular the thymus (19, 21). This discrepancy almost certainly reﬂected differences in reagent speciﬁcity and technique reliability, and eventually resolved in favor of the latter view (22). This new information on structure and expression of Aire prompted a number of early hypotheses on how it might operate to control autoimmunity: by driving the organization of thymic stroma (23); by somehow controlling thymocyte tolerization (19); by regulating peripheral B and T cell responses to antigenic stimuli (18); by provoking apoptosis of parenchymal cells and thereby enhancing cross-presentation of their antigens (24); or by promoting the differentiation of

CD4+ Foxp3+ regulatory T cells (Tregs) (24). Given the impossibility of rigorously evaluating such hypotheses in humans, investigators rapidly cloned the mouse equivalent of the AIRE gene, termed Aire, to develop an experimental model of APECED (25).

AIRE’S ROLE IN THE THYMUS The Major Mechanism: Promotion of Clonal Deletion of Self-Reactive Thymocytes Studies on mice have permitted extensive mechanistic dissection of how Aire operates to control autoimmunity. First, the ready availability of tissues and appropriate histological and cell-sorting reagents allowed a clearer delineation of just where it is expressed: primarily in lymphoid organs, above all in the thymus; and within the thymus, primarily in medullary epithelial cells (MECs) and secondarily in dendritic cells (DCs) (26–28). The location in MECs was particularly intriguing (a) because of suggestions that this cell type is involved in negative selection of mature CD4+ 8− and CD4− 8+ self-reactive thymocytes (29), and (b) because of the coincident emergence of a body of data establishing that transcripts encoding a diversity of peripheraltissue antigens (PTAs) are ectopically expressed speciﬁcally in MECs (reviewed in 30). Thus, the hypothesis arose that Aire regulates the thymic expression and presentation of PTAs and thereby controls thymocyte tolerization and consequently autoimmunity. This hypothesis could be directly evaluated in Aire-knockout (KO) mice (26, 31, 32). These animals have a rather normal immune system but are afﬂicted with multiorgan autoimmunity, manifested as both inﬂammatory inﬁltrates and serum autoantibodies. Although initial descriptions of the disease in Aire-KO mice on a mixed C57Bl/6(B6)X129 genetic background indicated that it is relatively mild, broader organ implication and greater severity were noted when the mutation was crossed onto different mouse strains (References 32–34; www.annualreviews.org • Aire

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and see below, Two Disease Peculiarities). The autoimmune manifestations in the KO animals tracked with Aire’s absence from thymic epithelial cells, as demonstrated in experiments employing either (a) radiation/bone marrow chimeras (disease partitioned with Aire deﬁciency in the radio-resistant rather than radiosensitive cells), or (b) thymus transfers (disease associated with an Aire-deﬁcient thymus rather than periphery) (26, 32, 35). Puriﬁcation of MECs from Aire-KO and Aire-wild-type (WT) littermates followed by gene-expression proﬁling revealed that Aire does indeed promote the expression of a battery of PTA gene transcripts (e.g., encoding insulin, salivary protein 1, and fatty acid–binding protein); interestingly, there were also a number of PTA transcripts (e.g., encoding C-reactive protein and GAD67) whose expression appeared to be Aire independent, an observation later substantiated on a broader scale (36). Aire also regulates the expression of a range of non-PTA genes in MECs, either positively or negatively (37, 38). The signiﬁcance of Aire control of PTA expression for the autoimmune manifestations in Aire-KO mice has been conﬁrmed by linking loss of this control with development of particular T cell and autoantibody speciﬁcities, speciﬁcally for the eye (39) and stomach (40). It remained to establish how Aire control of PTA expression in thymic MECs was translated into an effect on immunological tolerance. Through crossing of the Aire-KO mutation into T cell receptor (TCR)/neo-self-antigen double-transgenic mouse systems, investigators could demonstrate that, in the absence of Aire, self-reactive thymocytes escape the usual clonal deletion that keeps them from emerging into the periphery, resulting in some, but not all, cases in rapid and severe autoimmune disease (27, 35, 41). Interestingly, the Aire mutation in heterozygous state also provoked autoimmunity, though it was less aggressive than the disease of homozygous mutants. Although these studies clearly linked Aire control of PTA expression to clonal deletion, several reports have emerged describing situations in which a lack of Aire compromised tol-

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erance induction without affecting expression of the corresponding PTA (27, 32, 34). Thus, there is now considerable interest in deﬁning roles for Aire in tolerance induction beyond the control of PTA transcript levels in MECs. Evidence has been reported for an effect on the presentation of antigens by MECs (27). Although such a deﬁciency would certainly be consistent with observations that transcripts of a number of genes involved in antigen processing and presentation are also regulated by Aire (27, 37, 38), so far the actual molecular defect remains undeﬁned. In particular, it does not appear to reﬂect lower cell-surface display of major histocompatibility complex (MHC) or costimulatory molecules (27). The recent observation that Aire expression rapidly induces cells to die (42) has prompted the hypothesis that at least some of its inﬂuence on antigen presentation may reﬂect cross-presentation of Aire-induced apoptotic bodies (42), in line with the ﬁnding that thymic hematopoietic cells extend the range of clonal deletion by crosspresenting MEC-derived antigens (43). Our current view of Aire’s major function in the thymus is schematized in Figure 1.

An Additional Function in the Selection of Regulatory T Cells? As argued above, Aire clearly operates through induction of T cell tolerance in the thymus. A number of investigators have been uncomfortable with this notion, raising the question of how a thymic stromal cell population as rare as MECs could possibly purge the entire emerging T cell repertoire of self-reactive speciﬁcities. This issue is exacerbated by recent reports, based on polymerase chain reaction (PCR) analyses of individual MECs, that a given PTA transcript is expressed by only a subset of MECs (44, 45). Nonetheless, such skepticism might be considered ill-founded because fully mature thymocytes spend almost two weeks in the medulla (46, 47); thymocytes are extremely motile (48); and the T cell repertoire can be effectively cleansed by as few as a hundred dendritic or other hematopoietic cells (49, 50). An

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alternative or additional possibility is that Aire promotes positive selection of Tregs by MECs, on the basis of the same set or a subset of PTAs. Indeed, there are precedents that peptides expressed by thymic epithelial cells can drive the selection of Tregs (51, 52). Aire does not seem to inﬂuence the Treg compartment at a global level. Numbers of CD4+ Foxp3+ Tregs in Aire-KO mice are normal, and these cells are normally active in standard in vitro suppression assays (27, 32, 35, 53). In contrast, one group has reported a lower proportion of circulating CD4+ Foxp3+ cells in some established APECED patients (53). However, it is important to solidify this contention by analyzing patients at an early stage of disease to rule out downstream effects of other immune system abnormalities or of any therapy received. For example, the observed reduction in Tregs might be secondary to the chronic fungal infection and autoimmune inﬂammation in these individuals, e.g., by their sequestration in inﬂamed sites, as has been observed in the HIV context (54). Another argument against a global defect in Tregs in Aire-deﬁcient individuals stems from the very different nature and aggressivity of the autoimmune phenotypes in IPEX (immune dysregulationpolyendocrinopathy-enteropathy-X-linked inheritance) and APECED patients, or in scurfy- and Aire-KO mice. In addition, there is a clear synergy between mutations in Foxp3 and Aire: The disease in double-deﬁcient animals was substantially worse than that of either single mutant (55), indicating that Aire can still have an impact when Tregs have been removed from the equation. Evidence from Aire-WT/Aire-KO dual-thymus transfer experiments also argues against primary perturbations of dominant tolerance. If the autoimmune disease imparted by maturation of T cells in an Aire-deﬁcient thymus was due to defective Tregs, it should have been prevented by the regulatory cells generated by the cografted WT thymus, and this was not the case (27, 32). The issue of Aire inﬂuences on Tregs was also examined at the level of individual self-reactive TCR speciﬁcities, with pairs of

Apoptotic MEC

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Clonally deleted thymocyte Figure 1 Aire promotes clonal deletion of self-reactive thymocytes. Aire induces MEC expression of a broad repertoire of peripheral tissue antigens (PTAs), which are processed and then presented on surface-displayed MHC/HLA molecules. Soon after the induction of Aire and PTAs, MECs die by apoptosis. Mature thymocytes percolate through the medulla, and, if their TCRs recognize an MHC:PTA complex in the appropriate afﬁnity/avidity window, they will be overactivated and deleted from the repertoire. Thymocytes can recognize MHC:PTA complexes directly on MECs or indirectly on DCs that have engulfed apoptotic MECs or MEC fragments.

transgenes encoding a TCR and its cognate antigen crossed onto Aire-negative or Airepositive backgrounds. Results from several systems demonstrated that clonal deletion was affected by the absence of Aire, but that there was no signiﬁcant effect on the numbers of Foxp3+ Tregs generated (27, 35, 41). To address this issue in a different manner, Aschenbrenner et al. (56) generated a transgenic mouse line in which expression of an epitope from inﬂuenza hemagglutinin (HA) was directed to Aire-positive thymic MECs, relying on a transgene whose expression was driven by the Aire promoter/enhancer, and coupled it with an anti-HA TCR-transgenic line that can generate a robust population of Tregs in the presence of thymically expressed HA (52). They www.annualreviews.org • Aire

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observed an augmentation in the representation of Tregs recognizing the HA epitope. Although this study showed that Aire-positive MECs can select Tregs, it fell short of demonstrating that either Aire or Aire-positive MECs have a critical function in molding the Treg repertoire because it failed to establish that the ability of Aire-positive MECs to select Tregs actually depends on Aire and that Aire-positive MECs are required for selection. Indeed, many cell types, for example thymic cortical epithelial cells, can promote the emergence of Tregs in the HA system (51, 52). It would seem, then, that there has not yet been clear demonstration of an important effect of Aire on the selection of Tregs in the thymus, nor of a direct inﬂuence on the peripheral Treg compartment (free of any confounding autoimmunity, immunodeﬁciency, infection, or drug treatments). Nonetheless, because Aire does control PTA expression in thymic MECs, it remains an attractive idea that it might also drive clonal deviation of certain thymocytes into the Treg lineage, rather than their clonal deletion, depending, for example, on the afﬁnity/avidity of the TCR-MHC/PTA interaction.

An early argument in favor of this scenario was that a more diverse repertoire of PTAs was expressed by what was assumed to be the more mature MEC subsets (36). At present, the terminal differentiation model is generally considered to be the more accurate representation of MEC differentiation on the basis of results from several recent studies that weighed the major distinguishing features between the two models. First, it is now very clear that Aire-positive MECs represent a late stage of maturation—not only do MHC-IIlo and/or B7lo cells give rise to them (42, 59–61), but also they are a postmitotic, terminal population (42, 61). Second, single-cell PCR analyses of PTA expression in individual MECs failed to reveal preferential coexpression of transcripts characteristic of particular epithelial lineages—rather, transcripts were expressed in a probabilistic fashion, and often monoallelically (References 44, 45; and see further discussion in Molecular Mechanisms of Aire). In addition, MEC expression of PTAs depended on transcription factors and transcriptional start sites different from those employed in the relevant peripheral cells (45).

Some Function in MEC Differentiation?

AIRE’S ROLE IN THE PERIPHERY

Farr and coworkers (57, 58) have argued that Aire plays a role in the differentiation of thymic MECs. In their progressive restriction model, Aire and PTA expression are properties of immature MEC precursors, and turning Aire on drives differentiation of MECs into progressively restricted epithelial cell fates, with individual cells taking on different fates. The initial impetus for suggesting such a scenario was microscopic evidence of intriguing epithelial organoids in the thymus, e.g., structures resembling thyroid follicles (57). Alternatively, other investigators have championed a terminal differentiation model whereby increasingly broad PTA expression accompanies MEC maturation from the Aire− MHC-IIlo CD80lo to the Aire+ MHC-IIhi CD80hi phenotype, and Aire does not serve a differentiative function (30). 292

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Although Aire is primarily expressed in the thymus, AIRE/Aire gene transcripts have also been detected in peripheral tissues, in particular the peripheral lymphoid organs (e.g., 21, 26, 62–64). This ﬁnding has provoked speculation about an extrathymic role for Aire, either in antigen presentation or, more speciﬁcally, in peripheral tolerance induction.

Peripheral Hematopoietic Cells RNA transcripts encoding Aire have been found in monocytes and in an array of DC types (either ex vivo or derived in culture) from both mice and humans (26, 62, 64). Several lines of evidence support the argument that Aire in murine DCs is unlikely to contribute much to tolerance induction. First, although Aire gene expression was detectable with very sensitive

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PCR assays, signals were very low in DCs compared with in MECs: Transcripts were reduced by a factor of 10 or more according to quantitative analyses, and protein was undetectable by ﬂow cytometry using a sensitive and speciﬁc intracellular staining technique that gives a clear signal in MECs (26, 28; D. Gray, C. Benoist, and D. Mathis, unpublished results). In addition, Aire expression in mouse DCs seems to be of little consequence as far as PTA expression is concerned: There were minimal differences in the gene-expression proﬁles of AireWT and Aire-KO mice (65; E. Venanzi, C. Benoist, and D. Mathis, unpublished data), and transcripts that had a strong Aire dependence in MECs did not in DCs. Most directly, experiments on radiation/bone marrow chimeras demonstrated that the autoimmune manifestation characteristics of Aire-KO mice developed independently of an Aire defect in cells of the radio-sensitive hematopoietic lineage, instead partitioning with Aire-deﬁcient radio-resistant stroma (26, 32, 35). There have been a few reports of alterations in the antigen-presenting capabilities of DCs or macrophages from AireKO mice—albeit, paradoxically, as an enhanced effectiveness (65, 66). However, data from other investigators were not able to conﬁrm this conclusion (27). On the other hand, the root of APECED patients’ striking susceptibility to Candida infection, a disease hallmark, remains curiously obscure. Investigators have reported alterations in the antigen-presenting capabilities and transcriptional programs of DCs derived from APECED patients versus healthy controls, or of monocytes transfected or not with AIRE (67, 68), supporting the argument that Aire promotes human DC maturation or function. Again, it will be important to rule out any effects of confounding factors like those mentioned above (in the section entitled An Additional Function in the Selection of Regulatory T Cells?) before embracing the signiﬁcance of these divergences, but they do have the potential of explaining the increased susceptibility of APECED patients to fungal infections. And, again, contradictory data, providing no ev-

idence for a defect in antigen presentation, have been published (69). Putting together the two sets of data, admittedly derived from two different species, we think it possible that Aire expression in peripheral DCs can affect the presentation of antigens in the periphery, especially under inﬂammatory conditions, and thereby can inﬂuence susceptibility to fungal infections. But so far, Aire appears to be of little consequence for the establishment and maintenance of tolerance to self.

Peripheral Stromal Cells Two recent papers on the expression of Aire and PTA transcripts in the stromal cells of peripheral lymphoid organs have excited great interest. Lee et al. (70) described a population of lymph node stromal cells expressing a repertoire of PTA transcripts that overlaps quite a bit, but not perfectly, with that of thymic MECs. These stromal cells could directly present a transgenically targeted antigen to T cells, activating and eventually deleting them. Although the stromal cells also expressed low levels of Aire gene transcripts, the level of Aire protein was not assessed, nor was the Aire-dependence of endogenous-PTA expression. On the other hand, Gardner et al. (71) found PTA transcripts in stromal cells residing in both the lymph nodes and spleen. At least some of these cells also expressed the Aire gene at the protein level: Indeed, they were originally noticed as cells expressing a green ﬂuorescent protein reporter under the dictates of Aire transcriptional control elements. The repertoire of PTA transcripts made by these stromal cells appeared to be of limited diversity and seemed rather distinct from that of thymic MECs. An antigen transgenically targeted to the peripheral stromal cells could entice T cells to make sustained contact and provoke full-blown activation followed by death. Although they are potentially of substantial interest, it is important to keep in mind what these studies do and do not demonstrate. They do show that stromal cells in the peripheral lymphoid organs have the capacity www.annualreviews.org • Aire

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to present transgene-encoded antigens directly to T cells, provoking activation-induced cell death. They do not, however, establish that the endogenously encoded antigens ectopically expressed in these cells, often at lower levels, have the same capacities. Nor do they provide any information on how important this proposed mechanism really is in maintaining tolerance. In this regard, it should be kept in mind that thymus transplant experiments by multiple groups have provided no evidence for an important role for Aire-positive peripheral cells in warding off autoimmunity (26, 32, 35). Lastly, it is important to understand why the cells identiﬁed in the two studies appear to be so different: in the lymph nodes only versus in all peripheral lymphoid organs; UEA-1+ ERTR7+ gp38+ versus negative for all of these markers.

Peripheral Autoimmune Effector Mechanisms Information on just how defective tolerance induction in mice lacking Aire translates into pe-

AIRE IN THE REPRODUCTIVE ORGANS The testis and ovary stood out as parenchymal tissues with readily detectable Aire gene transcripts (26), and investigators have found Aire protein as well, speciﬁcally in spermatogonia and spermocytes (142). This is an intriguing ﬁnding because there has been a long history of the testis spuriously expressing transgene constructs. Might Aire promote promiscuous expression of batteries of proteins in the reproductive organs as well, and, if so, what might be their function? Mice lacking Aire are often infertile (26, 31), but this state likely reﬂects autoimmune attack on these organs. Schaller et al. (142) recently demonstrated that Aire-KO mice have perturbations in the waves of scheduled and sporadic germ cell apoptosis that normally take place during murine spermatogenesis. Given that such death is thought to eliminate cells with damaged DNA, this observation suggests that Aire might function somehow in enforcing germ line stability. If this does prove to be an important function, one will be led to question whether Aire was “borrowed” from the reproductive system by the immune system during evolution, or vice versa.

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ripheral autoimmune disease is beginning to emerge. DeVoss et al. (72) found that AireKO mice with an additional genetic deﬁciency in T cells were devoid of autoimmune manifestations. The CD4+ , but not CD8+ , T cell compartment was critical for disease development. The CD4+ T cells of Aire-KO mice were skewed to the T helper (Th) 1 phenotype, and Th1 cytokines were implicated in pathology. This group found minimal evidence for a role for B cells, but contradictory ﬁndings were reported by Gavanescu et al. (73), who found autoimmune pathology greatly muted in the absence of B cells, as well as a significant improvement in disease parameters after treatment with anti-CD20 monoclonal antibody. The most likely explanation for the divergent results is that the B cell–deﬁciency mutation relied on by DeVoss and colleagues (μMT−/− ) is leaky, with residual B cell numbers and antibody titers varying according to the particular genetic background and mouse colony (74). In neither study did autoantibodies appear to be directly pathogenic; rather, the impact of B cells was at the level of T cell priming and expansion. In both studies, however, the read-outs for an effect of transferred autoantibodies on disease development were conﬁned to an examination of organ inﬁltrates. Other inﬂuences remain possible, perhaps akin to the recently reported dampening of monocyte (and monocyte-derived DC) expression of genes responsive to type I interferons (IFNs) by the anti-IFN antibodies present at high titers in the serum of most APECED patients (75, 76). Deﬁnition of the peripheral autoimmune effector mechanisms in Aire-KO mice has potential implications for the treatment of APECED patients. It is hoped that the demonstrated therapeutic effects of anti-CD4 (72) or anti-CD20 (73) monoclonal antibodies on the progression of multiorgan autoimmune disease in mice can be translated to patients. This would represent an important advance, especially given the limited treatment options available at the present time.

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TWO DISEASE PECULIARITIES

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Target-Organ Heterogeneity An intriguing feature of APECED has always been the heterogeneity of clinical manifestations in different patients, even in cohabitating siblings with exactly the same genetic lesion. Three explanations come to mind. First, even though APECED is classiﬁed as a monogenic disorder, there may be disease-modifying genes that alter target-organ speciﬁcity. Second, environmental factors may impact disease manifestations. And, third, stochastic elements may come into play, an obvious example being the randomly generated T and B cell repertoires: A particular organ may not be targeted in a given individual simply because he/she did not generate high-afﬁnity T or B cells that recognize the relevant antigens. Given the small number of patients, distinguishing between these different explanations in the APECED context is not currently feasible. Fortunately, Aire-KO mice allow direct experimental evaluation of the different possibilities. Backcrossing of the Aire-null mutation onto different genetic backgrounds revealed important genetic inﬂuences on the organs targeted by autoimmunity (32–34). For example, B6-KO mice developed a relatively mild autoimmune disorder, their Balb/c counterparts showed a predominant gastritis, and nonobese diabetic (NOD)-KO mice had very severe disease, dominated by pancreatitis. Interestingly, different genetic loci controlled targeting of different organs. Some of the disease-modifying loci mapped to the MHC, whereas others did not. Among the latter were genes within genetic intervals (idd3, idd5 ) that control susceptibility to T1D in NOD mice. In contrast, there has been essentially no evidence of environmental inﬂuences on the organs targeted by the autoimmunity of Aire-KO mice. Neither a battery of innate immune system stimulants, nor a defect in a critical element of the majority of Tolllike receptor pathways (MyD88), nor germ-free conditions could augment or diminish disease on the B6 or NOD background, respectively (77).

Experiments assessing the effects of stochastic elements like the TCR or autoantibody repertoires on the Aire-KO autoimmune disease have not yet been reported but promise to be of great interest.

Target-Organ Choice Aire controls the expression of thousands of PTA transcripts in the thymus. Yet the autoimmunity that develops in Aire-deﬁcient mice or humans appears circumscribed, targeting a limited number of organs and a restricted set of antigens within each organ. Why is disease not more rampant? Part of the explanation must lie in the fact that autoimmunity, like any immune response, is HLA/MHC-restricted, so that not every self-antigen can be presented to the potentially self-reactive T cell repertoire. Another factor that may conﬁne the autoimmune attack is that peripheral mechanisms of T cell tolerance almost certainly keep many autoreactive T cells under control. However, surprisingly few additional organs were targeted when mutations in Aire and Foxp3 were combined (55), leaving open the possibility that regulatory cells other than Foxp3+ Tregs play a dominant role. Lastly, one is led to consider the importance of B cell tolerance, which may provide an additional ﬁlter for which autoimmune responses are ultimately fruitful.

AIRE AND OTHER AUTOIMMUNE DISEASES The multiorgan autoimmune manifestations of Aire-deﬁcient APECED patients, and the particular target organs involved, raise the possibility that genetic variation in the AIRE locus might also play a role in more common organspeciﬁc autoimmune diseases, such as T1D, thyroiditis, etc. First, full loss-of-function mutations, in the heterozygous state, might result in inefﬁcient presentation of self-antigens in the thymus and borderline tolerance, a state that might favor the development of certain of the organ-speciﬁc autoimmune diseases with a different etiology. Indeed, heterozygote effects have been documented in both humans www.annualreviews.org • Aire

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and mice (27, 41, 78). Alternatively, sequence polymorphisms affecting the coding region of AIRE or its expression, changes too subtle to elicit full APECED on their own, might generally augment the propensity to develop autoimmune disease. Relevant in this context is an Italian APECED family with a high prevalence of autoimmune thyroiditis, thyroid manifestations cosegregating with a heterozygous, apparently dominant-negative, mutation of AIRE (8, 20). Initial attempts to address this issue provided very limited evidence that genetic variation in AIRE has a signiﬁcant impact on more common autoimmune diseases. In a large study of individuals presenting with Addison’s disease, T1D, or autoimmune thyroiditis, Meyer et al. (79) searched for an overrepresentation of heterozygous carriers of two of the most common AIRE mutations (a 13–base pair deletion in exon 8, the R257X nonsense mutation). Neither mutation was overrepresented in cases relative to controls. Other investigators have looked for an association between autoimmune diseases and particular AIRE haplotypes, using tagging single nucleotide polymorphisms to distinguish variants segregating through the population. On the one hand, no signiﬁcant association was observed with vitiligo (80), Addison’s disease (81), or T1D (82). On the other hand, the situation with two autoimmune diseases, both skin disorders (alopecia and vitiligo), remains cloudy, with some studies arguing for a genetic association with certain AIRE alleles (73, 76, 83) and others against (80, 84). Divergent results such as these usually come from underpowered analyses and/or dissimilar test populations. Furthermore, the AIRE chromosomal region has not scored positively in any of the recent genome-wide association studies. Thus, if there is a contribution of AIRE variation to the genesis of common autoimmune diseases, it likely represents fairly rare cases, perhaps with family-speciﬁc mutations. This scenario awaits testing in large-scale resequencing studies. Although genetic analyses have not so far provided very strong evidence of an association of most organ-speciﬁc autoimmune dis-

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orders with particular AIRE haplotypes, there are data arguing that a breakdown in other elements of the pathway of Aire-mediated tolerance induction may be involved in certain of these diseases. After the HLA complex, the second human T1D susceptibility locus to be identiﬁed was the INS gene, which encodes insulin. The number of tandem repeats (VNTRs) in the INS promoter region is variable in different individuals, and these polymorphisms have been associated both with levels of INS transcripts in the thymus and with diabetes incidence: The greater the number of VNTRs, the more thymic transcripts, and the less diabetes (85–87). Although these studies were only correlative, experiments on INS-transgenic mouse models have conﬁrmed the validity of the overall conclusions (88). Similarly, Aire, in conjunction with interferon regulatory factor 8 (IRF8), controls MEC expression of the CHRNA1 gene, which encodes the α-subunit of the muscle acetylcholine receptor, implicated as an antigen in myasthenia gravis (89). CHRNA1 has a biallelic functional variant in the promoter region that is associated with early onset of myasthenia gravis.

HUMAN VERSUS MURINE AIRE Certain investigators have speculated that human Aire may serve a function different from, or in addition to, that of its murine counterpart. The most frequently cited argument in support of this notion is that the initially described autoimmune disease manifested by Aire-KO mice on the B6X129 mixed genetic background was milder than that of APECED patients and involved a dissimilar spectrum of organs (26, 31). In addition, one strain of mice lacking Aire (B6 Aire-KO) was found to have a proﬁle of autoantibodies different from that of Aire-defective humans (90). However, these arguments were substantially weakened when it was reported that the identity and number of organs subject to autoimmune attack in Aire-KO mice vary strikingly according to the genetic background, and autoimmune attack is very severe on certain backgrounds, notably the NOD, where it

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provokes rapid wasting and death by 15 weeks of age (32–34). Actually, it is not unexpected that mice and humans would show a different spectrum of target organs and antigens—after all, like essentially all autoimmune disorders, the Aire-KO disease results from an immune response, and, like any immune response, it is subject to MHC/HLA restriction, implying that any two mouse strains or human patients will not necessarily be able to respond to a given autoantigen. Indeed, as already mentioned, genetic analyses in both mice (33) and humans (14, 15) have been able to document MHC/HLA effects on the spectrum of organs targeted. Another reported difference between the two species is that some established APECED patients, but not Aire-KO mice on a single genetic background, have a defect in Tregs (53). But, as argued above (An Additional Function in the Selection of Regulatory T Cells?), it will be important to substantiate this difference in the absence of any complicating treatments or of the confounding Candida infection and at comparable levels of background autoimmunity. The Candida infection itself represents an interesting and striking divergence between humans and mice lacking Aire, but we do not yet know whether the apparent dissimilarity is a reﬂection of Aire functioning in a different manner in the two species or whether it relates more to differences in physiology (e.g., of the skin) or in the environment. Of course, infection by Candida should not occur in the speciﬁcpathogen-free environment in which most experimental mice live, so there is currently no real information on whether Aire-KO animals are or are not more susceptible to this (or any) fungal infection. We therefore suggest that, although it remains a theoretical possibility that there are signiﬁcant species-dependent disparities in Aire function, to date no inarguable evidence of such has been published.

MOLECULAR MECHANISMS OF AIRE As outlined above, Aire affects key aspects of the induction of immunological tolerance to

PTAs: their ectopic expression in the thymus; the efﬁciency of their presentation by MECs; and MEC apoptosis and turnover, which may serve to facilitate their cross-presentation. Aire has usually been thought of as a transcription factor (91), a function certainly compatible with such a broad range of activities. A number of other ﬁndings argue for a role for Aire in the regulation of transcription: It is located predominantly in the nucleus, typically in punctate structures reminiscent of, but distinct from, PML (promyelocytic leukemia) bodies (64, 92–95); its domain structure and organization are highly evocative of transcriptional regulators; it demonstrably associates with other transcription factors and can modulate the transcription of a variety of reporter and endogenous genes in cotransfection assays (94, 96–100); and chromatin immunoprecipitation experiments in transfected cells show it to be associated with the loci it transactivates (99). Thus, at ﬁrst glance, Aire appears to be a classical transcription factor.

Modalities of Aire-Mediated Gene Regulation: A Conventional Transcription Factor? However, we must take into account several unusual features of Aire’s inﬂuence on gene expression when considering its molecular mode of operation:

Recent bioinformatic reevaluations of the impact of Aire on MEC transcription have revealed that it inﬂuences the expression of even more genes than previously thought—several thousand rather than several hundred—representing a substantial fraction of the total genome (36; E. Venanzi, C. Benoist, and D. Mathis, unpublished results). By several metrics, this impact is 5–8 times broader than that of classic transcription factors such as Foxp3 or Runx. This breadth is difﬁcult to reconcile with the notion that Aire operates by interacting with speciﬁc DNA sequences, as it is hard to imagine www.annualreviews.org • Aire

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that its binding site occurs in thousands of promoters of such disparate structure and cell-type speciﬁcity (unless the bulk of transcriptional effects are actually secondary, e.g., through the mediation of more restricted sets of transcription factors or via microRNAs). Aire-regulated genes are clustered genomically. Such an organization is not unusual, as certain transcription factors are able to bind to promoters in arrayed members of duplicated gene families (e.g., the CIITA and MHC class II genes) or to activate the transcription of a whole chromatin loop through a locus control region. However, with Aire, induced, repressed, and unaffected loci are interspersed within a given cluster (36, 38). The transcriptional footprint of Aire varies profoundly with the cell type in which it is expressed. Comparative geneexpression proﬁles of Aire-positive and -negative MECs, of lymph node stromal cells, and of pancreatic islet β cells showed the same overall scope of Aire activity (a plethora of loci implicated, roughly twice as many activated as repressed), but there was only very limited overlap in the identity of the genes affected in the different contexts (71, 101, 102). The same observation has been made with transfected tissue culture cells ( J. Abramson, A. Koh, C. Benoist, and D. Mathis, unpublished observations). Such patterns would not be expected from a sequence-speciﬁc transcription factor, which would generally tend to transactivate the same set of promoters carrying its recognition motif, irrespective of cell type. The expression of PTA genes in MECs is independent of transcriptional regulators essential for activity of the same loci in their home cells in the periphery (45). In addition, the transcriptional start sites used in MECs often differ from those employed in peripheral tis-

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sues. For instance, initiation of Ins gene transcripts in MECs utilizes some of the start sites found in pancreatic β cells, but also a novel one located 30.9 kb upstream (45). Aire’s inﬂuence on ectopic expression of PTA genes in thymic MECs is not that of a simple on/off switch. Rather, its effect is usually only quantitative, with most PTAs expressed to some level in the absence of Aire. (Notably, the Ins gene is one of the rare exceptions, its ectopic expression appearing fully dependent on Aire.) And a number of PTA transcripts are not affected by Aire at all (26, 36, 38, 101). It has been suspected for some time that individual MECs express only a subset of the Aire-dependent PTA repertoire (103). Recent experiments exploiting single-cell PCR of isolated MECs have shown that the expression of a given PTA transcript is probabilistic (44, 45). In addition, monoallelic transcription was often observed, with only one of the two copies of a gene transcribed in any given cell. A monoallelic expression pattern again points to a stochastic determinism, as the probability of expression from each chromosome is independent of expression from the other [ruling out that stochastic PTA transcription reﬂects stochastic expression of the transcription factors controlling them, as posited by earlier models (104)]. One question left unanswered by these single-cell studies concerns the dynamic nature of Aire’s activity: Are the stochastic expression patterns stable for a single MEC, or do the snapshot views we have so far obtained correspond to a slice in time, the expression of Aire target genes in a chromosome or cell ﬂuctuating over time? However, the rapid cell death provoked by Aire expression (42) does tend to downplay this issue. Ectopic expression of PTAs in the thymus is noisy, and Aire accentuates this noise (101, 105). A comparison of

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gene-expression proﬁles from MECs of individual mice (strictly inbred and gender-matched littermates) revealed signiﬁcantly more interindividual variability in Aire-responsive PTA transcripts than in the bulk of Aire-neutral transcripts. In contrast, this variability was not true of Aire-responsive PTA transcripts in their home cells in the periphery. The degree of variation in MECs was not subtle, with up to a tenfold difference between extremes. Comparable interindividual variability in PTA expression was observed in the human thymus (105), although this ﬁnding is more difﬁcult to interpret because of the unavoidable genetic, age, and environmental diversity in the donors. From a mechanistic standpoint, this noise implies that the stochastic nature of ectopic gene expression at the single-cell level somehow extends to the entire thymus, a surprising concept as one would have expected cell-to-cell variation to be statistically smoothed out over the totality of MECs. Nonetheless, this variability may also play an important part in the stochastic element of susceptibility to autoimmune diseases. These various ﬁndings are not easily reconcilable with the view of Aire as a conventional, sequence-speciﬁc transcription factor. They support the argument that Aire does not exert domineering control on the genes whose expression it regulates, but instead accentuates or biases programs already in place.

Aire Structural and Functional Domains Aire contains several structural domains found in other transcription factors, most of which are well conserved in Aire proteins across phyla (106). The presence of APECED-causing mutations in these domains underscores their functional signiﬁcance (Figure 2).

The SAND domain (also known as the KDWK domain) is a component of several chromatin-associated transcriptional modiﬁers that have a wide range of functions across species: the Sp100 proteins (107); the glucocorticoid-modulatoryelement-binding protein (108) [GMEB-1 and −2; also known as Pif (109)]; the Ski modiﬁer of Smad signaling (110); the Drosophila DEAF1 gene product, involved in homeotic regulation, and its mammalian equivalent DEAF-1 (previously known as NURD), which is involved in developmental neural patterning through interactions with LMO4 (111). SAND domains are also found in transcriptional regulators in plants (112). The DNA-binding properties of the SAND domain, which have been analyzed at the structural level, are centered around the conserved KDWK core motif (108, 113). However, the DNA-binding properties and resultant speciﬁcity of Aire’s SAND domain remain unclear. It has been difﬁcult to obtain convincing evidence of particular target sequences because reported experiments

APECED AIRE

CARD/HSR

SAND

PHD1

PRR

PHD2

Figure 2 Domain structure of the Aire protein. Aire contains several identiﬁable domains, which have homology with domains of transcriptional control proteins from diverse phyla. The CARD (caspase-recruitment domain) overlaps with the dimerization region, once referred to as the homogeneously staining region (HSR) (although investigators must still determine whether all functions previously mapped to the HSR can be attributed to the CARD motif). The four LXXLL (where X is any amino acid) are shown as light gray bars. Red bars indicate the position of missense mutations in APECED patients (see http://bioinf.uta.fi/AIRE); the dominant-negative G228W mutation is in black. www.annualreviews.org • Aire

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have lacked certain of the controls required for validation or have yielded internally inconsistent results. Indeed, the SAND domain of Aire does not contain the canonical KDKW motif (108, 113). Interestingly, the DNA-binding speciﬁcity of SAND domains in other factors seems to be rather relaxed, with short recognition motifs and little inﬂuence from ﬂanking residues or from the exact spacing within the motifs: Sp100 recognizes repeats of unmethylated CpG dinucleotides (107), and GMEB binds loosely spaced PuCGPy motifs (109). Perhaps not surprisingly, then, recent data indicate that Aire binds to DNA in a rather nonspeciﬁc fashion (100). Two plant homeodomain (PHD) ﬁngers are also found in Aire. PHDs, members of the huge family of zinc-ﬁnger proteins, are thought to be restricted to the nucleus, and include transcriptional coactivators and chromatin-modulating elements (114). They are phylogenetically widespread, found from plants to mammals, and are fairly common: Computational searches have identiﬁed PHD structures in more than 100 mammalian proteins. PHDs are related to, but different from, the zinc ﬁngers with

P→L P→Q I P S G T

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E3-ubiquitin ligase activity characteristic of the RING subfamily. Indeed, this similarity was the impetus for a series of experiments that revealed that Aire’s PHD1 functions as an E3-ubiquitin ligase (115). However, the nuclear magnetic resonance solution structure of PHD1 and further functional assessments did not support this notion (116). In general, PHDs are considered to be primarily protein-protein interaction domains (114), which, when analyzed at the structural level or by mutagenesis, were seen to involve their loop2 region (114). Interestingly, this loop is precisely the site affected by several of the point mutations of APECED patients, highlighting its functional role (Figure 3). The functional relevance of Aire’s PHD domains is well established from transfection studies (96, 99, 100, 115, 117), although the relative importance of the two PHDs diverged markedly between the studies, likely reﬂecting the different transcriptional targets and other experimental conditions used. Certain of the interactions involving PHD domains occur between two transcription factors (118–120), but recently there have been several reports of liaisons with nucleosomal histones, in particular with their amino-terminal tails (121, 122). In some cases, PHDs bind methylated forms of histones, such as histone H3 trimethylated at lysine-4 (H3K4me3), one of the key marks associated with transcriptional activity. For example, PHDs in the proteins BPTF, Yng1, and ING2 have a marked preference for H3K4me3 (123–125). In other instances, PHDs speciﬁcally recognize the unmethylated form of the same histone stretch—for example, BHC80 binding to H3K4 is inhibited by methylation (126). In still other cases, the methylated and unmethylated histone forms are bound indiscriminately (127). Thus, PHD domains are important

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in reading the histone code and thereby in targeting transcription factors to particular active or inactive regions of chromatin. Interestingly, Aire PHD1 binds the amino-terminal tail of H3, but only in its unmethylated K4m0 form; in contrast, binding tolerates modiﬁcations at K9 and K14 (99, 100). Such speciﬁcity is normally considered to be characteristic of repressive factors, serving to lock inactive chromatin in its silent state. Because its major function is to derepress genes that would normally be silent, the operational logic for Aire might be precisely the opposite: Binding of the PHD1 domain to H3K4m0 may help to concentrate Aire in regions of inactive chromatin, where it can then have an activating inﬂuence. As is true of other PHD/histone complexes, the binding of Aire PHD1 to H3K4m0 is of low afﬁnity (in the 5–30 μM range). The fast off-rates implied by such afﬁnities suggest dynamic interactions rather than a stable, long-term ﬁxation. Aire contains four interspersed LXXLL sequences, a small motif found in many transcriptional coactivators, including CBP (CREB-binding protein) and STAT (signal transducers and activators of transcription) factors. This motif has been studied particularly well in the nuclear receptor family (128, 129), where it is thought to mediate protein-protein interactions. The importance and mode of action of the LXXLL sequences in Aire remain to be established. Finally, a CARD (caspase-recruitment domain) is found at the amino-terminus of Aire. This 100 amino acid stretch is the site of many APECED-causing mutations and was long referred to as a homogeneously staining region (HSR) domain. More recently, structure-based sequence analyses, which can detect similarities in the absence of primary sequence conservation, suggested that this region encompasses a CARD, as it harbors the key hydrophobic residues in proper spacing

(130). This prediction was tested functionally, revealing that the point mutations most likely to disrupt proper folding of the CARD structure were those with the strongest impact on Aire’s transactivation potential. Initially identiﬁed as key elements in the recruitment and activation of caspases during apoptosis, CARDs are now recognized to be involved in a wider variety of processes, many of them in the inﬂammatory realm, by mediating homo- or hetero-dimerization through homotypic interactions, as in the apoptosis cascade. Thus, Aire’s CARD may participate in dimerization or in interactions with other transcriptional control proteins, perhaps CBP (130).

Aire’s Partners Under normal conditions, Aire is found predominantly in punctate nuclear bodies, where it is likely to partner with a number of other proteins. Indeed, such interactions can be detected by gel ﬁltration of extracts from cultured cells, transfected Aire being incorporated into large complexes of >650 kD (131). Mutations in the SAND domain and PHD1 had little effect on such complexes, but alterations in the aminoterminal region (HSR or CARD) or truncation beyond PHD1 reduced their formation. Thus, distinct regions of Aire seem to participate in the protein-protein interactions subtending complex formation. The identiﬁcation of Aire’s partners promises to yield important clues about its mechanism of action. Certain putative interactors have been reproducibly identiﬁed, and others require conﬁrmation. But the emerging notion is that Aire is a highly collaborative protein, apparently associated with a score of partners that may participate in regulating different facets of the expression of Aire’s target loci. Currently thought to be of great interest are:

CBP (CREB-binding protein). This ubiquitous transcriptional activator was the ﬁrst Aire partner to be identiﬁed (96). CBP is a histone and nonhistone www.annualreviews.org • Aire

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acetylase that activates transcription. It can synergize with Aire to turn up expression of reporter constructs, colocalizing with it in nuclear bodies (95–97, 130). Formation of a complex with Aire may promote migration of CBP to the nucleus: RANK (receptor activator of NF-κB) induces Aire expression in the epithelial component of fetal thymic organ cultures, accompanied by migration of CBP from the cytoplasm into nuclear bodies (130). Aire seemed to be directly responsible for this translocation, as CBP remained largely cytoplasmic in RANKtreated Aire-deﬁcient thymus cultures. Thus, one of Aire’s functions may be to facilitate the import and/or retention of CBP in the nucleus of MECs. DNA-PK (DNA-dependent protein kinase). This protein is another Aire partner, initially identiﬁed in pull-down assays from Aire-transfected monocytes (132) and conﬁrmed in the broad massspectrometry screen discussed below. DNA-PK is a serine/threonine kinase, composed of two regulatory subunits (Ku70 and Ku80) and a large catalytic subunit. It is activated by double-stranded DNA breaks and is therefore a key player in nonhomologous end joining and is essential for the VDJ recombination characteristic of immune receptor genes. DNA-PK phosphorylates a number of proteins implicated in transcription, in particular RNA polymerase-II (Pol-II), but also Fos, Jun, and TBP. It can phosphorylate Aire in vitro, at two different positions in the amino-terminal region (positions 68 and 156), and alanine replacements at these positions result in a marked decrease in Aire’s transactivation activity in transfected cells. The role of DNA-PK in transcription is still incompletely understood, but one clue may lie in its involvement in resolution of the double-stranded breaks generated by DNA topoisomerase II beta (Topo IIβ) during transcriptional activa-

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tion (133). Topo IIβ produces a transient double-stranded break in nucleosomal DNA, which appears necessary for nucleosome displacement during gene activation. DNA-PK might facilitate histone displacement, in line with its acknowledged role in fostering histone H2AX replacement during DNA damage repair (134). This release of nucleosomal inhibition of transcription could inﬂuence either the initiation or elongation steps. P-TEFb. In a similar vein, an effect of Aire on transcriptional elongation was recently suggested by the observation that it interacts, in coprecipitation and pulldown assays, with the P-TEFb complex (135), a heterodimer of the Cdk9 and Ccnt1 cyclin (CycT1). P-TEFb is a key element in transcriptional elongation (136, 137), permitting Pol-II to be released from the initiation complex established at the promoter region. Recently, investigators realized that a surprisingly large proportion of the eukaryotic genes inactive in a given cell type actually have initiation complexes formed on their promoter regions: Pol-II initiates the transcription of a short RNA (∼30– 50 bases) but is unable to proceed further (138, 139; reviewed in 137). For these loci, then, gene expression is regulated by release of Pol-II from the promoter region rather than by differential formation of the initiation complex. Negative elongation factors associated with the initiation complex can block Pol-II’s progression, and/or positive factors such as P-TEFb are required to release Pol-II from its tight interactions within the initiation complex poised at the promoter region. Interestingly, Aire expression in cultured cells increased the proportion of Pol-II on the intragenic regions relative to the promoter region of its target genes and augmented the recruitment of P-TEFb, as determined by chromatin immunoprecipitation experiments (135). In addition, Aire expression increased the

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proportion of long transcripts relative to the short ones typical of stalled polymerases. However, one caveat to all of the results concerning P-TEFb is that these experiments employed cells that had been treated with trichostatin-A, a deacetylase inhibitor, so we do not know the extent to which they will prove to be reﬂections of an abnormal state of histone modiﬁcation. Many others. Most recently, a broad screen for protein-protein interactions involving Aire, relying on highthroughput mass spectrometry, yielded a large array of proteins, many of which were validated in subsequent coprecipitation or pull-down tests ( J. Abramson, C. Benoist, and D. Mathis, unpublished data). About 20 independent proteins were found to interact, either directly or indirectly, with Aire. Some were previously identiﬁed interactors (e.g., DNAPK), whereas others were newly revealed partners belonging to several biological pathways. Perhaps most striking was a set of proteins involved in the splicing and other processing of primary transcripts. Their functional relevance for Aire-mediated transcriptional activation was veriﬁed in RNAi-knockdown experiments. In keeping with this observation, Aire had a far more pronounced effect on the levels of spliced forms of transcripts than it did on levels of their unspliced nuclear precursors. These observations support the notion that Aire has a role in the maturation of primary transcripts.

Coda: A Speculative Perspective on Aire’s Influence on Transcription Given the known or suspected functions Aire’s structural domains exert in other transcription factors, and the known or suspected activities of its identiﬁed partners, can one frame a coherent (if highly speculative) hypothesis as to Aire’s mode of action (Figure 4)? Its recruitment to chromatin might involve rather non-

speciﬁc binding to DNA by the SAND domain, sharing the low level of speciﬁcity characteristic of other SAND domains. In particular, binding of Aire to regions rich in CpG dinucleotides (CpG islands enriched in the promoter regions of many genes) could facilitate its recruitment to a large number of loci, consistent with its wide impact on transcription. PHD1 would also contribute importantly to this recruitment, favoring inactive chromatin through interaction with H3K4me0 residues. These basic interactions might be viewed in a dynamic fashion, not so much as Aire binding to static chromatin but rather as it ferrying the targeted regions to nuclear bodies, perhaps to exploit transcription and/or RNA processing factories therein. From there, Aire might have several different but synergistic activities. Guiding the formation of initiation complexes is by no means ruled out by the existing data and is in line with the observation of distinct initiation sites for PTAs in MECs relative to their peripheral home cell types. More likely, by recruiting P-TEFb and other elongation factors, Aire might help in rescuing stalled Pol-II molecules, promoting their disengagement from otherwise sterile initiation complexes. By interacting with splicing complexes, Aire might favor the maturation and export of transcripts derived from its target genes. Thus, Aire’s interactions and functional activities would be multifaceted, addressing several steps in the generation of mature mRNAs. Such a broad role is consistent with the currently held view that transcriptional initiation, elongation, termination, and splicing are not events that occur sequentially and disconnectedly, but rather are performed in concert by large multimolecular complexes, transcripts being spliced before they are even completed (140). These effects seem rather nonspeciﬁc, as are the Aire interaction partners identiﬁed so far. How, then, might one account for Aire’s preferential impact on PTAs, rather than on ubiquitously expressed housekeeping genes? The key is likely to be that Aire activates primarily genes that need help: inactive loci packaged in closed chromatin marked by H3K4me0 genes, www.annualreviews.org • Aire

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Figure 4 A speculative and incomplete model of Aire transcriptional activation. As outlined in the text, Aire interacts functionally with several partners that it may recruit to transcriptional centers; the nature and function of these partners suggest mechanisms and points of impact for Aire’s activity. Interaction of Aire’s PHD1 with the unmethylated tail of histone H3 (H3K4me0, orange tails on the gray nucleosomes) preferentially recruits it to regions of inactive chromatin. DNA-PK affects transcription by participating in the transient doublestranded breaks and resolution thereof, catalyzed by Topo II, that lead to nucleosomal opening/displacement and release of topological constraints during transcription. Recruitment of P-TEFb, a key complex in transcriptional elongation, would facilitate the release of stalled Pol-II from the initiation complex. Finally, Aire would increase the availability (or modify the composition and speciﬁcity?) of splicing complexes to facilitate maturation of the transcripts (red strings).

or with stalled Pol-II on their promoters that need derepression, or ineffectively spliced transcripts. Active genes, bristling with properly methylated histones and with rapidly processive Pol-II complexes, would not be affected. This interpretation might explain Aire’s bewilderingly broad activity and the fact that its targets are almost completely different in different cell types. (One should note, however, that this interpretation of Aire as a generous helper of poor genes cannot be the whole story, as it does regulate some genes that are quite richly transcribed according to gene-expression proﬁles.) In summary, although we are beginning to get glimpses of the unusual mode of Aire’s effect on transcription, the precise mechanisms and interacting partners remain largely obscure. In addition, it seems unlikely that such a multitasking factor would not somehow exploit or inﬂuence the regulatory properties of noncoding 304

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RNAs. Although preliminary analyses showed no strong impact of Aire on the levels of a few miRNAs tested (M. Giraud, C. Benoist, and D. Mathis, unpublished observations), it is quite plausible that Aire would elicit derepression of PTAs by affecting the miRNAs that contribute to repress them.

WHAT REGULATES THE REGULATOR? Rather little is known about the transcriptional controls impinging on the Aire locus itself. There is a striking contrast between the loose, noisy, promiscuous gene expression that Aire promotes and the very tight control of its own expression. Although it is rather abundant in Aire-positive cells, with high mRNA levels and easily detectable protein, this expression is largely conﬁned to a single epithelial cell type

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in the thymus, in rare cells in the spleen and lymph nodes, and in the gonads (21–23, 26, 70, 71, 141, 142). Even in MECs, Aire is turned on only at the latest stage of differentiation, a few days before a cell’s demise (42, 61). Most likely, given its mode of action, Aire is too dangerous a protein to be expressed in a widespread manner or for extended periods of time. The molecules responsible for controlling Aire gene expression have not been explored to any signiﬁcant degree. The promoter regions of the human and mouse genes share stretches of conserved sequences (141), plausibly the target of similar transcriptional activators, but whose identity and importance remain to be established. Several connections have been made between members of the tumor necrosis factor (TNF) family and Aire expression. Lymphotoxins (LTs) were ﬁrst invoked as inducers of Aire, but data on this point have been conﬂicting. One group reported that signals through the LTβ receptor regulate Aire and PTA gene transcription, and not the composition or structure of the thymus epithelium (143, 144). In contradiction, Boehm et al. (145) concluded that LTβ receptor signals are required for proper MEC differentiation and organization but have no impact on Aire expression. The current consensus favors the second interpretation: A modest change in the number of MECs is observed in mice lacking the LTβ receptor, but the levels of Aire on a per-cell basis are not affected by this deﬁciency, nor are the levels and patterns of Aire-controlled PTA expression (60, 146, 147). Interestingly, LT may have a greater impact on the Aire-negative MEC population and PTA expression there (147). However, the RANK/RANK-ligand pair (60, 148–150), and its downstream signal trans-

ducers TRAF6 (TNF receptor–associated factor 6) and NIK (NF-κB inducing kinase) (150, 151), have a much more profound effect on the generation of the Aire-positive MEC compartment, with a severe reduction in Aire+ cells in the corresponding knockouts. Signaling through RANK also synergizes with CD40. The required soluble RANK ligand and surface-bound CD40 ligand signals needed for proper maturation of MECs are furnished by positively selected single-positive thymocytes in the adult, or by CD4+ CD3− lymphoidinducer cells during the fetal period (60, 149, 150), providing a molecular basis for the longrecognized cellular cross talk between the epithelial and lymphoid lineages that establishes a normal thymus architecture (152). The signal from mature thymocytes is not absolutely indispensable, though, because Aire can be detected in the rudimentary medulla of Rag−/− mice (23, 26, 60, 148). Here, again, it is difﬁcult to appreciate the direct impact of RANK signaling on the activation of Aire transcription, independently of the promotion of MEC differentiation.

CONCLUSIONS Aire is a fascinating protein that the immune system may have co-opted—from what could be a more primordial role in genomic remodeling of the germ line—to promote tolerance to self constituents. There are many mysteries left to solve concerning both its impact on lymphocyte populations and its role in the few nonthymic locales where it appears to reside. New surprises will almost certainly be encountered in unraveling the mechanisms underlying Aire’s unusual properties as a transcriptional regulator.

LITERATURE CITED 1. Walker LS, Abbas AK. 2002. The enemy within: keeping self-reactive T cells at bay in the periphery. Nat. Rev. Immunol. 2:11–19 2. Mathis D, Benoist C. 2004. Back to central tolerance. Immunity 20:509–16 3. Goodnow CC, Sprent J, de St Groth BF, Vinuesa CG. 2005. Cellular and genetic mechanisms of self tolerance and autoimmunity. Nature 435:590–97 www.annualreviews.org • Aire

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4. Peterson P, Pitkanen J, Sillanpaa N, Krohn K. 2004. Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED): a model disease to study molecular aspects of endocrine autoimmunity. Clin. Exp. Immunol. 135:348–57 5. Notarangelo L, Casanova JL, Conley ME, Chapel H, Fischer A, et al. 2006. Primary immunodeﬁciency diseases: an update from the International Union of Immunological Societies Primary Immunodeﬁciency Diseases Classiﬁcation Committee Meeting in Budapest, 2005. J. Allergy Clin. Immunol. 117:883–96 6. Betterle C, Greggio NA, Volpato M. 1998. Autoimmune polyglandular syndrome type 1. J. Clin. Endocrinol. Metab. 83:1049–55 7. Leonard M. 1946. Chronic idiopathic hypoparathyroidism with superimposed Addison’s disease in a child. J. Clin. Endocrinol. Metab. 6:493–95 8. Cetani F, Barbesino G, Borsari S, Pardi E, Cianferotti L, et al. 2001. A novel mutation of the autoimmune regulator gene in an Italian kindred with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, acting in a dominant fashion and strongly cosegregating with hypothyroid autoimmune thyroiditis. J. Clin. Endocrinol. Metab. 86:4747–52 9. Boe AS, Knappskog PM, Myhre AG, Sorheim JI, Husebye ES. 2002. Mutational analysis of the autoimmune regulator (AIRE) gene in sporadic autoimmune Addison’s disease can reveal patients with unidentiﬁed autoimmune polyendocrine syndrome type I. Eur. J. Endocrinol. 146:519–22 10. Kogawa K, Kudoh J, Nagafuchi S, Ohga S, Katsuta H, et al. 2002. Distinct clinical phenotype and immunoreactivity in Japanese siblings with autoimmune polyglandular syndrome type 1 (APS-1) associated with compound heterozygous novel AIRE gene mutations. Clin. Immunol. 103:277–83 11. Buzi F, Badolato R, Mazza C, Giliani S, Notarangelo LD, et al. 2003. Autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy syndrome: time to review diagnostic criteria? J. Clin. Endocrinol. Metab. 88:3146–48 12. Podkrajsek KT, Milenkovi´c T, Odink RJ, Claasen-van der Grinten HL, Bratanic N, et al. 2008. Detection of a complete autoimmune regulator gene deletion and two additional novel mutations in a cohort of patients with atypical phenotypic variants of autoimmune polyglandular syndrome type 1. Eur. J. Endocrinol. 159:633–39 13. Vogel A, Strassburg CP, Obermayer-Straub P, Brabant G, Manns MP. 2002. The genetic background of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and its autoimmune disease components. J. Mol. Med. 80:201–11 14. Gylling M, Tuomi T, Bjorses P, Kontiainen S, Partanen J, et al. 2000. β-cell autoantibodies, human leukocyte antigen II alleles, and type 1 diabetes in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. J. Clin. Endocrinol. Metab. 85:4434–40 15. Halonen M, Eskelin P, Myhre AG, Perheentupa J, Husebye ES, et al. 2002. AIRE mutations and human leukocyte antigen genotypes as determinants of the autoimmune polyendocrinopathy-candidiasisectodermal dystrophy phenotype. J. Clin. Endocrinol. Metab. 87:2568–74 16. Bjorses P, Aaltonen J, Vikman A, Perheentupa J, Ben Zion G, et al. 1996. Genetic homogeneity of autoimmune polyglandular disease type I. Am. J. Hum. Genet. 59:879–86 17. Aaltonen J, Bjorses P, Sandkuijl L, Perheentupa J, Peltonen L. 1994. An autosomal locus causing autoimmune disease: autoimmune polyglandular disease type I assigned to chromosome 21. Nat. Genet. 8:83–87 ¨ 18. Aaltonen J, Bjorses P, Perheentupa J, Horelli-Kuitunen N, Palotie A, et al. The Finnish-German APECED Consortium. 1997. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-ﬁnger domains. Autoimmune Polyendocrinopathy-CandidiasisEctodermal Dystrophy. Nat. Genet. 17:399–403 19. Nagamine K, Peterson P, Scott HS, Kudoh J, Minoshima S, et al. 1997. Positional cloning of the APECED gene. Nat. Genet. 17:393–98 20. Su MA, Giang K, Zumer K, Jiang H, Oven I, et al. 2008. Mechanisms of an autoimmunity syndrome in mice caused by a dominant mutation in Aire. J. Clin. Invest. 118:1712–26 21. Heino M, Peterson P, Kudoh J, Nagamine K, Lagerstedt A, et al. 1999. Autoimmune regulator is expressed in the cells regulating immune tolerance in thymus medulla. Biochem. Biophys. Res. Commun. 257:821–25

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Regulatory Lymphocytes and Intestinal Inﬂammation Ana Izcue, Janine L. Coombes, and Fiona Powrie Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK; email: [email protected], [email protected], ﬁ[email protected]

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Key Words

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

intestinal ﬂora, colitis, mucosa, intraepithelial lymphocytes (IEL), Foxp3

This article’s doi: 10.1146/annurev.immunol.021908.132657 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/08/0423-0313$20.00

Abstract The immune system is pivotal in mediating the interactions between host and microbiota that shape the intestinal environment. Intestinal homeostasis arises from a highly dynamic balance between host protective immunity and regulatory mechanisms. This regulation is achieved by a number of cell populations acting through a set of shared regulatory pathways. In this review, we summarize the main lymphocyte subsets controlling immune responsiveness in the gut and their mechanisms of control, which involve maintenance of intestinal barrier function and suppression of chronic inﬂammation. CD4+ Foxp3+ T cells play a nonredundant role in the maintenance of intestinal homeostasis through IL-10- and TGF-β-dependent mechanisms. Their activity is complemented by other T and B lymphocytes. Because breakdown in immune regulatory networks in the intestine leads to chronic inﬂammatory diseases of the gut, such as inﬂammatory bowel disease and celiac disease, regulatory lymphocytes are an attractive target for therapies of intestinal inﬂammation.

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INTRODUCTION IEC: intestinal epithelial cells IEL: intraepithelial lymphocytes PP: Peyer’s patches

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ILF: isolated lymphoid follicles

The intestine is the largest surface of contact between the body and the external environment. It represents a major gateway for potential pathogens but also contains dietary antigens and an extensive and diverse bacterial ﬂora that need to be tolerated. Given these requirements, we should not be surprised that the gut constitutes the largest lymphoid organ in the body. It contains an extensive network of secondary lymphoid organs and is home to an enormous number of lymphocytes, including several intestine-speciﬁc subpopulations (1) (Figure 1). Upon activation, the intestinal immune system can mount a range of host protective immune effector functions. To meet this challenge, several cell populations are tasked with keeping misdirected immune responses in check. Breakdown in the regulatory pathways results in chronic intestinal inﬂammation (2). Several reviews have addressed in detail the origin and function of intestinal immune cell populations (1). This article focuses on the essential role of lymphocytes in controlling intestinal immune responses. We describe the main characteristics of the intestinal immune system, putting into context the mechanisms that regulate intestinal inﬂammation. We then discuss the functional properties of different subtypes of lymphocytes and how their joint interaction contributes to the exquisite speciﬁcity characterizing efﬁcient intestinal immune function.

THE INTESTINAL ENVIRONMENT Intestinal immune homeostasis is determined by a dynamic interplay between the immune system, intestinal tissue, and the ﬂora and dietary compounds present in the lumen. Here, we brieﬂy review their main features.

Anatomy of the Intestinal Mucosa The intestine functions as a selective barrier that controls the entry of various external sub314

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stances into the body. This applies not only to nutrients, but also to dietary and commensal antigens that are sampled by the immune system. The intestinal epithelium is composed primarily of intestinal epithelial cells (IEC), which are connected by tight junctions and form an impermeable barrier between the body and the luminal contents. IEC are responsible for both importing luminal nutrients and releasing IgA into the lumen. In addition, IEC play an active role in immunity by producing antimicrobial peptides and proinﬂammatory cytokines in response to triggering of pattern-recognition receptors by microbes (3). These attributes allow for a dual function in ﬁghting pathogens: a direct antimicrobial function and a sentinel function in alerting the immune system. Indeed, several mouse models show that disrupted signaling into IEC increases susceptibility to intestinal inﬂammation (3). Besides IEC, the epithelium includes specialized cells such as goblet cells, which secrete the protective mucus layer, and Paneth cells, which reside in the crypts of the small intestine but not of the colon and secrete high amounts of antimicrobial peptides (4). In addition to the immune function provided by the IEC themselves, populations of immune cells can be found within the epithelial cell layer. These include dendritic cells that sample antigens directly from the lumen (5) and intraepithelial lymphocytes (IEL), which consist mostly of T cells. More immune populations are found in the lamina propria (LP), a layer of loose connective tissue supporting the epithelium.

Gut-Associated Lymphoid Tissue (GALT) Because it is a primary site of foreign antigen encounter, the intestine is associated with several types of lymphoid organs collectively referred to as GALT. Some of them, such as Peyer’s patches (PP) and the isolated lymphoid follicles (ILF), are within the mucosa itself. In addition, intestinal lymph drains into the mesenteric lymph nodes (MLN), which constitute a key

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Small intestine

Large intestine Bacterial antigen

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Figure 1 Intestinal immune environment. Intestinal immune responses take place in the epithelium, lamina propria, and gut-associated lymphoid tissue (GALT). The gut harbors cell populations specialized in antigen sampling and presentation, antimicrobial immunity, and maintenance of tolerance toward harmless exogenous antigens. (Abbreviations: IEL, intraepithelial lymphocytes; NKT, natural killer T cell; DC, dendritic cell; Treg, regulatory T cell.)

checkpoint to determine the anatomical location of tolerogenic or inﬂammatory responses. PP are secondary lymphoid organs that arise in the fetal small intestine independently of the

intestinal ﬂora (6). Their organization is comparable to that of lymph nodes, with large B cell follicles and T cell areas. ILF, in contrast, are distributed along the whole intestinal tract, and, www.annualreviews.org • Intestinal Immune Regulation

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GALT: gut-associated lymphoid tissue

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Triggering microorganism: a microorganism that colonizes normal hosts without inducing pathology but is colitogenic in susceptible hosts

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unlike PP, their development seems to be triggered by the intestinal ﬂora (7). Their number increases during chronic inﬂammation, stressing similarities between ILF and tertiary lymphoid structures. The existence of GALT not only allows for rapid immunity, but also for the response to be restricted to the gut environment. Under noninﬂammatory circumstances, intestinal bacteria elicit an intestinal, but not systemic, antibody response (8). Furthermore, the trafﬁcking of lymphocytes primed in the GALT can be regulated through speciﬁc upregulation of gut homing receptors. Populations of GALT DCs and the vitamin A metabolite retinoic acid are thought to be integral to this process (9, 10).

Intestinal Flora The contents of the intestinal lumen play a central role in intestinal homeostasis. Their composition changes along the intestinal tract, bacterial content being higher in the colon than in the small intestine. A number of studies have focused on the role of the intestinal ﬂora, which can modulate the immune system (11). In mice, the composition of the bacterial ﬂora in the gut strongly inﬂuences the outcome of the immune response. Some microorganisms seem to promote immune regulation (12), whereas others behave as triggering microorganisms: They are not pathogenic in normal circumstances but trigger intestinal inﬂammation in susceptible hosts. An example of this is the bacterium Helicobacter hepaticus (13). This organism, common in many animal facil-

ities, colonizes the cecum and intestine without inducing visible signs of inﬂammation in most mouse strains. However, unlike traditional ﬂora, it triggers the production of systemic speciﬁc antibodies (14), indicating a mild ongoing response that in susceptible hosts progresses to pathology (13). Studies in germ-free animals suggest that the normal state of the intestine is one of low basal inﬂammation induced by the commensal ﬂora. Indeed, in the absence of ﬂora, the GALT, spleen, and other lymphoid tissues are underdeveloped. These differences disappear upon colonization with conventional ﬂora or addition of Toll-like receptor (TLR) ligands to the drinking water (11, 15). The intestinal ﬂora is also responsible for the basal levels of heat shock proteins and proinﬂammatory cytokines found in the gut. This affects not only lymphocytes, but also IEC and the other mucosal cell types, whose steady-state activity is tuned to this low-intensity inﬂammation. Therapies altering the gut microbiota must take account of these interactions to avoid disrupting intestinal homeostasis.

INTESTINAL INFLAMMATION The balance between host and ﬂora is maintained through the cooperation of various regulatory mechanisms that prevent the immune system from reacting toward harmless exogenous antigens present in the intestine. This balance can be disrupted for several reasons (Figure 2). Typically, inﬂammation occurs when proinﬂammatory stimuli are increased, due for example to pathogenic infection.

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 2 Disruption of intestinal immune homeostasis. Under normal circumstances, the intestinal regulatory mechanisms outweigh inﬂammatory signals induced by the immune system reacting to the stimuli from the ﬂora (basal status). However, this balance can be switched for different reasons. While in most cases the ﬁnal result will be an accumulation of inﬂammatory mediators, the underlying causes can be various. (a) In the case of impaired regulation, the normal reaction of the immune system to the intestinal stimuli can result in chronic inﬂammation. (b) Disruption of the intestinal barrier exposes the immune system to a great amount of proinﬂammatory stimuli, which can overcome intestinal regulation. (c) Enhanced immune reactivity can also lead to colitis even if the levels of regulation and proinﬂammatory stimuli remain basal. (d ) Reduced immune reactivity can paradoxically also enhance colitis. Reduced immune responses would fail to control opportunistic pathogens, so that the microbial proinﬂammatory stimuli would accumulate and tip the balance, causing chronic inﬂammation despite a reduced basal immune reactivity. 316

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Regulatory activity Inﬂammatory activity

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Barrier disruption models of colitis: experimental models in which colitis is induced by disruption of the intestinal epithelial barrier plus inﬂammatory stimuli, e.g., DSS, TNBS, and oxazolone models T cell transfer model: experimental model in which Tregdepleted CD4+ T cells (e.g., CD45RBhigh ) transferred into immunodeﬁcient hosts react to the intestinal ﬂora inducing chronic colitis and wasting disease IBD: inﬂammatory bowel disease CD: Crohn’s disease UC: ulcerative colitis

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Similarly, the disruption of the intestinal barrier in certain animal models also leads to intestinal inﬂammation by increasing the proinﬂammatory stimuli in contact with the immune system (2). Inﬂammation may also result from defective immunoregulatory mechanisms, as in IPEX patients, or from excessive immune effector activity (2). Paradoxically, diminished effector activity can also yield intestinal inﬂammation, as reduced immune responses may allow for the growth of pathogens and thus increase the load of proinﬂammatory stimuli. Indeed, intestinal disorders are frequent manifestations of AIDS and primary immunodeﬁciencies in humans (16, 17). In animal models, intestinal inﬂammation is induced by altering these different parameters. Barrier disruption models often combine disruption of the intestinal barrier through ethanol and a haptenating agent, such as trinitrobenzene sulfonic acid (TNBS) or oxazolone, to produce acute colitis (2). Administration of ethanol alone does not sufﬁce to induce colitis, indicating that regulation prevails after a transient breach of the intestinal barrier. Administration of dextran sulfate sodium (DSS) in the drinking water also disrupts the barrier, inducing acute colitis. Experimental colitis can also be induced by overexpressing proinﬂammatory mediators, such as tumor necrosis factor (TNF)-α, or by removing regulatory elements (2). Among the latter, transfer of naive CD4+ T cells into immunodeﬁcient hosts (T cell transfer model) is widely used (18). The nature of the model is critical to the interpretation of experimental observations. For example, interleukin (IL)-23 promotes colitis in several models of exacerbated immune reactivity or reduced regulation, but it seems to protect from colitis in settings of barrier disruption, consistent with a role in defense against microorganisms (19). Analysis of human disease also suggests that intestinal inﬂammation can result from an inappropriate immune response by the host to an external factor. In humans, the most frequent causes of chronic noninfectious intestinal inﬂammation are celiac disease and inﬂammatory bowel disease (IBD), which includes Crohn’s Izcue

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disease (CD) and ulcerative colitis (UC). Celiac disease arises as a response to gluten in the food, and the symptoms often disappear upon adoption of a gluten-free regime (20). Celiac disease has a strong immune-associated genetic component. The main genetic risk factor is a susceptible HLA haplotype, whereas several other risk-associated loci map to genes involved in immune function such as chemokines and cytokines (21). IBD, considered to be a dysregulated response to the intestinal ﬂora, also has a strong genetic component. Among the genetic risk factors are polymorphisms in patternrecognition receptors such as NOD2, and the IL-23 pathway, which also controls colitis in several mouse models (22, 23). Although it has been suggested that pathogenic microorganisms initiate human IBD, this view is currently under debate, as several studies have failed to identify the putative pathogens. In animal models, Th1, Th2, and Th17 responses can induce intestinal inﬂammation (2, 19). In addition, several other immune pathways have been described to mediate various types of responses in the intestine, including B cells, CD8+ T cells, mast cells, and natural killer (NK) cells. Clearly, intestinal inﬂammation can be mediated by many different pathways, and intestinal regulatory mechanisms need to be correspondingly diverse to meet the variety of effector cell types.

REGULATORY MECHANISMS Tolerance in the intestine depends on a wide array of independent immunosuppressive mechanisms with partially overlapping functions. Although many types of regulatory lymphocytes exert their function in the intestine, this activity is mediated by a relatively small number of known mechanisms common to several cell lineages. The best characterized to date are the cytokines IL-10 and transforming growth factor (TGF)-β. In addition, new evidence is drawing attention to the regulatory properties of other nonproteic molecules, such as nucleotides or tryptophan. Furthermore, restoration of intestinal homeostasis depends not only on

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reducing inﬂammation, but also on the integrity of the intestinal barrier, and lymphocytes can also regulate inﬂammatory responses by interacting with IEC to enhance barrier function. This section summarizes the current knowledge on how these processes control intestinal inﬂammation. We have chosen to concentrate on the factors for which evidence suggests a direct immunosuppressive or protective effect. As a result, factors with an indirect effect are only mentioned where they promote a known mechanism. Indirect pathways include proinﬂammatory cytokines that also induce responses that limit bystander damage, or deviation of the Th response toward a more easily regulated response. There are a number of confounding factors when trying to assess the relevance of a speciﬁc regulatory pathway in intestinal inﬂammation. One of them is the partial redundancy of tolerogenic mechanisms. As a consequence, functional effects may only become apparent in conditions of high immune reactivity or stimulatory load, and thus differences in the research model or the presence of triggering microorganisms can generate conﬂicting results between different research groups. A further issue arises when analyzing mice deﬁcient in a regulatory pathway critical for systemic tolerance. In most cases, genetically induced colitis only manifests several weeks after weaning (see below). In contrast, deletion of key pathways in prevention of autoimmunity can lead to a rapid multiorgan disease with death around weaning. In the latter case, the intestine is rarely among the primary target organs, but this does not mean that the pathway is not important in regulating intestinal inﬂammation. Rather, it appears that the requirement for regulation of the intestinal immune response is low early in life and increases when weaning diversiﬁes the dietary antigens and ﬂora, raising the immunogenic proﬁle of the gut.

IL-10 IL-10 is a homodimeric cytokine with broad anti-inﬂammatory activities. It can be pro-

duced by most hematopoietic and some nonhematopoietic cell types, and it acts on lymphocytes and myeloid cells to suppress a wide range of immune responses (24). Even though the effects of IL-10 are predominantly anti-inﬂammatory, IL-10 can enhance CD8mediated cytotoxicity and some B cell responses, such as IgA secretion, and seems to play an important pathogenic role in systemic lupus erythematosus. The IL-10 receptor is formed by the IL10R1 and IL-10R2 chains, the latter also forming part of the IL-22 receptor. It is expressed by most hematopoietic cells, although the level varies with activation. It can be induced on some nonhematopoietic cells, and it is constitutively expressed in the colonic epithelium (24). Even though IL-10 can modulate many different inﬂammatory pathways, IL-10 knockout or IL-10R2 knockout mice do not suffer from severe lethal autoimmune syndrome, but instead tend to develop colitis in the presence of triggering microorganisms (Figure 3) (25, 26). IBD in humans is also thought to arise from interplay between genetic and environmental factors and, signiﬁcantly, a recent report has identiﬁed IL10 as a susceptibility locus for human UC (27). This ﬁnding suggests that similar mechanisms regulate chronic intestinal inﬂammation in humans and mice. Colitis arising in IL-10 knockout mice is mediated by CD4+ T lymphocytes, as RAG deﬁciency but not B cell deﬁciency inhibits disease development, and the pathology could be transferred by intestinal CD4+ cells (28). However, the crucial role of IL-10 in preventing colitis may not be exerted directly on T cells, but rather on a myeloid population through the ability of IL-10 to reduce the antigen-presenting capacity of monocytes and DCs (24). Accordingly, mice with a myeloid-speciﬁc ablation of STAT3, a mediator in IL-10 signaling, develop a similar colitis to IL-10 knockout mice (29). IL-10 has also been described to inhibit cytokine and chemokine production by monocytes and macrophages and to induce the production of soluble antagonists of IL-1β and TNF-α, www.annualreviews.org • Intestinal Immune Regulation

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Figure 3 Synergy of genetic susceptibility and triggering ﬂora in the induction of colitis. (a) Under normal circumstances, the intestinal regulatory mechanisms outweigh the inﬂammatory signals induced by the immune system reacting to the stimuli from the ﬂora. Even the presence of triggering organisms such as Helicobacter hepaticus among the ﬂora does not result in intestinal inﬂammation. (b) When the intestinal regulatory mechanisms are reduced (for example in IL-10 knockout mice), the basal immune activity and normal ﬂora may not be enough to induce inﬂammation. However, if the inﬂammatory stimuli are increased (for example, through the presence of triggering ﬂora), the balance switches to inﬂammation and colitis.

two key mediators in intestinal inﬂammation (24). Because of IL-10’s anti-inﬂammatory role in the intestine, provision of exogenous IL-10 has been used as a strategy to restore intestinal homeostasis in animal models. Injection of recombinant IL-10 or IL-10 transgenic T cells inhibits colitis after naive T cell transfer (30, 31). At a more local level, feeding intestinal bacteria genetically engineered to produce 320

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IL-10 ameliorates gut inﬂammation in IL-10deﬁcient mice or in mice developing chronic colitis in a barrier disruption model (32). Indeed, polysaccharide A produced by the intestinal commensal Bacteroides fragilis can prevent colitis in the T cell transfer model in an IL10-dependent manner (12). In contrast to its role in promoting tolerance to the ﬂora, IL-10 seems to be dispensable for the induction of oral tolerance (33).

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TGF-β TGF-β is a pleiotropic cytokine with essential functions in many cellular pathways. There are three forms of TGF-β in mammals: TGF-β1, TGF-β2, and TGF-β3 (34). The TGF-β receptor is widely expressed on hematopoietic and nonhematopoietic cells. TGF-β1 is secreted as a latent complex and needs to be activated via conformational change or protein cleavage. Several molecules can mediate this activation, including furin, metalloproteinases, and the integrins αvβ6 and αvβ8. The essential role of TGF-β1 in controlling immune responses became apparent when TGF-β1 knockout mice were generated and died within a few weeks after birth as a consequence of extended multiorgan autoimmunity (34). T cells are key for this autoimmunity, as depletion of CD4+ or CD8+ lymphocytes prolongs survival, and lymphocyte-deﬁcient mice do not develop autoimmunity (35–37). Indeed, mice speciﬁcally lacking TGF-β responsiveness in T cells develop a similar inﬂammation to TGF-β1-deﬁcient mice (38, 39). TGF-β also has proinﬂammatory effects through the induction of Th17 cells (40). Interestingly, a recent report demonstrated that high levels of TGF-β could inhibit Th17 induction, suggesting that TGF-β could promote or inhibit Th17 cells in a dose-dependent manner (41). The autoimmune syndrome initiated by TGF-β1 deﬁciency or by a lack of TGF-β responsiveness in T cells develops quickly, and the intestine is not severely affected, probably because of the early onset of the autoimmune disease. Indeed, mice in which T cell responsiveness to TGF-β is severely impaired but not completely abolished develop a slower disease at around 2 months of age, and intestinal inﬂammation is observed (42). Mice with a T cell-speciﬁc deletion of furin, which has been involved in activating TGF-β, develop spontaneous autoimmune disease including colitis at six months of age (43). Similarly, mice expressing TGF-β that cannot bind integrins recapitulate the phenotype of TGF-β1-deﬁcient

mice, and mice in which myeloid cells or DCs are deﬁcient for the integrins αv or β8, respectively, develop colitis at around 4 months of age (44–46). Although these results should be interpreted with caution, they ﬁt in with a role for activated TGF-β in maintaining intestinal tolerance. Further support that TGF-β plays a role in intestinal homeostasis comes from mice with a defective TGF-β signaling pathway; these mice develop rectal prolapse and intestinal inﬂammation (47, 48). In addition to its functions as a suppressor of immune responses, TGF-β is also thought to play an important role in the homeostasis of IEC, thereby contributing to the maintenance of the intestinal barrier and the reduction of proinﬂammatory stimuli. Indeed, mice with reduced TGF-β signaling in goblet cells or IEC show increased susceptibility to barrier disruption colitis (49, 50). It remains to be assessed whether this susceptibility is linked to defects in the barrier function or to a lack of immune regulation.

Indoleamine 2,3-dioxygenase (IDO) IDO is an enzyme that degrades tryptophan to kynurenine. It reportedly mediates immune tolerance in various models (51). The putative mechanism involves the depletion of the essential amino acid tryptophan, which is necessary for T cell proliferation, and production of the toxic catabolite kynurenine. IDO can be expressed by several cell types, including DCs and macrophages. No autoimmune phenotype has been found in IDO-deﬁcient mice, although this could be due to functional redundancy, as there are two related IDO genes whose respective functions remain to be characterized (51). IDO is highly expressed in the intestine, and this expression is further increased upon inﬂammation in mice and humans (52–55). One report using a barrier disruption model showed increased colitis in mice treated with an IDO inhibitor, and IDO knockout mice seem more susceptible to colitis associated with graftversus-host disease (GVHD) (52, 56). In contrast, IDO knockout mice are more resistant

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to Citrobacter rodentium–induced inﬂammation, presumably because of enhanced antibacterial immunity in the absence of IDO-mediated regulation (53).

Apoptosis of Effector Cells

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In several models, resolution of intestinal inﬂammation is observed in conjunction with the apoptosis of effector cells (57, 58). Indeed, induction of apoptosis plays an essential role in terminating immune responses, and mice with defects in apoptotic pathways exhibit increased autoimmunity (59). In addition, apoptosis may trigger tolerance by promoting immunosuppressive pathways (60). Thus, phagocytosis of apoptotic lymphocytes can trigger TGF-β production by DCs and macrophages, leading to inhibition of immune responses (61). Several mechanisms are responsible for apoptosis. Two of them, cytotoxicity and cytokine deprivation, have lately become the focus of studies on regulation. Cytotoxicity involves active killing of the target cell. Considered a major feature of CD8+ and NK cells, cytotoxicity has also been described in other cell types. Although several reports have shown killing of effector subsets by regulatory lymphocytes in vitro, the exact extent of these mechanisms in vivo is not known (62). In vivo, cytotoxic regulation could involve killing of the effector cell or the antigenpresenting cell (APC); the latter would allow for regulation of immune responses in an antigenspeciﬁc way. Activated immune cells undergo apoptosis when inﬂammatory cytokines are withdrawn in vitro. The same mechanism likely operates in vivo, for example during the termination of immune responses, when the levels of proinﬂammatory cytokines drop and inﬂammatory cells are no longer required. Cytokine withdrawal by competitive uptake or by blockade with a therapeutic antibody has been put forward as a mechanism preceding and inducing the termination of immune responses (57, 58). This hypothesis, however, is hard to test owing to the difﬁculty in determining whether apoptosis is the cause 322

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or the consequence of the terminated immune response.

Barrier Mechanisms To abrogate intestinal inﬂammation, it is necessary not only to suppress immune activity, but also to reduce to a minimum the stimulatory load that is driving the inﬂammation. Restoring the barrier function is an essential component of intestinal homeostasis. This can be achieved by promoting IEC homeostasis or wound restitution (the migration of epithelial cells to reseal an injury), and also through the induction of antimicrobial factors that create a microbicidal barrier on the epithelium, such as defensins. Intestinal trefoil factor (TFF3), produced by goblet cells, can play a role in epithelial cell homeostasis. In the intestine, TFF3 is involved in IEC restitution and protection from apoptosis (63); mice deﬁcient for TFF3 are more sensitive to DSS-induced colitis, presumably because of poor epithelial repair. IL-6 can trigger TFF3, and, consistently, mice with defects in IL-6 signaling are also more susceptible to DSS-mediated colitis (64). Apart from TFF3, several growth factors can promote IEC growth and differentiation, including Reg proteins and the keratinocyte growth factor (KGF). Administration of recombinant KGF can ameliorate colitis not only after barrier disruption, but also in the T cell transfer model (65). In addition to IL-6, other cytokines such as IL-17 and IL-22 can promote barrier function. IL-22 is mainly produced by T lymphocytes, and it reportedly induces restitution, proliferation, and production of proinﬂammatory cytokines and antimicrobial peptides by epithelial cells (66). In mice, IL-22 protects hepatocytes during acute immune responses. IL-22-deﬁcient mice do not develop spontaneous colitis, but they show increased susceptibility to intestinal challenges, compatible with a defect in the intestinal barrier (67, 68). Human IBD is also associated with high IL-22 levels in the intestinal lesions, but the signiﬁcance of this observation remains to be established (69).

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CD4+ FOXP3+ REGULATORY T CELLS IN INTESTINAL RESPONSES Among the cells mediating dominant tolerance, CD4+ Foxp3+ regulatory T cells (Tregs) play an essential role in intestinal homeostasis. Tregs are characterized by constitutively high levels of the transcription factor Foxp3, which is considered to confer their regulatory activity (70, 71). In humans, and perhaps also in mice, low levels of Foxp3 are also transiently expressed by other CD4+ T cell subsets upon activation, although the signiﬁcance of this is unclear. Patients with mutations affecting the FOXP3 gene develop immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX) (72). IPEX is a fatal autoimmune syndrome starting early in life and affecting, among other organs, the skin and endocrine glands. However, the most frequently affected organ is the intestine, highlighting the importance of Tregs in maintaining intestinal tolerance (73). In the mouse, deﬁciency of the Foxp3 gene triggers a rapid fatal multiorgan autoimmune disease (71). Probably because of the rapid course of disease, these mice do not tend to develop intestinal inﬂammation. With the exception of IPEX, intestinal inﬂammation is not generally associated with a decrease in Tregs. Indeed, absolute Treg numbers seem to be increased in the mucosa of patients with IBD or celiac disease, probably as a consequence of the ongoing inﬂammation (74–76). The identiﬁcation of Foxp3 as a marker for Tregs sheds light on the identity of a population of “naturally arising” regulatory T cells that have the ability to block colitis and several autoimmune diseases upon T cell transfer in vivo. In mice, this population is enriched within the CD45RBlow antigen-experienced CD4+ T cell population, especially in the CD25+ subset (18, 71). Transfer of this subset can prevent colitis induced by T cell transfer or innate immune activation (77). The protective activity is now considered to be due to Tregs, as they account

for about 90% of the CD4+ CD45RBlow CD25+ population (71). Foxp3 is a more speciﬁc Treg marker than CD45RB and CD25. The CD4+ CD45RBlow subset also includes nonregulatory antigen-experienced cells, and CD25 is expressed by recently activated cells in addition to Tregs. Despite these deﬁciencies, Treg isolation based on the CD45RBlow CD25+ phenotype is a useful tool to study Treg function.

Treg: CD4+ Foxp3+ regulatory T cell

Treg Development Tregs can acquire Foxp3 expression and their regulatory activity in the thymus. Differentiation into the Treg lineage is T cell receptor (TCR)-driven and is believed to take place in response to high-afﬁnity recognition of thymic antigens (78). Indeed, transfer of CD4+ CD25+ thymocytes is able to prevent the development of intestinal inﬂammation in the T cell transfer model (79). However, it is not known whether Tregs are activated by endogenous self-antigens to mediate bystander suppression of responses directed against the ﬂora, or whether they directly recognize microbial antigens. In addition to the thymic development of Tregs, activation under certain conditions can also induce Foxp3 expression and regulatory function on naive peripheral T cells. In vivo, Tregs emerge in the intestinal LP and MLN following oral administration of antigen (80). This occurs in both the presence and the absence of a normal thymically derived Treg repertoire (81). Furthermore, after systemic transfer of naive polyclonal T cells, Tregs were detected primarily in the small intestinal LP and MLN, suggesting that the GALT may be a preferential site for the peripheral generation of Tregs (81). This may reﬂect the relatively high availability of foreign antigenic material at these sites. Generation of Tregs in the intestine may also reﬂect other local environmental conditions. For example, expression of Foxp3 in naive peripheral T cells in both in vitro and in vivo systems depends on TGF-β signaling, and the intestine is widely acknowledged to be a TGFβ-rich environment (82). Furthermore, recent data show that retinoic acid, a metabolite of www.annualreviews.org • Intestinal Immune Regulation

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the dietary component vitamin A, can act as a cofactor to TGF-β in the induction of Foxp3 (81, 83–85). Targeting of antigen to DCs in vivo can result in the peripheral generation of Tregs (86). In addition, small intestinal LP and MLN DCs deriving from the intestine are better at driving Foxp3 expression than their splenic or lymph node–resident counterparts (81, 84, 85, 87). Intestinal LP macrophages may also perform this function (88). This may correlate with their ability to produce or activate TGF-β or to metabolize vitamin A. Accordingly, intestinal DC-mediated induction of Foxp3 can be inhibited by blocking antibodies to TGF-β or by retinoic acid receptor antagonists. Oral feeding of antigen was shown a number of years ago to induce a regulatory CD4+ T cell subset acting through TGF-β. This subset was called Th3 cells (89). They have been recently reported to express and be dependent on Foxp3, suggesting that Th3 cells and induced Tregs may in fact be the same cells (90). The need to prevent destructive immune responses against foreign but harmless antigen may necessitate the generation of Tregs speciﬁc for these antigens to complement the repertoire of what are thought to be largely self-reactive Tregs generated in the thymus. Although Tregs generated in vitro or through oral antigen administration can have regulatory function in both in vitro suppression assays and disease models, it remains unclear what contribution they may make to intestinal immune regulation in normal hosts (91, 92). Complications in differentiating thymically derived and peripherally induced Tregs in normal animals will make this a complex question to address, as no markers allowing the distinction between thymically and peripherally generated Tregs have been identiﬁed so far.

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Effector Pathways Studies analyzing the regulatory function of CD4+ CD45RBlow , CD4+ CD25+ , and CD4+ Foxp3+ T cells have identiﬁed several independent mechanisms that can mediate 324

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their regulatory activity. These mechanisms can be cytokine-mediated (Figure 4) or cytokine-independent. None of them seems obligatory for Treg function, but rather it seems that Tregs can make use of an array of immunosuppressive mechanisms with partially redundant effects. Importantly, Tregs can regulate not only T cell responses, but also the activity of the innate immune system. One speciﬁc feature of intestinal Tregs is their ability to produce IL-10. Several studies have shown that Tregs isolated from the spleen or MLN produce very little IL-10 under steady-state conditions. However, Tregs are the main CD4+ T cell population producing IL-10 in the colonic LP, and they account for about one-third of the IL-10-producing CD4+ T cells in the LP of the small intestine (75, 93, 94). Although IL-10 deﬁciency does not impair the ability of Tregs to prevent colitis in the T cell transfer model, IL-10 knockout Tregs are less able to abrogate established colitis or prevent inﬂammation induced by the triggering microorganism Helicobacter hepaticus (75, 77). These results suggest that regulation of intestinal responses does not absolutely require IL-10, but Treg-produced IL-10 becomes mandatory in the presence of a strong microbial stimulus or established intestinal inﬂammation. In accordance with these data, Treg-speciﬁc ablation of IL-10 is sufﬁcient to confer susceptibility to ﬂora-triggered colitis (95). The colitis is less severe than in complete IL-10 knockout mice, indicating that IL-10 produced by other cell populations contributes to intestinal tolerance. Nevertheless, this result highlights the nonredundant role of Tregs in controlling intestinal inﬂammation. Interestingly, mice lacking the transcription factor Blimp in all T cells had reduced IL-10 production in Tregs (96, 97). These mice also showed hyperreactive effector T cells and developed colitis in a way reminiscent of IL-10-deﬁcient mice. Besides its role in peripheral generation and homeostasis of Tregs, TGF-β is also a Treg effector molecule. T cells that cannot respond to TGF-β are refractory to Treg-mediated regulation, but the sources of TGF-β are still not

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Epithelium

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Figure 4 Cytokines in the Treg network. The cytokine TGF-β induces Treg development, whereas IL-6, IL-21, IL-23, and IL-27 can inhibit this process. IL-2 produced by activated effector cells maintains Tregs. Tregs can produce IL-10 and TGF-β. IL-10 acts on innate immune cells to reduce inﬂammation, whereas TGF-β inhibits effector cells directly and promotes epithelial homeostasis. In addition, TGF-β can synergize with other cytokines to promote the differentiation of Th17 cells that promote IL-17 and IL-22, which also contributes to the barrier function. Effector cells can enter into a state that is refractory to suppression in the absence of TGF-β signals or in the presence of IL-6.

well deﬁned (79). Tregs can produce TGF-β, and a T cell–speciﬁc deletion of TGF-β1 in mice yielded colitis after 4 months of age (98). However, studies on the relevance of TGF-β production by Tregs to control intestinal inﬂammation have produced contradictory results (79, 98). This discrepancy could be due to the presence or absence of triggering microorganisms in the two experiments, as occurs in IL-10-deﬁcient mice. If this is the case, TGFβ1 and IL-10 production could be regarded as independent mechanisms employed by Tregs to maintain intestinal tolerance. Tregs are versatile enough to compensate for the lack of one of these factors to prevent colitis. However, when the proinﬂammatory stimuli in the colon are increased (for example by triggering ﬂora), Tregs deﬁcient in one of these mechanisms will be

unable to regain the tolerant state, and chronic colitis will ensue. It is unclear how T cell–produced TGFβ1 exerts its protective functions and why it plays such a decisive role when TGF-β1 produced by other sources is readily available in the intestinal environment. Treg production of TGF-β1 could be critical for dampening T or innate cell responses or for the recovery of epithelial barrier function. Tregs have been shown to promote Th17 differentiation in a TGF-βdependent manner (40). Th17 cells can then produce IL-22, which may provide a mechanism through which Tregs reinforce the epithelial barrier. Another cytokine that may mediate some of the effects of Tregs is IL-35, which consists of IL-12p35 and IL-27 EBI3 (99). Although www.annualreviews.org • Intestinal Immune Regulation

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mice deﬁcient for p35 or EBI3 do not develop spontaneous autoimmunity or colitis, Tregs deﬁcient for any of these subsets prove less efﬁcient in curing colitis in the T cell transfer model. To date, no data are available as to whether IL-35 is also expressed by human Tregs or whether it is associated with intestinal inﬂammation. Prevention of the intestinal immune response has been associated with apoptosis of the effector T cells (57, 58). Indeed, several reports have shown that Tregs can express cytotoxic molecules and induce effector cell apoptosis in vitro, and cytokine withdrawal has been proposed as mediating Treg function in vitro and in vivo (58, 62, 100). As discussed above, the causative role of apoptosis in regulation is hard to test, and more experimental evidence is needed to support these hypotheses. Cytotoxic T lymphocyte antigen (CTLA)4 is an inhibitory receptor induced on naive T cells upon activation, but it is expressed on a high proportion of Tregs under steady-state conditions (101, 102). Blocking Treg-expressed CTLA-4 with antibodies abrogates their protective capacity, inducing colitis in the T cell transfer model (103). CTLA-4-deﬁcient Tregs are functional in vivo, but unlike their wildtype counterparts, they cannot prevent colitis induced by CTLA-4 knockout T cell transfer, suggesting that autoimmunity is a combination of defects in both arms of the immune response (103). A recent report showed that, unlike Foxp3 knockout, mice with a Foxp3speciﬁc CTLA-4 ablation survived into adulthood (104). These knockouts then succumbed to lethal autoimmunity, conﬁrming the important role of CTLA-4 in Treg-mediated immune regulation. Interestingly, enterocolitis is the most frequent adverse reaction after blocking CTLA-4 during clinical trials, suggesting that in humans as in mice CTLA-4 plays an important role in Treg control of intestinal homeostasis (105). Although the importance of CTLA-4 in intestinal regulation is well established, the mechanisms involved are poorly understood. CTLA4 has been reported to induce IDO in DCs

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and macrophages, and Tregs may use this same mechanism to achieve tolerance (106). However, another possibility is that CTLA-4 selectively downregulates costimulatory molecules on the APC, thus limiting the activation of pathogenic T cells (107). Finally, CTLA-4 is involved in controlling T cell trafﬁcking (108). Clearly, more research is needed to discern which of the CTLA-4 functions is essential for Treg activity. Several other mechanisms have been put forward as mediators of Treg activity. One of these is the control of the extracellular levels of adenosine. Adenosine can act as a signal of tissue stress and can induce immune tolerance, and a deﬁciency in the adenosine receptor A2A can impair Treg function and decrease the susceptibility of effector cells to Treg signals in the T cell transfer model of colitis (109). Paradoxically, knockout mice for this adenosine receptor do not develop spontaneous disease. An independent report has also found that Tregs deﬁcient for CD39, which participates in extracellular adenosine generation from ATP, are less efﬁcient in vitro and in preventing skin graft rejection in vivo (110). The physiological role of adenosine in intestinal homeostasis remains to be ascertained.

Control of Tregs To allow for the induction and resolution of host protective immune responses, Treg development and activity is tightly linked to the inﬂammatory response. On one hand, Treg control needs to be restrained to allow immune responses to proceed. On the other hand, Tregs have to keep pace with ongoing immune responses to restore the immune balance once inﬂammation is no longer required. Indeed, inﬂammation is often associated with increased numbers of Tregs. Several proinﬂammatory cytokines can inhibit peripheral Treg induction. IL-6, IL-21, and IL-27 suppress TGF-β-mediated Treg generation, whereas IL-23 is key for the inhibition of Treg induction during intestinal inﬂammation (111–114). In contrast, the

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proinﬂammatory cytokine IL-2 is also essential for Treg homeostasis, suggesting a possible mechanism to link Treg numbers to activated T cell responses (115). Indeed, IL-2-deﬁcient mice develop intestinal inﬂammation after several months of age, indicating that IL-2 is more important for regulation than for inﬂammation in the intestinal setting (116). Tregs that cannot respond to IL-2 can develop, but they fail to accumulate in the periphery. In vitro data indicate that IL-2 is also important for Treg homeostasis in humans, and, interestingly, a patient with mutations in the IL-2R gene developed an IPEX-like syndrome despite possessing intact Foxp3 (117). Glucocorticoid-induced tumor necrosis factor receptor (GITR) is another Treg-associated molecule. Although it is expressed on Foxp3− T cells upon activation, its expression in Tregs, like CTLA-4, is high under steady-state conditions (118, 119). Unlike the latter, GITR seems to neutralize Treg function, as agonistic antibodies against GITR abrogate Treg activity and allow for the development of colitis (119, 120). Wiskott-Aldrich syndrome protein (WASP) is implicated in TCR-mediated cytoskeletal reorganization and it is mutated in WiskottAldrich syndrome. Paradoxically, patients suffer both from immunodeﬁciency and from an autoimmune syndrome that includes IBD among its manifestations. Accordingly, WASP knockout mice develop colitis by 4 months of age (121). Interestingly, WASP knockout Tregs develop normally but fail to compete with wildtype Tregs and are unable to prevent colitis in a T cell–transfer model (122–124). Impaired function of human Tregs from Wiskott-Aldrich syndrome patients has also been demonstrated, suggesting that this may be a causative factor in their autoimmunity and intestinal inﬂammation. DCs not only play an important role in Treg generation, but they can also control Treg activity by the secretion of cytokines and through direct interactions. For example, in the T cell transfer model, Tregs require CD103 expression by DCs to prevent colitis (125).

Treg Location Another control point in Treg activity is their trafﬁcking. It is unclear whether Tregs need to migrate into the gut itself to control intestinal inﬂammation or if they can do so by inhibiting lymphocyte activation in the spleen and MLN. Transfer of T cell subsets has shown that Tregs and effector T cells preferentially accumulate in the lymphoid organs during prevention of colitis. In contrast, Tregs transferred into recipients with established colitis extensively migrated to and proliferated in the gut, suggesting that Tregs can control inﬂammation in situ (126). Indeed, Tregs can prevent intestinal inﬂammation in mice lacking spleen, MLN, and PP, indicating that these sites are not essential for prevention of colitis (127). Chemokines are important mediators of cell migration, and Tregs deﬁcient for chemokine receptors present defects in regulation of immune responses in speciﬁc organs (128). The chemokine receptor CCR9 and the integrin α4β7 direct migration into the small intestine. The effects of CCR9 on Treg maintenance of intestinal homeostasis have yet to be assessed. However, a study using Tregs deﬁcient for the β7 molecule, which pairs with α4 to generate gut-tropic receptors, showed that they are not impaired in prevention of colitis in the T cell transfer model (129). This prevention could, however, be taking place in the lymphoid tissues, as the same report indicated that β7deﬁcient Tregs have impaired accumulation in the intestine. Interestingly, a blocking antibody against α4 integrin is approved for CD treatment; its efﬁciency is supposedly linked to the inhibition of pathogenic T cell migration into the colon. However, studies in a mouse model suggest that blocking α4 could also increase colitis, maybe by inhibiting Treg migration into the colon (130). Other chemokine receptors implicated in migration to the intestine are CCR2 and CCR5, which have been reported to control trafﬁcking during inﬂammatory responses; however, both CCR2- and CCR5-deﬁcient Tregs are able to prevent colitis in the T cell transfer

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model (131). By contrast, Tregs deﬁcient for CCR4 cannot prevent colitis in this model, and CCR7-deﬁcient Tregs show a modest decrease in their potency (131, 132). The elucidation of the factors regulating Treg localization during immune responses will be key for future therapeutic treatments.

FOXP3− CD4+ T CELLS

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Aside from Tregs, other CD4+ subsets can also regulate immune responses. CD4+ Foxp3− T cells are also able to produce TGF-β and IL-10; they can also express CTLA-4 and IFN-γ and thus presumably activate IDO (24, 34, 51). In addition, they have been reported to express adenosine-producing ectoenzymes, although at lower levels than do Tregs (110). They can also produce cytokines that promote barrier integrity, such as IL-17 and IL-22 (133). Despite this variety of potential tolerogenic mechanisms, most reports dealing with the regulatory effects of CD4+ Foxp3− T cells have focused on their ability to produce IL-10. Many different types of CD4+ lymphocytes can produce IL-10. Among the most studied are Tr1 cells, which were ﬁrst described as IL-10-producing and IFN-γ-producing T cells that arose in vitro after culture with IL-10 (134). These cells were shown to inhibit a number of inﬂammatory diseases, including T cell transfer colitis via an IL-10-dependent mechanism (134, 135). However, the lack of markers for Tr1 cells has hampered assessment of their physiological role. Indeed, it is now evident that most CD4+ Th subsets (Th1, Th2, Th17) can produce IL-10 if stimulated under certain conditions (136, 137), raising the possibility that Tr1 cells represent chronically stimulated Th cells that have downregulated their proinﬂammatory cytokines but maintained IL10 production (136). Thus, T cell–derived IL10 represents an important negative regulator that limits the inﬂammatory reaction, avoiding extensive damage to the tissue and even allowing for the survival of a restricted population of pathogens to refuel protective memory responses. 328

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Such a negative-feedback loop among antigen-speciﬁc T cells is also evident in the intestine, as IL-10-secreting bacteria-reactive CD4+ T cells can prevent immune pathology in Helicobacter hepaticus infection (138). However, in this study it was not formally proven that the active population was Foxp3− . Nevertheless, experiments involving IL-10 ablation in speciﬁc subsets indicate that IL-10 production by non-Tregs is important for intestinal homeostasis (95). In celiac disease, immune suppressive gliadin-speciﬁc Tr1 cells were isolated from intestinal biopsies, suggesting a potential role for these cells in controlling the inﬂammatory response to dietary antigens (139). Indeed, IL-10 production by non-Treg CD4+ cells may be particularly important in the small intestine because, compared with the colon, most IL-10-producing cells in the small intestine were Foxp3− CD4+ (94). Interestingly, the induction of an IL-10 reporter gene in intestinal T cells was unaffected by the absence of IL-10, suggesting that these cells do not develop in the same way as Tr1.

CD8+ CELLS CD8+ T cells can also have regulatory properties and contribute to the maintenance of intestinal homeostasis (140). Both CD8αβ+ and CD8αα+ cells with regulatory function have been described. CD8αβ is primarily considered a lineage marker of MHC class I–restricted TCRαβ+ T cells with cytotoxic effector function, whereas CD8αα can be expressed alone or alongside CD4 or CD8αβ on TCRαβ+ T cells and also on TCRγδ+ T cells. Many of the described CD8+ regulatory populations express a restricted TCR repertoire and recognize nonclassical MHC class I molecules. CD8+ regulatory T cells have been suggested to control immune responses through a variety of regulatory mechanisms, including the production of TGF-β and IL-10 or killing of effector T cells or APC. Early studies of CD8+ T cell–mediated regulation revealed an important role for the murine nonclassical MHC class I molecule

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Qa-1 (HLA-E in humans) (141). It is thought that upregulation of Qa-1 and presentation of TCR-derived peptides on activated CD4+ effector T cells allows for their speciﬁc lysis by Qa-1-restricted CD8+ regulatory T cells. Recently, a distinctive population of Qa-1-restricted CD8αα+ T cells that regulate through this pathway has been cloned (142). However, many subpopulations of CD8+ regulatory T cells have been described that regulate immune responses via a number of different mechanisms, and whether Qa-1 plays a role in all these cases remains unclear. Good evidence exists that the intestinal environment can support the differentiation of CD8+ regulatory T cells. For example, regulatory CD8+ cells can be expanded in the presence of human IEC in a CD1d-dependent manner. These cells were characterized by TCR Vβ5.1 expression (143). CD8+ T cells also regulate inﬂammatory responses in the intestine. CD8+ CD28− T cells isolated from the spleen and LP prevented colitis in the T cell transfer model. Analogous to what has been demonstrated for Tregs, their regulatory function depended on their ability to secrete IL-10 and on the ability of the CD45RBhigh population to respond to TGF-β (144). Intriguingly, CD8+ T cell–mediated immune regulation may be defective in IBD patients. CD8+ T cells with regulatory function could be isolated from normal human LP, but not from the LP of IBD patients, where the proportion of TCR Vβ5.1– expressing T cells was also reduced (145). In addition to these populations, the IEC layer is home to a prominent population of CD8αα+ TCRαβ+ T cells (146). In this location, CD8αα can either be expressed on more conventional CD4+ or CD8αβ+ T cells, or on a distinct population of self-reactive cells. This latter population can have regulatory properties, although how it relates to the CD8αα+ clones described above remains an open question. For example, in a model in which mice with lymphocytic choriomeningitis virus (LCMV)-speciﬁc CD8αα+ IEL are infected with LCMV, antigen recognition does not appear to result in the upregulation of

cytotoxic effector function, but rather in the enhanced expression of TGF-β (147). Furthermore, IL-10 is produced by both TCRαβ+ and TCRγδ+ CD8αα+ IEL in the intestine (94). The idea that these cells are involved in preventing aberrant immune responses in the intestine is further supported by the ﬁnding that they prevent colitis in the T cell transfer model. This suppressive effect was IL-10 dependent (148). A minor population of CD8+ Foxp3+ cells has been described, but its physiological function remains to be clariﬁed (70).

γδ T CELLS TCRγδ+ T cells are also present in the IEC layer and may contribute to the maintenance of intestinal homeostasis either through direct regulation of the immune response or maintenance of the epithelial cell layer (149). These cells may also express the CD8αα receptor. TCRγδ+ IEL are not restricted by classical MHC molecules, but rather they are considered to recognize nonclassical MHC I molecules and other endogenous stress-induced ligands. Many TCRγδ+ IEL also express NK receptors such as NKG2D, which may modulate their functional properties. Furthermore, the γδ TCR and NKG2D appear to have common ligands, such as the human nonclassical MHC molecules MICA and MICB. Increased numbers of TCRγδ+ IEL have been observed in celiac disease, although the functional signiﬁcance of this observation remains unclear (150). Interestingly, CD8+ TCRαβ+ IEL have been suggested to kill IEC in an NKG2D-MICAdependent manner during celiac disease (151). TCRγδ+ IEL might be activated by the same stimulus to induce protection, but more evidence is needed to determine whether TCRγδ+ T cells are protective or pathogenic during celiac disease. A role for TCRγδ+ IEL in IEC homeostasis was described some time ago (149). These IEL positively regulate IEC turnover, likely as a result of their ability to produce KGF. This activity appears to have a protective www.annualreviews.org • Intestinal Immune Regulation

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effect in barrier disruption models of colitis. Thus, DSS- or TNBS-treated TCRδ−/− mice develop more severe colitis and are impaired in their ability to repair the intestinal epithelium when compared with wild-type mice (149). In agreement with this ﬁnding, transgenic expression of MICA in the intestine attenuated DSS colitis (146), although it was unclear whether this effect was mediated by TCRγδ+ , TCRαβ+ , or NK cells. In addition to the suggested role in epithelial repair of KGF derived from TCRγδ+ IEL, TCRγδ+ T cells secrete TGF-β, which is important both in epithelial repair and regulation of the immune response (152). Indeed, the presence of TCRγδ+ cells in barrier disruption colitis has been associated with decreased production of IFNγ, introducing the possibility that TCRγδ+ IEL may contribute directly to regulation of the immune response (152). Consistent with these ﬁndings, one report showed that a population of TCRγδ+ IEL isolated from celiac patients increased their expression of TGF-β1 following ligation of the inhibitory NK receptor, NKG2A. These cells were able to inhibit expression of molecules associated with cytotoxity in TCRαβ+ IEL through a mechanism partially dependent on TGF-β (153). Together, these ﬁndings suggest that TCRγδ+ IEL may play a protective role in intestinal injury or inﬂammation through enhancing epithelial repair and regulating immune responses. However, analysis of TCRγδ+ IEL function in triggering ﬂora-driven models of colitis resulted in rather contradictory ﬁndings. The triggering ﬂoradriven UC-like disease observed in TCRα−/− mice was signiﬁcantly attenuated in the absence of TCRγδ+ cells, suggesting that TCRγδ+ IEL actually exacerbate intestinal inﬂammation (149).

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NATURAL KILLER T CELLS (NKT) AND MUCOSAL-ASSOCIATED INVARIANT T CELLS (MAIT) The intestinal LP is also home to populations of T cells with invariant TCR repertoires that have a putative role in immune homeostasis. 330

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NKT cells are CD1d restricted and can be divided into populations expressing an invariant TCRα chain (Vα14-Jα18 in mice or Vα24-Jα18 in humans) or those expressing a more diverse TCR repertoire. Invariant NKT cells are reactive to α-GalCer presented on CD1d, whereas variant NKT cells are not. Despite being rare in the intestine, these cells may have a role in intestinal immune homeostasis (154). Notably, activation of NKT cells through α-GalCer has been reported to have some protective effects against DSS-induced colitis. Furthermore, DX5+ NKT cells can prevent colitis in the T cell transfer model in a CD1d-dependent manner. The proposed mechanism for the protective function of these cells was the PDL1-mediated killing of colitogenic cells, although this has not been demonstrated in an in vivo setting (155). However, NKT cells are thought to play a pathogenic role in oxazolone-induced colitis, commonly considered a Th2-driven model (156). CD1d can be expressed both by the intestinal epithelium and by hematopoietic cells such as B cells, macrophages, and DCs residing in the intestine. Interestingly, in addition to the putative direct regulatory function of NKT cells, ligation of CD1d on IEC could result in IL-10 production and protection of barrier function (157). This effect may also play a role in the regulatory function of other CD1d-expressing cells, such as B cells. A further population with a putative role in control of intestinal immune responses are the mucosal-associated invariant T (MAIT) cells. Like NKT cells, these cells are characterized by expression of an invariant TCRα chain, which utilizes the Vα7.2-Jα33 combination (Vα19Jα33 in mice) (158). They are restricted by the nonclassical MHC class I molecule MR1 (159). MAIT cells are abundant in the small intestinal LP, but absent from the thymus, spleen, and bone marrow. Furthermore, in humans gut tropic α4β7+ MAIT are found circulating in blood (159). The preferential accumulation of MAIT in the intestinal LP, and their dependence on the commensal ﬂora suggests a speciﬁc role for these cells either in

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protection against intestinal pathogens or in the maintenance of tolerance toward the commensal ﬂora. A recent study demonstrated that MR1-restricted Vα19 transgenic T cells rapidly produced IFN-γ, IL-4, IL-5, and IL-10 following TCR ligation. Interestingly, the NK1.1− subpopulation was the highest producer of IL10 (160). It may therefore be interesting to test the role of these cells in intestinal homeostasis.

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B CELLS B cells play an important role in immune responses through antibody production, APC function and cytokine secretion. The intestine harbors a population of B cells that follow a distinct differentiation pathway and are specialized in IgA production. Interestingly, many of the factors involved in tolerance, such as IL-10, TGF-β, and retinoic acid, also promote IgA secretion, highlighting the complementarity of barrier function and mucosal tolerance (161). IgA can also mediate antigen sampling, and it may contribute to pathogenesis in settings such as celiac disease by increasing the antigenic stimuli imported into the mucosa (162). B cells can produce the regulatory cytokines IL-10 and TGF-β and play a role in the termination of immune responses (163). Importantly,

they also reportedly limit colitis in a Th2 model via an IL-10- and CD1d-dependent mechanism (164). In addition, B cells could also inhibit colitis in a model of innate intestinal inﬂammation (165). However, to date there are no data on the functional role of B cell–mediated regulation in human intestinal inﬂammation.

CONCLUDING REMARKS To achieve tolerance to food antigens and commensal ﬂora, intestinal homeostasis relies on a network of different regulatory populations. Defects in key components of this network can result in exaggerated responses to innocuous antigens, inducing intestinal inﬂammation. Therapies have traditionally attempted to restore the balance by reducing the proinﬂammatory stimuli. This approach can have negative side-effects, not only because of an increased susceptibility to opportunistic pathogens, but also because it can simultaneously decrease the activity of regulatory lymphocytes. A better understanding of the key cellular components of the intestinal regulatory network, how they control inﬂammation, and how they are themselves controlled will allow researchers to design improved targeted therapies to treat intestinal disorders.

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We are grateful to Michael Barnes and Alessandra Geremia for critical reading of the manuscript. The authors are supported by the Wellcome Trust. LITERATURE CITED 1. Mayer L. 2005. Mucosal immunity. Immunol. Rev. 206:5 2. Strober W, Fuss IJ, Blumberg RS. 2002. The immunology of mucosal models of inﬂammation. Annu. Rev. Immunol. 20:495–549 3. Artis D. 2008. Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nat. Rev. Immunol. 8:411–20 4. Cash HL, Whitham CV, Behrendt CL, Hooper LV. 2006. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313:1126–30 www.annualreviews.org • Intestinal Immune Regulation

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147. Saurer L, Seibold I, Rihs S, Vallan C, Dumrese T, Mueller C. 2004. Virus-induced activation of selfspeciﬁc TCRαβ CD8αα intraepithelial lymphocytes does not abolish their self-tolerance in the intestine. J. Immunol. 172:4176–83 148. Poussier P, Ning T, Banerjee D, Julius M. 2002. A unique subset of self-speciﬁc intraintestinal T cells maintains gut integrity. J. Exp. Med. 195:1491–97 149. Nanno M, Shiohara T, Yamamoto H, Kawakami K, Ishikawa H. 2007. γδ T cells: ﬁreﬁghters or ﬁre boosters in the front lines of inﬂammatory responses. Immunol. Rev. 215:103–13 150. Halstensen TS, Scott H, Brandtzaeg P. 1989. Intraepithelial T cells of the TcRγ/δ+ CD8− and Vδ1/Jδ1+ phenotypes are increased in coeliac disease. Scand. J. Immunol. 30:665–72 151. Hue S, Mention JJ, Monteiro RC, Zhang S, Cellier C, et al. 2004. A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease. Immunity 21:367–77 152. Inagaki-Ohara K, Chinen T, Matsuzaki G, Sasaki A, Sakamoto Y, et al. 2004. Mucosal T cells bearing TCRγδ play a protective role in intestinal inﬂammation. J. Immunol. 173:1390–98 153. Bhagat G, Naiyer AJ, Shah JG, Harper J, Jabri B, et al. 2008. Small intestinal CD8+ TCRγδ+ NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients with celiac disease. J. Clin. Invest. 118:281–93 154. Zeissig S, Kaser A, Dougan SK, Nieuwenhuis EE, Blumberg RS. 2007. Role of NKT cells in the digestive system. III. Role of NKT cells in intestinal immunity. Am. J. Physiol. Gastrointest. Liver Physiol. 293:G1101–5 155. Hornung M, Farkas SA, Sattler C, Schlitt HJ, Geissler EK. 2006. DX5+ NKT cells induce the death of colitis-associated cells: involvement of programmed death ligand-1. Eur. J. Immunol. 36:1210–21 156. Heller F, Fuss IJ, Nieuwenhuis EE, Blumberg RS, Strober W. 2002. Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity 17:629–38 157. Colgan SP, Hershberg RM, Furuta GT, Blumberg RS. 1999. Ligation of intestinal epithelial CD1d induces bioactive IL-10: critical role of the cytoplasmic tail in autocrine signaling. Proc. Natl. Acad. Sci. USA 96:13938–43 158. Treiner E, Lantz O. 2006. CD1d- and MR1-restricted invariant T cells: of mice and men. Curr. Opin. Immunol. 18:519–26 159. Treiner E, Duban L, Bahram S, Radosavljevic M, Wanner V, et al. 2003. Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature 422:164–69 160. Kawachi I, Maldonado J, Strader C, Gilﬁllan S. 2006. MR1-restricted Vα19i mucosal-associated invariant T cells are innate T cells in the gut lamina propria that provide a rapid and diverse cytokine response. J. Immunol. 176:1618–27 161. Cerutti A. 2008. The regulation of IgA class switching. Nat. Rev. Immunol. 8:421–34 162. Matysiak-Budnik T, Moura IC, Arcos-Fajardo M, Lebreton C, Menard S, et al. 2008. Secretory IgA mediates retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease. J. Exp. Med. 205:143–54 163. Fillatreau S, Gray D, Anderton SM. 2008. Not always the bad guys: B cells as regulators of autoimmune pathology. Nat. Rev. Immunol. 8:391–97 164. Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg RS, Bhan AK. 2002. Chronic intestinal inﬂammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity 16:219–30 165. Gerth AJ, Lin L, Neurath MF, Glimcher LH, Peng SL. 2004. An innate cell-mediated, murine ulcerative colitis-like syndrome in the absence of nuclear factor of activated T cells. Gastroenterology 126:1115–21

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Contents

Volume 27, 2009

Frontispiece Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Translating Molecular Insights in Autoimmunity into Effective Therapy Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Structural Biology of Shared Cytokine Receptors Xinquan Wang, Patrick Lupardus, Sherry L. LaPorte, and K. Christopher Garcia p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Immunity to Respiratory Viruses Jacob E. Kohlmeier and David L. Woodland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 61 Immune Therapy for Cancer Michael Dougan and Glenn Dranoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 83 Microglial Physiology: Unique Stimuli, Specialized Responses Richard M. Ransohoff and V. Hugh Perry p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p119 The Liver as a Lymphoid Organ Ian Nicholas Crispe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Immune and Inﬂammatory Mechanisms of Atherosclerosis Elena Galkina and Klaus Ley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p165 Primary B Cell Immunodeﬁciencies: Comparisons and Contrasts Mary Ellen Conley, A. Kerry Dobbs, Dana M. Farmer, Sebnem Kilic, Kenneth Paris, Soﬁa Grigoriadou, Elaine Coustan-Smith, Vanessa Howard, and Dario Campana p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 The Inﬂammasomes: Guardians of the Body Fabio Martinon, Annick Mayor, and Jürg Tschopp p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p229 Human Marginal Zone B Cells Jean-Claude Weill, Sandra Weller, and Claude-Agn`es Reynaud p p p p p p p p p p p p p p p p p p p p p p267

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Aire Diane Mathis and Christophe Benoist p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p287 Regulatory Lymphocytes and Intestinal Inﬂammation Ana Izcue, Janine L. Coombes, and Fiona Powrie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p313 The Ins and Outs of Leukocyte Integrin Signaling Clare L. Abram and Clifford A. Lowell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p339

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Recent Advances in the Genetics of Autoimmune Disease Peter K. Gregersen and Lina M. Olsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p363 Cell-Mediated Immune Responses in Tuberculosis Andrea M. Cooper p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p393 Enhancing Immunity Through Autophagy Christian Munz ¨ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p423 Alternative Activation of Macrophages: An Immunologic Functional Perspective Fernando O. Martinez, Laura Helming, and Siamon Gordon p p p p p p p p p p p p p p p p p p p p p p p p451 IL-17 and Th17 Cells Thomas Korn, Estelle Bettelli, Mohamed Oukka, and Vijay K. Kuchroo p p p p p p p p p p p p p p485 Immunological and Inﬂammatory Functions of the Interleukin-1 Family Charles A. Dinarello p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p519 Regulatory T Cells in the Control of Host-Microorganism Interactions Yasmine Belkaid and Kristin Tarbell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p551 T Cell Activation Jennifer E. Smith-Garvin, Gary A. Koretzky, and Martha S. Jordan p p p p p p p p p p p p p p p591 Horror Autoinﬂammaticus: The Molecular Pathophysiology of Autoinﬂammatory Disease Seth L. Masters, Anna Simon, Ivona Aksentijevich, and Daniel L. Kastner p p p p p p p p p621 Blood Monocytes: Development, Heterogeneity, and Relationship with Dendritic Cells Cedric Auffray, Michael H. Sieweke, and Frederic Geissmann p p p p p p p p p p p p p p p p p p p p p p p p669 Regulation and Function of NF-κB Transcription Factors in the Immune System Sivakumar Vallabhapurapu and Michael Karin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p693

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The Ins and Outs of Leukocyte Integrin Signaling Clare L. Abram and Clifford A. Lowell Program in Immunology, Department of Laboratory Medicine, University of California, San Francisco, California 94143-0451; email: [email protected]

Annu. Rev. Immunol. 2009. 27:339–62

Key Words

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

inside-out, outside-in, kinases, Rap GTPase, ITAM

This article’s doi: 10.1146/annurev.immunol.021908.132554

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0339$20.00

Integrins are the principal cell adhesion receptors that mediate leukocyte migration and activation in the immune system. These receptors signal bidirectionally through the plasma membrane in pathways referred to as inside-out and outside-in signaling. Each of these pathways is mediated by conformational changes to the integrin structure. Such changes allow high-afﬁnity binding of the receptor with counteradhesion molecules on the vascular endothelium or extracellular matrix and lead to association of the cytoplasmic tails of the integrins with intracellular signaling molecules. Leukocyte functional responses resulting from outside-in signaling include migration, proliferation, cytokine secretion, and degranulation. Here, we review the key signaling events that occur in the inside-out versus outside-in pathways, highlighting recent advances in our understanding of how integrins are activated by a variety of stimuli and how they mediate a diverse array of cellular responses.

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INTRODUCTION leukocyte function– associated antigen 1 (LFA-1): integrin αL β2 , CD11a/CD18 Mac-1: integrin αM β2 , CD11b/CD18

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very late antigen 4 (VLA-4): integrin α4 β1 leukocyte adhesion deficiency (LAD): a variety of human immunodeﬁciency syndromes characterized by defects in integrin function Integrin avidity: combination of afﬁnity changes and clustering that leads to increased ligand binding and signaling BCR: B cell receptor TCR: T cell receptor Integrin affinity: alterations in integrin conformation from bent inactive form to intermediate and high-afﬁnity active ligand binding forms

340

At sites of inﬂammation or infection, leukocytes exit the vasculature through a cascade of events that involves a series of adhesion and homing receptors (1). Integrins play a central role in this cascade, mediating ﬁrm arrest of leukocytes on the inﬂamed endothelium and coordinating transmigration through the basement membrane to allow homing to the site of infection or inﬂammation. Integrins are cell surface receptors composed of α and β chain heterodimers of type I transmembrane glycoproteins with short cytoplasmic tails. There are 18 different α and 8 different β subunits, which associate in pairs to form at least 24 distinct αβ receptors (see Reference 2 and table therein). Common integrins expressed on leukocytes include leukocyte function–associated antigen 1 (LFA-1 or αL β2 ), Mac-1 (αM β2 ), and very late antigen 4 (VLA-4 or α4 β1 ). The ligands (or counter-receptors) for LFA-1 are the intercellular adhesion molecules (ICAMs) 1–5 that are expressed primarily on vascular endothelial cells. Mac-1 recognizes a more diverse set of ligands, including extracellular matrix proteins such as ﬁbrinogen and ﬁbronectin, as well as activated complement proteins such as iC3b. VLA-4 recognizes the vascular intercellular adhesion molecule (VCAM-1), whereas the major integrin on platelets, αIIb β3 , binds primarily ﬁbrinogen. In leukocytes, these integrins are involved in slow rolling, adhesion strengthening, and transendothelial migration, and they transmit signals that facilitate respiratory burst, complement-mediated phagocytosis, cytokine production, proliferation, survival, differentiation, degranulation, and cellular polarization. In platelets, integrins are required for stable clot formation and hemostasis. From a cell biologic viewpoint, the primary function of integrins is as a link between the actin cytoskeleton of the cell (or platelet) and the extracellular matrix. The key role integrins play in immune function is evidenced by several human conditions in which defects in integrin expression or function occur. These conditions are collectively known

Abram

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Lowell

as leukocyte adhesion deﬁciencies (LAD) and are summarized in Table 1. In mice, all the leukocyte integrin-encoding genes have been knocked out, and several ﬂoxed alleles have been reported. In addition, investigators have engineered mice containing speciﬁc point mutations in different integrins in order either to study the role of speciﬁc pathways in vivo or to try to mimic human disease (those discussed in the text are summarized in Table 2).

INSIDE-OUT SIGNALING Inside-out signaling is deﬁned as those events that induce conformational changes in the integrin leading to increased ligand binding afﬁnity (integrin activation) and clustering of integrins in the membrane, which together result in avidity modulation allowing cell attachment. Circulating leukocytes generally maintain their integrins in a nonadhesive state in which the integrin ectodomains are held in a bent or folded conformation that restricts their ability to bind ligands. Chemoattractants or cytokines stimulate receptors, leading to rapid integrin activation and clustering. In lymphocytes, integrin avidity is also modulated by stimulation through the B cell receptor (BCR) and T cell receptor (TCR). Signaling through other receptors, such as CD14 on monocytes and CD40 on B cells, also affects integrin-mediated adhesion. The ﬁrst step in integrin activation is separation of the cytoplasmic tails. This ultrastructural change is relayed throughout the length of the integrin and results in unfolding of the ectodomain of the receptor (2). The extended conformation of the integrin becomes a high-afﬁnity receptor for its ligand and facilitates clustering of the extended receptors at the cell surface. This process enables leukocytes to adhere and de-adhere rapidly to vascular endothelium during inﬂammatory cell responses. Experimentally, inside-out signaling leading to integrin activation can be measured by binding of activation state-speciﬁc antibodies or attachment to ligands such as ICAM. Integrin afﬁnity modulation is also involved with the

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Table 1

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Human genetic mutations associated with leukocyte adhesion deficiency (LAD)

Syndrome

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5 December 2008

Phenotype

Defect

LADI

recurrent bacterial infections, impaired wound healing, defects in multiple cell types

loss or reduced β2 on cell surface, defects in β2 signaling, mutations in β2

http://bioinf.uta.fi/ITGB2base

107

LADII

infections (periodontitis), decreased chemotaxis, and neutrophilia

mutations in selectin glycosylation (absence of SLeX)

mutations found in Golgi guanosine diphosphate (GDP)-fucose transporter

108

LADIII (variant LADI)

recurrent bacterial infections, impaired wound healing, defects in multiple cell types

defects in GPCRmediated activation of multiple integrins

mutations in CalDAG-GEFI identiﬁed

109

Glanzmanns thrombasthenia

autosomal recessive bleeding disorder

mutations in αIIb or β3 , platelet defects

http://sinaicentral.mssm.edu/ intranet/research/glanzmann

110

Wiskott-Aldrich syndrome

increased susceptibility to pathogens, defects in various immune cell types

mutations in WASp

http://homepage.mac.com/ kohsukeimai/wasp/WASPbase.html

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initial stages of leukocyte rolling on vascular endothelium (1). Analysis of integrin mutants that cause either constitutive integrin activation or failure to bind ligands, along with analysis of structures obtained by X-ray crystallography and electron microscopy, have deﬁned regions important in integrin activation (2, 3). It is clear from FRET analysis of αL and β2 that the cytoplasmic domains are close together in the resting inactive state, but spatial separation is detected upon integrin activation by a variety of intracellular signals, including activation of protein kinase C (PKC), G protein–coupled receptors (GPCRs), or transfection of the talin head domain. How do signals from divergent cell surface receptors converge to mediate these structural changes in integrins? The cytoplasmic tails of integrins are fairly short (Figure 1). α integrin cytoplasmic tails contain a conserved GFFKR amino acid sequence that is critical for a salt bridge–mediated interaction with the β chain; deletion or mutations of the GFFKR sequence lead to impaired association with the β chain and constitutive integrin activation both in vitro

Mutations

References

and in vivo (2). The β tails also contain amino acids important for the salt bridge with the α chain and contain two NPXY/F motifs that mediate protein binding. Several phosphorylation sites have been identiﬁed in α and β tails, and a number of proteins speciﬁcally associate with either tail (Figure 2).

Rap GTPases in the Inside-Out Pathway The Rap GTPases have been implicated as major regulators of the inside-out pathway in lymphocytes (4). Rap1 and Rap2 are activated downstream of TCR/BCR or chemokine signaling, leading to LFA-1 and α4 integrin-dependent adhesion (5–7). Activation of either Rap1 or Rap2 in myeloid cells has not been reported. Sebzda et al. (8) made a transgenic mouse expressing a constitutively active form of Rap1 in T cells, which leads to unregulated activation of LFA-1. Expression of dominant-negative Rap1 inhibits TCRmediated LFA-1 activation in Jurkat T cells (9), whereas overexpression of the Rap1-speciﬁc www.annualreviews.org • Leukocyte Integrin Signaling

protein kinase C (PKC): a serine/ threonine kinase that has several different isoforms activated (variably depending on isoform) by diacylglycerol (DAG) and Ca2+ GPCR: G protein– coupled receptor

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Table 2

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Engineered mouse mutants used to study integrin function

Integrin β1

Pairs with

Mouse model

α1-11 , αV

Knockout Floxed-β1 x Mx-Cre Y763A, Y775A Y763F, Y775F D739A

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β2

β3

α L , α M , αX , αD

αV , αIIb

αIIb

β3

αL (CD11a)

β2

113

Knockout

viable but platelet defects and osteosclersosis, pathological bleeding selectively disrupts outside-in signaling disrupts binding of talin, ﬁlamin, and other proteins, disrupts inside-out signaling disrupts binding with talin speciﬁcally, disrupts inside-out signaling, protection from pathological bleeding

114

embryonic lethal disrupts binding with paxillin, mice viable and fertile, impaired mononuclear cell recruitment in thioglycollate-induced peritonitis defects in hematopoiesis

115 20

no knockout made, but treatment with blocking antibodies that do not block αV β3 show same phenotype as β3 -null

117

viable, defect in leukocyte adhesion and migration (decrease in airpouch model) constitutively activated integrin, impaired deadhesion and migration, impaired T cell activation and neutrophil chemotaxis

118

defect in neutrophil binding and degranulation, but no effect on neutrophil emigration

120

Knockout Deletion of GFFKR

Knockout

guanine nucleotide exchange factor (GEF): activates GTPases by catalyzing exchange of GDP for GTP

342

GTPase-activating protein (GAP) SPA1 (signal-induced proliferation-associated protein 1) inhibits rapid ICAM-dependent adhesion of primary T cells to vascular endothelium (7). In B cells, expression of constitutively active Rap2 enhances B cell adhesion, whereas expression of a Rap-speciﬁc GAP leads to a reduction in LFA-1- and VLA-4-dependent adhesion (5). Mice that lack Rap1b show embryonic and perinatal lethality owing to a

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43

viable but chronic dermatitis, T cell defects, increased neutrophil number, leukocytosis, increased susceptibility to bacterial infections

Knockout Y991A

β2

43, 44 43, 44

112

Floxed- α4 x Mx-Cre

αM (CD11b)

111 26

viable, fertile, mild granulocytosis

L746A

β1 , β7

References

Gene targeting, hypermorph Knockout

Y747F, Y759F Y747A

α4

Phenotype Embryonic lethal normal hematopoietic development, mild delay in platelet responses phenocopies β1 knockout no gross phenotype, mild defect in outside-in integrin signaling in platelets no phenotype

Lowell

45 34 34

116

119

bleeding disorder, which may be attributable to poor activation of αIIb β3 on platelets (10). Despite high similarity between Rap1a and Rap1b, mice that lack Rap1a are viable, but both lymphoid and myeloid cells manifest integrin-dependent adhesion defects (11, 12). Several GEFs that activate Rap GTPases have been identiﬁed; in particular, modulation of CalDAG-GEF1 activity leads to changes in cell adhesion and migration (13). These GEFs

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Human β1 Mouse β1 Human β2 Mouse β2 Human β3 Mouse β3

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IIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK IIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK IAAIVGGTVAGIVLIGILLLVIWKALIHLSDLREYRRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES VAAIVGGTVVGVVLIGVLLLVIWKALTHLTDLREYRRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES ILVVLLSVMGAILLIGLAALLIWKLLITIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT ILVVLLSVMGAILLIGLATLLIWKLLITIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT

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Human α2 Mouse α2 Human α4 Mouse α 4 Human α IIb Mouse α IIb Human α V Mouse α V Human α L Mouse α L Human α M Mouse α M Human α X Mouse α X Human α D Mouse α D

1103 1103 984 985 997 992 994 990 1090 1086 1106 1107 1106 1115 1085 1082

TGVIIGSIIAGILLLLALVAILWKLGFFKRKYEKMTKNPDEIDETTELSS 1152 TGVIIGSIIAGILLLLAMTAGLWKLGFFKRQYKKMGQNPDEMDETTELNS 1152 IVIISSSLLLGLIVLLLISYVMWKAGFFKRQYKSILQEENRRDSWSYINSKSNDD 1038 IIIITISLLLGLIVLLLISCVMWKAGFFKRQYKSILQEENRRDSWSYVNSKSNDD 1039 IWWVLVGVLGGLLLLTILVLAMWKVGFFKRNRPPLEEDDEEGE 1039 VWWVLVGVLGGLLLLTLLVLAMWKAGFFKRNRPPLEED-EEEE 1033 VWVIILAVLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET 1048 VWVIILAVLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET 1044 LYLYVLSGIGGLLLLLLIFIVLYKVGFFKRNLKEKMEAGRGVPNGIPAEDSEQLASGQEAGDPGCLKPLHEKDSESGGGKD 1170 LHVYVLSGIGGLVLLFLIFLALYKVGFFKRNLKEKMEADGGVPNGSPPEDTDPLAVPGEETKDMGCLEPSGRVTRTKA 1163 LPLIVGSSVGGLLLLALITAALYKLGFFKRQYKDMMSEGGSPGAEPQ 1152 VPLIVGSSIGGLVLLALITAGLYKLGFFKRQYKDMMNEAAPQDAPPQ 1153 TPLIVGSSIGGLLLLALITAVLYKVGFFKRQYKEMMEEANGQIAPENGTQTPSPPSEK 1163 VPLIVGSSVGGLLLLAIITAILYKAGFFKRQYKEMLEEANGQFVSDGTPTPQVAQ 1169 IPIIMGSSVGALLLLALITATLYKLGFFKRHYKEMLEDKPEDTATFSGDDFSCVAPNVPLS 1145 VFLMVFSSVGGLLLLALITVALYKLGFFKRQYKEMLDLPSADPDPAGQADSNHETPPHLTS 1142

Figure 1 Alignment of transmembrane and cytoplasmic regions of leukocyte integrins. Data are taken from UniProt (http://www.uniprot.org), and amino acid numbering does not include the signal sequence. The solid orange box indicates the transmembrane domain predicted by the Hidden Markov model (121). The open red box indicates residues that were experimentally determined to be within the lipid bilayer (122). Residues boxed in yellow are important for the salt bridge linking the α and β integrin tails. The blue boxes indicate the two NPXY/F motifs in the β integrin tails.

are activated by a variety of upstream stimuli, such as PKC or PKD activation, cAMP, and Ca2+ , suggesting that multiple pathways can converge on Rap GTPases.

Proteins that Interact with Integrin α Chains During Inside-Out Signaling The Rap effector RapL binds to a site consisting of two lysine residues following the GFFKR motif in the αL chain (14). As shown in Figure 1, only αL contains these lysine residues, so the role of RapL in activation of other integrins is unclear, although reduction of RapL protein affects VLA-4mediated adhesion (15). Lymphocytes and dendritic cells (DCs) isolated from RapL-deﬁcient mice display reduced adhesion in response to chemokines in vitro and homing and migration defects in vivo (16). RapL forms a complex with the serine/threonine kinase Mst1. Rap1 activation leads to recruitment of both RapL

and Mst1 to LFA-1 and activation of Mst1 kinase activity. Mst1 activation is not seen in RapL-deﬁcient cells. When Mst1 expression is knocked down using siRNA, integrin activation in response to TCR or chemokine stimulation is reduced (17). Paxillin binds to a sequence within the α4 cytoplasmic tail; Y991A or E983A mutations within the α4 tail abolish this interaction (18). Binding is also blocked by phosphorylation of S988 within this sequence; mutation of this residue to a phosphomimetic amino acid, S988D, disrupts paxillin binding and causes increased spreading and decreased migration. Conversely, mutation of this residue to S988A results in enhanced binding of paxillin to α4 and reduced spreading (19). Mice expressing α4 that lacks the paxillin-binding site owing to a Y991A mutation are viable and fertile, but show impaired mononuclear cell recruitment in thioglycollate-induced peritonitis (20). These observations are consistent with an www.annualreviews.org • Leukocyte Integrin Signaling

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Activated open integrin

Inactive closed integrin

KR GF F RapL

Paxillin

Filamin

MstI

PLCγ PKC

ADAP

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Radixin

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Chemokines

Kindlins

α-actinin

RIAM

Skap55

Rap1 GTP CalDAG GEFI

Actin cytoskeleton

PKD

Figure 2 Integrin inside-out signaling. The ﬁgure outlines the key signaling events that occur downstream of chemokine and T and B cell receptors that lead to integrin activation. Inactive integrins exist in a bent conformation, and the α and β cytoplasmic tails are held in close proximity by a salt bridge between residues found in the membrane-proximal region of the tail. Activation of a variety of signaling pathways results in the recruitment of GTP-bound Rap1 and activated talin to the integrin, leading to tail separation. The conformational change in the cytoplasmic region is transmitted through the integrin transmembrane domain and results in structural changes in the extracellular region, leading to an open conformation that can bind ligand with high afﬁnity. The C-terminal rod domain of talin interacts with the actin cytoskeleton to provide physical coupling of the integrin to the actin network of the cell. Many other molecules interact with integrin cytoplasmic tails, but exactly how these interactions are coordinated with integrin activation is unclear.

inhibitory role for paxillin association with α4 integrin. Phosphorylation of integrin α chains can affect integrin activation, but the residues involved are found only in human and not in mouse integrins. Phosphorylation of the αL cytoplasmic tail occurs on S1140 in T cells, stimulated either by antibody crosslinking of the TCR or by treatment with phorbol ester. A mutant S1140A αL chain cannot be activated in T cells by chemokines or expression of constitutively active Rap1 (21). Integrin αM is 344

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constitutively phosphorylated on S1126 in human neutrophils, and when this site is mutated neutrophils can no longer be activated to bind ICAM-1 or -2, and they fail to migrate into target organs in vivo (22).

Proteins that Interact with Integrin β Chains During Inside-Out Signaling Talin is a large cytoskeletal protein consisting of head and rod domains that are separated by a calpain cleavage site. In human

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T cells, siRNA knockdown of talin leads to defects in TCR-mediated LFA-1 activation (23), whereas in CHO cells that express integrin αIIb β3 there is reduced binding of an activation state–speciﬁc antibody (24). In phagocytes, talin is required for complement-mediated, but not FcRγ-mediated, phagocytosis; lack of talin impairs particle binding (25). Mice deﬁcient in talin die during embryogenesis, but platelets that lack talin (generated using a talinloxP strain, crossed with the Mx-cre line) fail to activate integrins in response to many different ligands. These mice are resistant to arterial thrombosis mediated by platelet β1 and β3 integrins (26, 27). The head domain of talin contains a FERM domain that interacts with β1 , β2 , and β3 integrin cytoplasmic tails. The FERM domain contains three subdomains: F1, F2, and F3, and the latter, the F3 subdomain, contains a phosphotyrosine-binding (PTB)-like domain that binds to the membrane-proximal NPXY/F motif found in β integrin cytoplasmic tails (28). Expression of the talin N-terminal head domain leads to increased ligand binding of several integrins, including αM β2 and αIIb β3 (25, 29). However, the talin-integrin interaction is complex and requires activation of full-length talin via mechanisms such as calpain-mediated cleavage, binding of phosphoinositol phosphates, and phosphorylation (29). Recent structural studies have tried to determine accurately the nature of the talin-integrin interaction. Investigators have suggested that talin constitutively associates with inactive integrins, but its cleavage alters the binding in such a way that integrin tail unfolding occurs, initiating integrin activation. Talin then dissociates from the β integrin tail, and α-actinin binds in its place (30). β3 integrins that contain mutations in the talin-binding site fail to activate, although some of these mutations also prevent binding of other proteins, such as ﬁlamin (24). Association of any PTB-containing protein to the integrin NPXY motif alone is not sufﬁcient to cause activation. Other regions of the talin head domain associate with more membraneproximal residues in the integrin tail with a

lower afﬁnity, and these disrupt the salt bridge between the α and β tails, allowing integrin activation (31). These other requirements for talin binding may be distinct for different integrins. For example, the F3 domain of talin by itself will activate β3 but not β1 integrins, where portions of the F1 head domain are also required (32). Hato et al. (33) found that a chimera consisting of β3 integrin with a β1 tail is constitutively active. Two mutations in β3 also lead to constitutive activation (I719M and E749S), and conversely reverse mutations in β1 inhibit its activation. The activity of the mutants depends on the presence of talin. Conﬁrmation that the talin interaction with β integrin tails controls inside-out signaling came from knock-in mice expressing β3 integrin that contains point mutations in the cytoplasmic tail. A Y747A mutation disrupts binding of talin, ﬁlamin, and several other binding proteins, whereas L746A disrupts binding speciﬁcally of talin (34). Platelets from both strains of mice show impaired agonist-induced ﬁbrinogen binding and platelet aggregation, hallmarks of integrin inside-out signaling. When integrins are activated exogenously by addition of Mn2+ , platelets are able to spread and initiate outside-in signals. In in vivo models of thrombosis, the Y746A mouse is resistant to pulmonary thromboembolism and ferricchloride-induced vascular injury. Furthermore, this mouse does not show the anemia and gastrointestinal bleeding seen in β3 -null mice, suggesting that different functions of integrins could be targeted therapeutically. Cytohesin-1, a GEF for ARF-GTPases, is predominantly expressed in hematopoietic cells, where it interacts with the cytoplasmic tail of β2 integrin and regulates adhesion (35). Mutation of membrane-proximal residues WKA723-725TRG of the β2 cytoplasmic tail abolishes binding. Expression of this mutant in a T cell line that lacks endogenous β2 integrins results in reduced adhesion to ICAM when compared with expression of wild-type β2 . The exchange activity of cytohesin-1 is not required for integrin activation, as measured by binding of an integrin activation state–speciﬁc www.annualreviews.org • Leukocyte Integrin Signaling

FERM domain: named after the domain found in 4.1 protein, ezrin, radixin, and moesin; molecules containing this domain act as linkers between the membrane and the actin cytoskeleton phosphotyrosinebinding domain (PTB): typically binds NPXY motifs and can be dependent or independent of tyrosine phosphorylation

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antibody, but it is required for spreading, suggesting that cytohesin plays different roles in inside-out versus outside-in integrin signaling (see below). Overexpression of cytohesin1 leads to LFA-1-dependent arrest of T cells stimulated by stromal cell–derived factor 1α (SDF1α). The actin-binding protein, α-actinin, binds to β2 integrin tails in neutrophils following activation (30). Experiments suggest that following talin activation, tail displacement opens up the α-actinin-binding site in a membrane-proximal region of the β2 tail, which may stabilize the integrin open conformation, allowing for ﬁrm adhesion. α-actinin binding is not mediated by a PTB-NPXY interaction. Activation state– speciﬁc antibodies have demonstrated that αactinin colocalizes with intermediate-afﬁnity extended integrins found at the leading edge of migrating T cells rather than with highafﬁnity integrins found at ﬁrm adhesion points (36). These results suggest that there may be a cascade of protein interactions with integrin tails that stabilize different conformations during inside-out signaling. Kindlin family members are FERM domain–containing proteins that interact with integrin β1 and β3 tails at the membrane distal NPXY motif, i.e., a site distinct from the talin interaction domain. The FERM domain of the kindlins is most closely related to that of talin, but expression of the kindlin-2 FERM domain alone cannot activate integrins in the same way that talin does (37). Kindlins instead seem to work as coactivators of integrins with talin because coexpression of the talin and kindlin-2 FERM domains has a synergistic effect on integrin activation (38). Kindlin-1 and -2 are ubiquitously expressed, whereas kindlin-3 is restricted to hematopoietic cells. Mutations in kindlin-1 are associated with skin disease; perhaps mutations in other kindlins could cause LAD. Kindlin-3-null mice die shortly after birth owing to severe bleeding from a platelet dysfunction; inside-out αIIb β3 integrin signaling in platelets is completely defective in these animals (39). Loss of kindlin-2 in mice results in peri-implantation lethality, but

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embryonic stem cells from these mice show decreased adhesion on a variety of integrin ligands (38). Several other FERM domain–containing proteins bind to β integrin tails. Radixin, like talin, is held in a closed conformation by an intramolecular interaction. Expression of the head domain alone leads to increased adhesiveness of CHO cells containing αM β2 and increased binding of an activation state–speciﬁc antibody. The interaction between radixin and β integrins does not require either of the β2 tail NPXF motifs. Instead, radixin binds to a more membrane-proximal region that overlaps with the cytohesin-1-binding site (40). Other FERM domain–containing proteins that interact with β integrin chains include Myosin X, ICAP-1a, Dok1, and Numb (41, 42). These proteins may act in the same way as kindlins to promote integrin activation through talin or inhibit integrin activation by competing with talin. Several phosphorylation sites have been reported in β integrin tails. The tyrosine residues found in both NPXY motifs of β1 and β3 integrins are obvious targets. Whereas binding of talin to integrins is phosphorylationindependent, other proteins show preference for phosphorylation of the tyrosine contained within the NPXY motif. Genetically engineered mice have been generated to address the role of the tyrosine residues contained within the NPXY motifs of the integrin β tail (Table 2). Mutation of these tyrosines to phenylalanines prevents phosphorylation of the tail but would not be expected to interfere with proteins that bind in a phosphorylationindependent manner. Mutation of these tyrosines to alanines would be expected to interfere with all NPXY motif–binding proteins. Mice that express β1 containing both Y783A and Y795A mutations phenocopy the β1 -null phenotype, implying that the NPXY motifs are critical for β1 integrin function. Mice that express β1 containing both Y783F and Y795F mutations do not show any obvious phenotype, suggesting that tyrosine phosphorylation of these motifs is not required (43, 44). However, platelets from these mice do have mild

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spreading defects, suggesting that tyrosine phosphorylation of the β integrin tails is important for outside-in signaling. Similar results were seen in mice containing analogous mutations in β3 integrin (Y772F, Y784F); platelets showed no defect in inside-out signaling but failed to aggregate (45). A Y772A mutation in the β3 cytoplasmic tail disrupts talin binding to the proximal NPXY motif and prevents integrin activation, but it also disrupts binding of other PTB-containing proteins such as ﬁlamin. It is interesting to note that β2 integrins contain phenylalanine residues instead of tyrosine in both their NPXY/F motifs, and therefore they would not be regulated by tyrosine phosphorylation. β2 chain phosphorylation on T758, which is the ﬁrst of three threonine residues found between the two NPXF motifs, occurs in response to TCR stimulation and phorbol ester treatment and creates a binding site for 14-3-3 proteins (21). Mutation of this residue to alanine seems to affect cell spreading, which may suggest an outside-in signaling defect, possibly linking integrins to Rac- and Cdc42-mediated actin cytoskeletal changes. Filamin also binds to a region of the β2 tail that includes T758, but only when the threonine is unphosphorylated. Talin and ﬁlamin, and therefore 14-3-3 proteins, can all compete for the same binding site on β2 integrin tails (46).

The Sequence of Events in Inside-Out Signaling Although it is clear that many proteins associate with integrin cytoplasmic tails and impact both inside-out and outside-in signaling, the kinetics of binding of all these proteins and the cascade of protein associations that lead to integrin conformational change and phosphorylation are poorly understood. Moreover, the pathways regulating inside-out signaling can be common to several integrins or speciﬁc to others depending on cellular context. Han et al. (47) used CHO cells expressing αIIb and β3 to reconstruct integrin inside-out signaling. They observed increased binding of an αIIb β3 activation state–speciﬁc antibody in response to phor-

bol 12-myristate 13-acetate (PMA) if cells also expressed levels of talin and PKCα equivalent to levels found in platelets. Although talin is a substrate for PKC, phosphorylation of talin by PKC is not required for PMA-stimulated αIIb β3 activation. The authors also found that activated Rap1 could substitute for PMA and PKCα. PKC (activated by PMA or through TCR stimulation) can activate and relocalize Rap1 by phosphorylating PKD1; PKD1 associates with Rap1 via its PH domain, which leads to membrane relocalization and association with the cytoplasmic tail of β1 integrin (48). Rap1 is activated by diacylglycerol (DAG) and cytosolic Ca2+ and is therefore likely to be downstream of PLC. Indeed, TCR-mediated activation of LFA-1 and Rap1 is dependent on PLCγ (6). Strong evidence for a role of the Rap1 GEF CalDAG-GEFI in the activation of Rap1 has come from in vivo mouse and human data. Platelets from CalDAG-GEFI-null mice showed greatly reduced activation of αIIb β3 as measured by activation state–speciﬁc antibodies and fail to aggregate (49). Neutrophils from these mice also show defects in Rap1 activation and, consequently, in β1 and β3 activation (13). These mice show defects similar to those of patients with LAD type III, in which mutations in CalDAG-GEFI were found (50). The ability of Rap1 to activate αIIb β3 is dependent on talin interacting with the β3 tail. Rap1 induces the formation of an integrin activation complex containing talin and the Rap1 effector RIAM (Rap1-GTP-interacting adapter molecule), which binds and activates αIIb β3 (51). RIAM-mediated recruitment of talin unmasks the β3 integrin–binding site in the talin F3 domain. RIAM also links Rap1 to ADAP (adhesion and degranulation– promoting adapter protein) and Skap55, which are essential for TCR-induced integrin activation (52). Downregulation of Skap55 by siRNA in T cells impairs TCR-mediated LFA-1 clustering and T cell–APC conjugation (53). T cells from Skap55-deﬁcient mice show defects in β1 and β2 integrin adhesion, clustering, and downstream functions. A similar phenotype is seen in mice lacking ADAP, which also lack www.annualreviews.org • Leukocyte Integrin Signaling

phospholipase C (PLC): a class of enzyme recruited to plasma membrane through lipid binding pleckstrin homology domain that converts PIP2 into two important signaling second messengers, DAG and IP3

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Skap55 expression (54). Membrane-targeting ADAP and Skap55 can increase T cell adhesiveness in the absence of TCR stimulation, indicating that this complex may help to recruit active Rap1 to the membrane (55). These observations suggest that, in T cells, formation of a Rap1/RIAM/ADAP/Skap55 complex at the membrane is an early step in the pathway leading to talin activation and integrin tail unfolding. How this pathway intersects with PKC/PKD-mediated activation of Rap1 is unclear. Related molecules, such as the ADAP/Skap55 homologs PRAM-1 and SkapHom, may function in a similar fashion in myeloid cells.

Open Questions in the Inside-Out Pathway In summary, it is clear that a pathway involving Rap GTPases and talin is critical for insideout signaling, but there are many unanswered questions. The requirement for the different pathway components downstream of classical immunoreceptors such as the TCR and BCR compared with GPCRs still needs to be clariﬁed. For example, the role of Rap GTPases and ADAP-RapL-RIAM proteins in myeloid cells downstream of chemokine receptors is unknown. Whether these pathways differ in their ability to activate different integrins is also unclear. For example, in human T cells, inactivation of Rap1 blocks SDF1α-stimulated LFA-1-mediated binding to ICAM-1, but not VLA-4-mediated binding to VCAM. Similarly, silencing of CalDAG-GEFI inhibits SDF1αand PMA-stimulated adhesion to ICAM but not VCAM. These data suggest that different pathways are used to activate LFA-1 and VLA4 (56). However, in B cells, inactivation of Rap2 decreases both LFA-1- and VLA-4-mediated adhesion to ligands (5). Experiments have suggested that different chemokines show speciﬁcity for activation of different integrins. Neutrophil chemotaxis to fMLP is dependent on Mac-1 and mediated by p38MAPKs, whereas IL-8 is dependent on LFA-1 and mediated by phosphatidylinositol-3-kinase (PI3K). In the 348

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presence of both chemokines, fMLP is dominant unless p38MAPKs are inhibited (57). Are both these pathways activating Rap GTPases to the same extent? There is recent evidence that Rap GTPases are also involved in the maintenance of basal integrin activity. Intermediateafﬁnity VLA-4 is found on resting eosinophils, allowing attachment to the endothelium under shear ﬂow, and this requires Rap GTPase activity, PLC, and homeostatic Ca2+ concentrations, but not p38MAPK (58). Lastly, if a similar pathway is activating integrins downstream of chemokines and TCR or BCR stimulation, is the signaling additive? Given that, in general, chemokines promote migration, whereas TCR or BCR stimulation requires stable cellcell contacts found in immunological synapses, how are these two outcomes achieved through a similar pathway? Imaging the localization of integrins at different stages of activation upon treatment with diverse stimuli could help to address this.

The Pathway to Clustering Inside-out signaling pathways are known to stimulate integrin clustering, which contributes to increased integrin avidity. This appears to be particularly important for LFA-1, which undergoes dramatic relocalization in T and B cells during the formation of immunological synapses, allowing cells to respond to much lower concentrations of ligand (59). Whether clustering is stimulated by the same signaling pathways as changes in integrin afﬁnity is unclear, although stimuli that affect either clustering or afﬁnity selectively have been observed (60). Luo et al. (61) identiﬁed mutations in the transmembrane domains of αIIb and β3 that selectively affect either afﬁnity upregulation or clustering; molecules such as Skap55 are clearly required for clustering only (53). Upon PMA stimulation of T cells, LFA-1 becomes more mobile within the lipid bilayer and is clustered in rafts. Clustering of LFA-1 into rafts alone using cholera toxin or crosslinking antibodies against raft-associated CD24 increases binding to ICAM, and this effect is dependent

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on PI3K activity (62). Cholesterol depletion prevents cholera toxin– or PMA-mediated increases in integrin ligand binding, but importantly it does not affect increased ligand binding mediated by extracellular Mg2+ (62, 63). More recently, single particle tracking has been used to determine that different integrin conformations have distinct diffusion proﬁles within the membrane, implying that different activation states are uniquely tethered (64). LFA-1 may exist in clusters on some cells allowing them to respond more rapidly to stimuli. Cambi et al. (65) suggest that the presence of LFA-1 in nanoclusters allows monocytes to bind ICAM-1, whereas DCs cannot. Clearly, how integrin afﬁnity modulation by inside-out signaling affects integrin clustering remains to be deﬁned. Clustering represents the ﬁnal steps in the inside-out pathway leading to a transition to outside-in signaling events that are associated more with leukocyte functional activation.

OUTSIDE-IN SIGNALING The signaling steps that occur following ligandinduced clustering of leukocyte integrins are referred to as the outside-in pathway (Figure 3). In leukocytes, much of the signaling from integrins themselves occurs in the context of other stimulatory events involved in inside-out pathways, such as engagement of the TCR or activation of GPCRs. Hence, it can be difﬁcult to separate outside-in signaling events unique to the integrin versus those that are also induced by additional stimuli. In this regard, especially in lymphocytes, we often think of the outsidein pathway as being mainly an ampliﬁer, or costimulus, to other signaling reactions. However, it is now well deﬁned in lymphocytes, myeloid cells, and especially platelets that there are speciﬁc signaling pathways induced by integrin engagement that lead to well-deﬁned functional responses of cells. Using agents that bypass inside-out signaling such as PMA or Mn2+ , investigators can isolate outside-in signaling. For the sake of discussion, we deﬁne outside-in signaling responses from integrins as those events that lead to ﬁrm cell adherence, cell spread-

ing owing to actin cytoskeletal rearrangement, and aspects of cell migration. These cellular responses in turn lead to functional leukocyte activation. In T cells, integrin outside-in signaling responses include activation of proliferation, IL-2 secretion, and stabilization of T cell/APC contacts. In neutrophils, integrin-induced signaling results in degranulation and activation of the NADPH oxidase, leading to ROS production, whereas in macrophages integrin signaling induces stabilization of cytokine mRNAs, in particular IL-1β, and differentiation. Outsidein integrin signaling responses in platelets are classically studied by examination of ﬁlopodial and lamellipodial extensions that produce full spreading and ﬁrm adhesion, leading in vivo to thrombus formation (66).

Initiating Events in the Outside-In Pathway

Syk: predominantly hematopoieticexpressed cytoplasmic tyrosine kinase containing two N-terminal SH2 domains; Syk is related to ZAP-70, which is restricted to T and natural killer cells Src family kinases: a family of cytoplasmic tyrosine kinases containing an N-terminal unique domain, followed by SH3 and SH2 domains; Src, Hck, Fgr, Lyn, Blk, Fyn, and Lck are expressed in hematopoietic cells

Perhaps the earliest biochemical event in the outside-in signaling response is activation of tyrosine kinases, particularly those of the Src and Syk families. Rapid activation of these enzymes has repeatedly been demonstrated in lymphocytes, myeloid cells, and platelets following β1 , β2 , and β3 integrin engagement (67). The requirement for these kinases in mediating outside-in signaling responses has been most clearly deﬁned using knockout mouse models, in particular in studies of myeloid cells and platelets where integrin signaling events can be clearly distinguished from other signaling events. Deﬁciency of the major Src family kinases expressed in myeloid cells (Hck, Fgr, and Lyn) produces a complete block in neutrophil degranulation and ROS production following engagement of β2 and β3 integrins. Similarly, ﬁrm adhesion, cell spreading, and phosphorylation of speciﬁc downstream substrates (such as Vav, Cbl, and other molecules described below) are defective in macrophages lacking Src family kinases. Platelets isolated from Src−/− Hck−/− Fgr−/− Lyn−/− combinatorial mutant mice show complete defects in spreading responses following plating on ﬁbrinogencoated surfaces, which are mediated by the β3 www.annualreviews.org • Leukocyte Integrin Signaling

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Activated open integrin

NPXY

ITAM

K SF

Cytohesin D/E

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RapL

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NPXY

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FK

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Integrin ligand

PLCγ

Syk

JAB-1

ADAP/PRAM SLP76 Vav

Rap1 GTP

Rap1 GTP

FAK/Pyk2 Cbl

WASp

Rho GTPases

PI3K

ARF GTPases Transcription

Actin polymerization

Figure 3 Integrin outside-in signaling. The ﬁgure details integrin-mediated signaling events that occur downstream of ligand binding. Zhu et al. (87) have shown that outside-in signaling requires structural changes with the cytoplasmic region of integrin tails. Activation of Src family kinases (SFKs) is a key step, although the exact mechanism by which this occurs is unclear and results in phosphorylation of a variety of downstream molecules. These include ITAM-containing adapters that, when phosphorylated, lead to the recruitment and activation of Syk or ZAP-70 kinases. These kinases in turn phosphorylate various substrates, including SLP76 and Vav. This leads us to propose that integrin outside-in signaling is analogous to signaling downstream of immunoreceptors, as indicated by the molecules in bold (85, 86). Vav activates Rho GTPases, leading to actin cytoskeletal reorganization. SFKs can also activate FAK (focal adhesion kinase) and Pyk2 kinases, leading to Cbl phosphorylation and recruitment and activation of PI3K. Association of other molecules such as JAB ( Jun-activating binding protein) and cytohesin with the integrin cytoplasmic tails activates other downstream signaling pathways. In this ﬁgure, signaling is depicted as happening in lipid rafts (indicated as red coloration in the membrane), although the role of rafts in integrin signaling differs in various cell types.

integrin (66). That deﬁciency of these kinases affects primarily the outside-in versus insideout integrin signaling pathways has been somewhat difﬁcult to prove. Indeed, there are many examples in the literature of Src family kinases being implicated in integrin activation events. In part, this distinction depends on the assays being used. For example, classic leukocyte adhesion assays, which are generally assumed to reﬂect integrin afﬁnity modulation required for attachment, may also reﬂect changes in outsidein pathways such as cell spreading and actin 350

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cytoskeletal rearrangement. Deﬁciency of Src family kinases appears not to affect cell attachment following stimulation by fMLP or P- or E-selectin (68, 69). Instead, integrin receptor events leading to cell spreading, ﬁrm adhesion, and resistance to shear force (i.e., sustained adhesion) are primarily lost in neutrophils isolated from mice lacking Hck, Fgr, and/or Lyn kinase, demonstrating that these kinases are involved in the outside-in pathway. In Jurkat T cells, it is easier to distinguish signaling events that are speciﬁcally mediated by β2 integrins using

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monoclonal antibodies that directly crosslink and cluster the integrin. Such experiments result in formation of a focal area of polymerized actin (referred to as the actin cloud) that contains many of the adapters and downstream signaling molecules implicated in outside-in integrin signaling (70). Formation of this structure is dependent on the Src kinase Lck in Jurkat cells, suggesting that Lck functions predominately in the outside-in pathway, as these experiments are done in the absence of agonists that would affect integrin afﬁnity modulation. Ultimately, the question of whether loss of Src family kinases affects the inside-out versus outside-in pathway will necessitate development of conformation-speciﬁc monoclonal antibodies that recognize activated or clustered versions of the mouse β2 or β3 integrins. Such antibodies have been invaluable in the study of integrin activation in human leukocytes. The second kinase family implicated in the outside-in pathway is the Syk/ZAP-70 class of kinases. Syk is expressed in myeloid cells and platelets and ZAP-70 in T and natural killer cells. The strongest evidence implicating Syk/ZAP-70 comes from genetic studies. Deﬁciency of Syk kinase results in a complete block in β1 , β2 , and β3 integrin signaling events in neutrophils and macrophages (71). Similarly, Syk-deﬁcient platelets manifest a complete block in ﬁrm adhesion and spreading over ﬁbrinogen-coated surfaces (66, 72). In contrast, deﬁciency of this enzyme does not block GPCR signaling events in neutrophils or mast cells, supporting the notion that the integrin signaling defect resides within the outside-in pathway (73). Deﬁciency of ZAP-70 in T cells also abrogates signaling events from the β1 integrin in Jurkat T cells and can, in part, be separated from the signaling defects in CD3/TCR-mediated events caused by deﬁciency of this kinase (74). Although there is strong evidence that these kinases are required for leukocyte integrin signaling events leading to cellular activation (especially in myeloid cells), the role of these kinases in leukocyte migration, which is classically thought to be the primary function of integrins, is somewhat confused (67). Whereas

monocytes and macrophages lacking Src family kinases or Syk display signiﬁcant migratory defects both in vitro and in vivo, kinasedeﬁcient neutrophils seem to migrate normally in both standard transwell chemotaxis assays and during migration into the inﬂamed peritoneum. In contrast, migration of neutrophils or eosinophils into the lung during inﬂammatory or allergic reactions is dependent on Src family kinase activity (67). Similar differences seem to exist for Syk-deﬁcient neutrophils. In the inﬂamed cremaster muscle model, Syk−/− cells show profound defects in adhesion to the vessel wall and transmigration (75). In contrast, in the Schwartzman skin hemorrhagic vasculitis model, Syk-deﬁcient (and Src family kinase–deﬁcient) neutrophils readily enter the inﬂammatory site but fail to undergo Mac-1mediated activation and hence cause very little tissue damage (76). Thus, one is left with the conclusion that the requirement for integrin outside-in signaling during leukocyte migration is dependent on both the cell type and the inﬂammatory tissue site. Indeed, the recent report from Lammermann et al. (77) suggests that leukocyte integrins are not required at all for DC migration either into 3D collagen gel matrices in vitro or into lymph nodes and skin in vivo. Of course, β2 integrins are essential for neutrophil migration, as both mice and humans lacking this receptor present with LAD (Table 1 and 2). Hence, the notion that integrin outside-in signaling is a required component of leukocyte migration is too broad and needs to be assessed in the context of the speciﬁc type of inﬂammatory response involved. The other principal tyrosine kinases implicated in leukocyte integrin signaling are focal adhesion kinase (FAK) and its homolog Pyk2. Based on models in ﬁbroblasts, these kinases are generally thought to act downstream of Src family members. FAK has a number of tyrosine residues that are phosphorylated by Src and are required for FAK function. Because FAK deﬁciency results in early embryonic lethality, FAKloxP mice crossed to the LysM-cre strain have been used to study the effect of FAK deletion in myeloid cells. Macrophages www.annualreviews.org • Leukocyte Integrin Signaling

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lacking FAK fail to form stable lamellipodia and as a result have marked defects in directional chemotaxis and impaired migration into the peritoneum in vivo (78). Macrophages derived from Pyk2−/− mice also exhibit altered cell polarization and diminished chemokine-induced motility, similar to the FAK-deﬁcient cells (79), suggesting that these kinases may have partially overlapping functions in macrophage motility. In neutrophils, Pyk2 is implicated in integrin outside-in signaling using pharmacologic inhibitors or by transduction of inhibitory Tat-Pyk2 fusion peptides into human cells (80). Macrophages and neutrophils lacking Src family or Syk kinases show impaired activation/phosphorylation of FAK and Pyk2 following integrin engagement, whereas the reverse tends not to be the case, supporting the notion that FAK and Pyk2 act further downstream in the integrin outside-in pathway (81). Further studies of the role of these kinases using newly available knockout mouse models are needed.

Coupling Kinases to the Integrins As in other signaling cascades, it is believed that physical clustering of the integrins leads to clustering of associated Src and Syk kinases, which can phosphorylate each other and initiate the outside-in pathway. This raises the unanswered question of how the kinases become coupled to integrins. Most leukocyte integrins do not have any obvious interaction motifs for the various protein domains (such as SH2 or SH3) involved in protein kinase associations. However, direct interaction between both Src family kinases and Syk kinase and the cytoplasmic domain of β2 and β3 integrins has been reported (82). In most cases, these interactions have been deﬁned either by coimmunoprecipitation or by analysis of binding by recombinant proteins encompassing domains of the kinases and integrins. However, in CHO cells it is possible to observe physical interactions between the β3 tail and Src using FRET reporter methods (83). The interaction between the kinase and the integrin cytoplasmic tail is found in resting cells and, in the case of Syk, occurs independently 352

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of kinase activation. This supports the general model that resting, inactive integrin tails have low levels of associated Src and Syk kinases. Following integrin activation/clustering, these enzymes become activated (often recruiting more kinase molecules to the complex) and downstream signaling is initiated (66). More recently, studies from several other groups have suggested that molecules containing immunoreceptor tyrosine-based activation motifs (ITAMs) are also involved in the coupling of kinases to integrins (72, 84). Neutrophils isolated from mice lacking the two ITAM adapter proteins FcRγ (the ITAMcontaining signaling chain of the IgG and IgE Fc receptors) and DAP12 (which is associated with a number of activating receptors in natural killer and myeloid cells) are deﬁcient in β2 and β3 integrin signaling. The function of ITAMcontaining molecules in classical immunoreceptor signaling pathways, such as the TCR, BCR, or FcR, has been well deﬁned; these proteins are phosphorylated by Src family kinases and serve as docking sites for Syk or ZAP-70, which in turn initiate downstream signaling responses (85). Using retroviral-mediated gene transduction of hematopoietic stem cells, the two studies demonstrate that Syk SH2 domain binding to phosphorylated ITAMs is critical for outside-in signaling from β2 and β3 integrins in neutrophils and platelets, respectively. In this regard, initiation of the integrin outsidein pathway seems to mimic the steps involved in initiation of immunoreceptor signaling (86). Although at ﬁrst glance these models of initiation of outside-in signaling may appear conﬂicting, it is possible that both models represent different phases of the signal. Integrinassociated kinase activity may be involved in helping the integrin cytoplasmic tails open up, allowing association of ITAM adapters such as DAP12 and FcRγ, which in turn amplify the signal, leading to actin cytoskeletal rearrangement, cell spreading, and activation of effector function. An obvious question in the ITAM model is how the signaling adapter becomes associated with the cytoplasmic tail of β2 or β3 . Is the association constitutive in resting cells or

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is it induced in activated cells? Integrins lack charged residues in their transmembrane domains that are generally required for association of DAP12 or FcRγ with other receptors, and to date there is no genetic or biochemical evidence for direct association of the adapters with the integrins. An intriguing recent study from Zhu et al. (87) demonstrates that physical separation of the α and β tails of the αIIb β3 integrin is required for initiation of intracellular outside-in signaling events. In this study, the investigators introduced a disulﬁde bond between the α and β chains that locks the transmembrane regions together. When expressed in CHO cells, this mutant integrin displays normal inside-out signaling responses in that it adopts an extended conformation and allows cell attachment to ﬁbrinogen-coated surfaces. However, subsequent outside-in signaling steps of cell spreading, actin polymerization, and focal adhesion formation are blocked. Other mutations in the integrin β3 tail have been deﬁned that impair outside-in signaling speciﬁcally, although whether they affect structural changes in the cytoplasmic tails is unclear (88). It is interesting to speculate that this tail separation is required for association of ITAM-containing adapters with the integrin tail to allow propagation and expansion of the outside-in signal. Another potential mechanism of coassociation of ITAM-containing proteins and kinases with leukocyte integrins may be through their joint recruitment to lipid raft membrane domains following adhesion. Disruption of lipid raft structures inhibits T cell adhesion via both LFA-1 and VLA-4 (63). The reverse is also true: Enforced clustering of T cell lipid rafts will induce LFA-1-mediated adhesion (62). In contrast, lipid rafts seem not to be involved in αIIb β3 integrin outside-in signaling in platelets (89), whereas treatment of neutrophils with methyl-β-cyclodextrin primes the cells for enhanced adhesion and integrin signaling (90). Clearly, the details in this area remain to be explored. Because the Src family kinases are well-known components of lipid raft domains, some colocalization of ITAM adapters, integrins, and potentially other downstream sig-

naling molecules within these domains is likely contributing to the outside-in signal. Recent observations have shown that these ITAM-containing adapters also play a role in signaling through the P/E-selectin receptor PSGL-1 (P-selectin glycoprotein ligand-1) to modulate integrin afﬁnity states, allowing the neutrophil to respond more rapidly to endothelial-expressed chemokines (91). The homology between the signaling events involved in PSGL-1-mediated integrin activation and subsequent integrin-mediated functional responses in leukocytes is perhaps the best example of the overlap between inside-out and outside-in signaling pathways.

Lipid raft: cholesterol- and sphingolipid-enriched microdomain found in the plasma membrane that allows compartmentalization of different signaling molecules

Downstream Proteins in the Outside-In Pathway A number of adapter proteins that serve as scaffolds for protein-protein interactions have been implicated in integrin outside-in signaling. Perhaps the most well-studied example is SLP76. Deﬁciency of SLP76 leads to a severe block in β2 and β3 integrin outside-in signaling events in neutrophils, DCs, and platelets (92). Mice lacking SLP76 speciﬁcally within the myeloid lineage (using SLP76loxP mutants crossed to the LysM-cre strain) are completely protected in the Schwartzman skin hemorrhagic vasculitis model, similar to mice lacking the myeloid Src family kinases or Syk, demonstrating the in vivo signiﬁcance of SLP76 for integrin outside-in signaling. Although SLP76 is also implicated in immunoreceptor signaling, it is interesting that speciﬁc mutations within SLP76, which affect its ability to bind downstream proteins such as Gads, can impair TCR signaling without affecting integrin signaling. Likewise, targeting SLP76 to membrane lipid rafts restores the ability of this adapter protein to function in the immunoreceptor pathway but does not restore normal β2 integrin signaling events in Slp76−/− neutrophils (72). This suggests that differential association of downstream signaling molecules with SLP76 may serve to distinguish immunoreceptor from integrin signaling responses in leukocytes. www.annualreviews.org • Leukocyte Integrin Signaling

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The adapter protein ADAP and its myeloidspeciﬁc homolog PRAM-1 have also been implicated in outside-in integrin signaling. The major function of ADAP in T cells is the activation of β1 and β2 integrins following engagement of the TCR, as described above. In neutrophils, however, ADAP seems to play a role in the outside-in pathway, since ADAP-deﬁcient cells show defects in adhesion-induced superoxide production (92). Similar results have been observed with PRAM-1-deﬁcient neutrophils, suggesting an overlapping role for these two adapters in myeloid integrin outside-in pathways (93). The dual role of SLP76, ADAP, and its associated molecules in inside-out pathways in T cells, versus outside-in events in myeloid cells, illustrates the cell speciﬁcity of these signaling cascades. Cytohesin-1 also appears to serve a dual function in T cell integrin signaling. Though initially studied in the inside-out pathway of integrin activation, as described above, direct involvement of cytohesin-1 in the outside-in pathway is suggested by the observation that engagement of LFA-1 in Jurkat T cells leads to threonine phosphorylation of cytohesin-1, whereas signaling through the TCR does not. Expression of a dominant-negative form of cytohesin-1 blocks LFA-1-mediated ERK activation and impairs IL-2 production following costimulation of LFA-1 and the TCR (94). JAB-1 ( Jun-activating binding protein 1), a coactivator of the c-Jun transcription factor, is implicated in LFA-1 outside-in signaling. JAB-1 is found in both the nucleus and cytoplasm, where a fraction of the JAB-1 pool associates with LFA-1. Engagement of LFA-1 causes dissociation of JAB-1 from LFA-1 with a concomitant increase in the nuclear pool of JAB-1, leading to formation of c-Jun complexes and activation of AP-1 transcriptional responses (94). Expression of dominant-negative forms of JAB-1 speciﬁcally blocks c-Jun phosphorylation and results in impaired IL-2 production, similar to the effects of cytohesin-1 inhibition. In myeloid cells, the dual adapter and ubiquitin ligase protein c-Cbl has been implicated in integrin outside-in signaling responses

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through study of Cbl-deﬁcient macrophages (95). Following engagement of integrin β2 in neutrophils or β1 in macrophages, c-Cbl is rapidly tyrosine phosphorylated by Src family kinases and becomes associated with PI3K, where it may assist in translocation of a fraction of intracellular PI3K to the membrane (96). Macrophages from c-Cbl−/− mice display reduced adhesion, poor podosome formation, and impaired migratory responses (95). Hence, part of the role of Cbl in the outside-in pathway may involve its ability to bring PI3K to the membrane to generate lipid products needed to activate downstream signaling components. PKC family members also function in both inside-out and outside-in pathways. Recently, both PKCβ and PKCθ have been implicated in αIIb β3 outside-in signaling responses (97, 98). Platelets deﬁcient in PKCβ or PKCθ do not spread on ﬁbrinogen-coated surfaces, although agonist-induced binding of soluble ﬁbrinogen occurs normally (indicating normal β3 integrin activation via inside-out pathways). Both PKC isoforms associate with β3 integrin: PKCβ following integrin ligand binding and PKCθ constitutively. The substrates of these PKC isoforms are unknown, and their roles in inside-out or outside-in pathways remain to be explained.

Connecting Integrin Outside-In Signaling to the Actin Cytoskeleton Following integrin clustering, a number of biochemical events are induced downstream that culminate in modulation of the actin cytoskeletal network. Many of these reactions function to modulate Rho GTPases. Activation of the Vav family of Rho-GEFs is a key step downstream of integrin clustering (99), and the most deﬁnitive studies have come from examination of Vav family–deﬁcient mice. Neutrophils lacking all three Vav family members show profound defects in β2 integrin signaling, including poor adhesion, spreading, and activation of oxidative burst, which mimics the phenotype of Src family kinase– or Syk-deﬁcient cells (100). As predicted, β2 -induced activation of Cdc42, Rac1,

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and RhoA is defective in Vav1/3-deﬁcient neutrophils. Like Src- or Syk kinase–deﬁcient cells, chemokine-induced attachment and initial adhesion of Vav-deﬁcient neutrophils to inﬂamed endothelium is unaffected, supporting the argument that integrin afﬁnity modulation is intact in these cells. However, because of impaired signaling events to the Rho GTPases and poor cell spreading, Vav-deﬁcient neutrophils show an inability to maintain ﬁrm adhesion under shear ﬂow conditions. The defective activation of oxidative burst activity as well as of other integrin signaling events likely contributes to the severely compromised host defense against bacterial pneumonia seen in Vav-deﬁcient mice (100). In a similar fashion, platelets lacking Vav1 and Vav3 show poor adhesion and spreading responses following engagement of αIIb β3 (101). Another major function of Vav family members is regulation of PLCγ isoforms and downstream Ca2+ signaling, particularly in immunoreceptor pathways in T cells and B cells (99). Recent studies in neutrophil integrin signaling show a similar role for Vav proteins; Vav-deﬁcient neutrophils fail to activate PLCγ2, leading to poor Ca2+ responses that likely contribute to impaired activation of oxidative burst (100). This same phenotype is seen in Vav1/3-deﬁcient platelets (101). These observations are supported by the fact that PLCγ2-deﬁcient neutrophils and platelets show defects in integrin-mediated activation events similar to those in Vav-deﬁcient cells (100, 101). The mechanism by which PLC isoforms modulate integrin signaling is unclear but likely involves their association with SLP76 and Vav proteins. As in the immunoreceptor pathway, integrin ligation induces a SLP76/Vav/PLCγ complex that is required for PLCγ phosphorylation and activation. Mutations in SLP76 that block this association also block integrin outside-in responses (92). An equally likely role for PLCγ is production of the lipid mediator IP3 , which is known to have direct effects on Rho GTPase activation. The primary effectors of the integrin outside-in signaling pathway leading to the

actin cytoskeletal rearrangements needed for cell spreading and ﬁrm adhesion are the Rho GTPase family members, which include Rac, Rho, and Cdc42. The literature on these GTPases in integrin signaling is vast, primarily in nonhematopoietic cells. Most studies of the Rho GTPases in leukocyte integrin signaling involve their role in polarization and migration following adhesion (102). A recent example involves phosphorylation of the integrin α4 chain following VLA-4 ligand binding. This releases paxillin and the GTPase ARF6 from the membrane, leading to accumulation of active Rac at the leading edge (103). Ultimately, the establishment of cell polarity and directed migration will be the sum of chemoattractant, mechanical (caused by shear stress in the vasculature), and integrin signals, which will obviously vary depending on the migratory site and the inﬂammatory stimulus involved. Of the many effector molecules downstream of the Rho GTPases, the Wiskott-Aldrich syndrome protein (WASp) has been deﬁned as playing a major role in integrin signaling in lymphocytes, myeloid cells, and platelets (104). WASp is activated by a number of stimuli, such as association with GTP-bound Cdc42 and tyrosine phosphorylation. Activated WASp assumes an unfolded conformation that allows the molecule to interact with the Arp2/3 complex to nucleate actin polymerization. Lack of WASp, in both humans and mice, results in impaired leukocyte (and platelet) adhesion, poor spreading responses, reduced migration, and impaired lymphocyte activation. WASp deﬁciency results in poor clustering of β2 integrins in macrophages and DCs, resulting in impaired podosome formation and reduced migration both in vitro and in vivo. Both mouse and human neutrophils lacking WASp show similar defects in β2 integrin clustering, leading to poor polarization following adhesion, impaired transendothelial migration in shear ﬂow, and reduced activation of degranulation/respiratory burst (105). At face value, reduced integrin clustering may sound more like an inside-out signaling impairment; however, given that WASp www.annualreviews.org • Leukocyte Integrin Signaling

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is downstream of Cdc42 GTPase activation, we interpret this ﬁnding as indicating that the defective actin polymerization responses in the WASp-deﬁcient leukocytes results in a reduced ability to form secondary adhesion structures (e.g., podosomes in mononuclear cells) needed for ﬁrm adhesion and spreading. In other words, loss of this distal step in outside-in signaling leads to a reduction of subsequent insideout steps as leukocyte adhesion progresses, suggesting that integrin signaling is really a circular pathway.

CONCLUSION The bidirectional nature of integrin signaling, ﬁrst envisioned by Richard Hynes (106), is now

INTEGRIN-DIRECTED THERAPIES IN THE CLINIC There are several integrin antagonists used in clinical studies. These fall into three groups: humanized antibodies, synthetic peptides, and nonpeptide small molecules (reviewed in 123, 124). All target the extracellular region of integrins and interfere with ligand binding. Antibodies have been made to target several different integrins including Efalizumab (Raptiva®), which antagonizes αL β2 and is used to treat psoriasis, and Natalizumab (Tysabri or Antegren®), which binds to the α4 chain and antagonizes α4 β1 and α4 β7 integrins and is used to treat multiple sclerosis and Crohn’s disease. The antibodies MEDI-522 (Vitaxin or Abegrin) and CNTO95 target integrin αV and are currently being tested as inhibitors of angiogenesis in several cancer trials (125). Abciximab (ReoPro®) is a Fab fragment that binds αIIb β3 and is used to inhibit platelet aggregation. Examples of cyclic RGD peptide mimetics include Cilengitide that inhibits αV β3 and αV β5 and Eptiﬁbatide (Integrilin®) that inhibits αIIb β3 . Nonpeptide inhibitors include Tiroﬁban (Aggrostat®), which targets αIIb β3 and is also used to inhibit platelet aggregation, and Valategrast, a small molecule inhibitor of α4 β1 for treatment of asthma. With the increased understanding of integrin conformational changes, other modulators of integrin function have been generated and are being evaluated in preclinical models. Whether inhibitors could be designed that target the intracellular portion of integrins, potentially distinguishing between different signaling pathways, has not yet been explored.

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a ﬁrmly established fact. The conformational changes that β2 and β3 integrins undergo following activation by inside-out signals are becoming well understood at a molecular level. The diversity of signals originating from these receptors, when clustered by their respective ligands, is also being unraveled, cell type by cell type. In reality, the two phases of integrin signaling almost certainly overlap, and it is often difﬁcult to separate them experimentally. Some molecules are clearly involved in the activation of different integrins in different cell types, such as talin and Rap GTPases, but inside-out pathways leading to LFA-1 activation in T cells are different from those that activate αIIb β3 in platelets. Likewise, it is clear that many molecules are involved in both inside-out and outside-in pathways; in many cases this results in a circular pathway that propagates signaling, which is likely needed for rapid adhesion and cell spreading, particularly in response to intravascular shear stress. This type of regulation has recently been coined as an adhesionstrengthening function of integrins (1). In a general sense, for leukocytes, this dynamic regulation makes sense. Leukocyte adhesion needs to be carefully regulated to allow rapid migration to sites of infection/inﬂammation, to modulate immune responses in secondary lymph nodes, or in the case of platelets to quickly establish hemostasis. So given this multitude of functional requirements, it is not surprising that integrin signaling is complex. Yet general themes do exist in all integrins, such as the conformational changes in the cytoplasmic domains induced by binding of cytoplasmic proteins such as talin or the activation of tyrosine kinases as initiators of outside-in responses. If we are ever going to design therapeutics that target integrin signaling events (and given some of the initial positive clinical results with inhibitors that block integrin/ligand interactions, therapeutic modulation of integrin function may hold great clinical promise—see side bar), then a greater understanding of how these events are regulated will be an area of active research in the future.

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DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS This work was supported by NIH grants AI068150 and AI065495. We apologize to all colleagues whose work we could not cite owing to space limitations.

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LITERATURE CITED 1. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. 2007. Getting to the site of inﬂammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7:678–89 2. Luo BH, Carman CV, Springer TA. 2007. Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25:619–47 3. Arnaout MA, Goodman SL, Xiong JP. 2007. Structure and mechanics of integrin-based cell adhesion. Curr. Opin. Cell Biol. 19:495–507 4. Kinashi T. 2005. Intracellular signalling controlling integrin activation in lymphocytes. Nat. Rev. Immunol. 5:546–59 5. McLeod SJ, Shum AJ, Lee RL, Takei F, Gold MR. 2004. The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J. Biol. Chem. 279:12009–19 6. Katagiri K, Shimonaka M, Kinashi T. 2004. Rap1-mediated lymphocyte function-associated antigen-1 activation by the T cell antigen receptor is dependent on phospholipase Cγ1. J. Biol. Chem. 279:11875–81 7. Shimonaka M, Katagiri K, Nakayama T, Fujita N, Tsuruo T, et al. 2003. Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under ﬂow. J. Cell Biol. 161:417–27 8. Sebzda E, Bracke M, Tugal T, Hogg N, Cantrell DA. 2002. Rap1A positively regulates T cells via integrin activation rather than inhibiting lymphocyte signaling. Nat. Immunol. 3:251–58 9. Katagiri K, Hattori M, Minato N, Irie S, Takatsu K, Kinashi T. 2000. Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase. Mol. Cell. Biol. 20:1956–69 10. Chrzanowska-Wodnicka M, Smyth SS, Schoenwaelder SM, Fischer TH, White GC II. 2005. Rap1b is required for normal platelet function and hemostasis in mice. J. Clin. Invest. 115:680– 87 11. Li Y, Yan J, De P, Chang HC, Yamauchi A, et al. 2007. Rap1a null mice have altered myeloid cell functions suggesting distinct roles for the closely related Rap1a and 1b proteins. J. Immunol. 179:8322–31 12. Duchniewicz M, Zemojtel T, Kolanczyk M, Grossmann S, Scheele JS, Zwartkruis FJ. 2006. Rap1Adeﬁcient T and B cells show impaired integrin-mediated cell adhesion. Mol. Cell. Biol. 26:643–53 13. Bergmeier W, Goerge T, Wang HW, Crittenden JR, Baldwin AC, et al. 2007. Mice lacking the signaling molecule CalDAG-GEFI represent a model for leukocyte adhesion deficiency type III. J. Clin. Invest. 117:1699–707 14. Tohyama Y, Katagiri K, Pardi R, Lu C, Springer TA, Kinashi T. 2003. The critical cytoplasmic regions of the αL/β2 integrin in Rap1-induced adhesion and migration. Mol. Biol. Cell 14:2570–82 15. Parmo-Cabanas M, Garcia-Bernal D, Garcia-Verdugo R, Kremer L, Marquez G, Teixido J. 2007. Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin α4β1. J. Leukoc. Biol. 82:380–91 16. Katagiri K, Ohnishi N, Kabashima K, Iyoda T, Takeda N, et al. 2004. Crucial functions of the Rap1 effector molecule RAPL in lymphocyte and dendritic cell trafﬁcking. Nat. Immunol. 5:1045–51 17. Katagiri K, Imamura M, Kinashi T. 2006. Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion. Nat. Immunol. 7:919–28 www.annualreviews.org • Leukocyte Integrin Signaling

10. Genetic evidence that Rap1 GTPases are required for integrin inside-out signaling.

13. Genetic evidence that mutation of CalDAG-GEFI leads to symptoms of LADIII.

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26 and 27. Genetic evidence that talin is required for platelet inside-out integrin signaling.

38 and 39. First demonstration of role for kindlin family members in integrin inside-out signaling in vivo.

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108. Etzioni A, Doerschuk CM, Harlan JM. 1999. Of man and mouse: leukocyte and endothelial adhesion molecule deﬁciencies. Blood 94:3281–88 109. Alon R, Aker M, Feigelson S, Sokolovsky-Eisenberg M, Staunton DE, et al. 2003. A novel genetic leukocyte adhesion deﬁciency in subsecond triggering of integrin avidity by endothelial chemokines results in impaired leukocyte arrest on vascular endothelium under shear ﬂow. Blood 101:4437–45 110. Salles II, Feys HB, Iserbyt BF, De Meyer SF, Vanhoorelbeke K, Deckmyn H. 2008. Inherited traits affecting platelet function. Blood Rev. 22:155–72 111. Stephens LE, Sutherland AE, Klimanskaya IV, Andrieux A, Meneses J, et al. 1995. Deletion of β1 integrins in mice results in inner cell mass failure and peri-implantation lethality. Genes Dev. 9:1883–95 112. Wilson RW, Ballantyne CM, Smith CW, Montgomery C, Bradley A, et al. 1993. Gene targeting yields a CD18-mutant mouse for study of inﬂammation. J. Immunol. 151:1571–78 113. Scharffetter-Kochanek K, Lu H, Norman K, van Nood N, Munoz F, et al. 1998. Spontaneous skin ulceration and defective T cell function in CD18 null mice. J. Exp. Med. 188:119–31 114. Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H, Crowley D, et al. 1999. β3-integrindeﬁcient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J. Clin. Invest. 103:229–38 115. Arroyo AG, Yang JT, Rayburn H, Hynes RO. 1996. Differential requirements for α4 integrins during fetal and adult hematopoiesis. Cell 85:997–1008 116. Scott LM, Priestley GV, Papayannopoulou T. 2003. Deletion of α4 integrins from adult hematopoietic cells reveals roles in homeostasis, regeneration, and homing. Mol. Cell. Biol. 23:9349–60 117. Smyth SS, Reis ED, Vaananen H, Zhang W, Coller BS. 2001. Variable protection of β3-integrin– deﬁcient mice from thrombosis initiated by different mechanisms. Blood 98:1055–62 118. Ding ZM, Babensee JE, Simon SI, Lu H, Perrard JL, et al. 1999. Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration. J. Immunol. 163:5029–38 119. Semmrich M, Smith A, Feterowski C, Beer S, Engelhardt B, et al. 2005. Importance of integrin LFA-1 deactivation for the generation of immune responses. J. Exp. Med. 201:1987–98 120. Lu H, Smith CW, Perrard J, Bullard D, Tang L, et al. 1997. LFA-1 is sufﬁcient in mediating neutrophil emigration in Mac-1-deﬁcient mice. J. Clin. Invest. 99:1340–50 121. Luo BH, Springer TA, Takagi J. 2004. A speciﬁc interface between integrin transmembrane helices and afﬁnity for ligand. PLoS Biol. 2:e153 122. Armulik A, Nilsson I, von Heijne G, Johansson S. 1999. Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits. J. Biol. Chem. 274:37030–34 123. Ulbrich H, Eriksson EE, Lindbom L. 2003. Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inﬂammatory disease. Trends Pharmacol. Sci. 24:640–7 124. Yonekawa K, Harlan JM. 2005. Targeting leukocyte integrins in human diseases. J. Leukoc. Biol. 77:129– 40 125. Avraamides CJ, Garmy-Susini B, Varner JA. 2008. Integrins in angiogenesis and lymphangiogenesis. Nat. Rev. Cancer 8:604–17

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Recent Advances in the Genetics of Autoimmune Disease Peter K. Gregersen and Lina Olsson Robert S. Boas Center for Genomics and Human Genetics, The Feinstein Institute for Medical Research, Manhasset, New York 11030; email: [email protected], [email protected]

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Key Words

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

genome-wide association (GWA) study, interferon, NF-κB, autophagy, autoantigen

This article’s doi: 10.1146/annurev.immunol.021908.132653 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0363$20.00

Abstract Extraordinary technical advances in the ﬁeld of human genetics over the past few years have catalyzed an explosion of new information about the genetics of human autoimmunity. In particular, the ability to scan the entire genome for common polymorphisms that associate with disease has led to the identiﬁcation of numerous new risk genes involved in autoimmune phenotypes. Several themes are emerging. Autoimmune disorders have a complex genetic basis; multiple genes contribute to disease risk, each with generally modest effects independently. In addition, it is now clear that common genes underlie multiple autoimmune disorders. There is also heterogeneity among subphenotypes within a disease and across major racial groups. The current crop of genetic associations are only the start of a complete catalog of genetic factors for autoimmunity, and it remains unclear to what extent common variation versus multiple rare variants contribute to disease susceptibility. The current review focuses on recent discoveries within functionally related groups of genes that provide clues to novel pathways of pathogenesis for human autoimmunity.

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INTRODUCTION

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Over the past several years, there has been an explosion of new information on genetic risk factors for human autoimmune diseases. This progress has resulted from a conﬂuence of remarkable advances in human genetics generally, including the development of commercial genotyping platforms that allow for the production of hundreds of millions of genotypes in a rapid and cost-effective manner. In addition, along with the completion of the ﬁnished human genome sequence in 2003, a worldwide effort by the International HapMap Project (1) has provided us with a rich resource for understanding the nature and extent of human genetic diversity that is buttressed by the development of sophisticated statistical tools for analysis. Finally, there has been a renewed appreciation of the genetic complexity of many human diseases, and this has catalyzed collections of very large population data sets for study, thereby providing the required statistical power to ﬁnd and conﬁrm genetic associations that were previously undetectable. These trends in human genetics have had a major impact on the study of autoimmune diseases, and indeed, the new ﬁndings in autoimmunity are a gratifying demonstration that the rigorous application of modern genetic tools can lead to exciting new insights and hypotheses about disease pathogenesis, in spite of the extraordinary genetic complexity of these disorders. Many of the recent genetic discoveries have resulted from the application of genome-wide association (GWA) scans, an approach that is generally geared toward the detection of relatively common genetic risk variants (2). It is important to realize that we are still in the relatively early stages of gene discovery, and even when a putative risk gene has been identiﬁed, in most cases the actual causative genetic variants have not been clearly deﬁned. In addition, other forms of genetic variation are emerging as relevant for autoimmunity, including rare variants and copy number variation, and we are still far from understanding the role of epigenetic factors and somatic genetic changes. There-

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fore, to interpret the signiﬁcance of present and future genetic ﬁndings, an understanding of the strengths and weaknesses of current genetic mapping approaches, as well as those on the horizon, is essential.

Why Map Genes for Human Autoimmunity? The answer to this question may seem obvious, but it arises surprisingly often, especially in clinical settings. This is because many of the new genetic associations with autoimmunity are extremely modest, often with odds ratios in the range of 1.1–1.5. For the most part, there is no obvious clinical utility to this information, and the predictive value for future development of disease is low. Of course, this may change as knowledge becomes more complete, but currently only the rare autoimmune and inﬂammatory diseases have a strong genetic component with Mendelian patterns of inheritance. In these cases, knowledge of the underlying genetic defect can be crucial for proper diagnosis and treatment. The periodic fever disorders reviewed in this volume are a good example of this [see the review by Kastner and colleagues (3), in this volume]. However, for most autoimmune diseases this is not the case. Therefore, the most compelling reason for identifying the genetic underpinnings of common autoimmune disorders is to generate new hypotheses about disease mechanisms and pathogenesis. As discussed in this review, the ﬁrst wave of results has generated a plentiful harvest.

Approaches to Identifying Disease Genes To evaluate new genetic ﬁndings, it is useful to understand some of the conceptual and analytic issues in genetic mapping. Until very recently, three basic approaches have been used to identify genetic variants that may contribute to any human phenotype, including autoimmune disorders. These approaches are (a) candidate gene association studies, (b) linkage analysis in multiplex families, and (c) GWA studies. Candidate

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gene studies have been a mainstay for human genetic studies for several decades, and they will continue to play an important role. However, early candidate gene case-control studies often suffered from insufﬁcient statistical power owing to inadequate sample sizes and to a lack of appreciation for the importance of careful matching of cases and controls. The strong publication bias for initial positive ﬁndings has been clearly documented (4), and reports of candidate gene associations should be viewed with caution until multiple replications have been carried out. Candidate gene studies are usually done to address a plausible hypothesis, but plausibility should not mitigate the requirement for robust and reproducible statistical evidence. Genetic linkage analysis depends on the cosegregation of chromosomal regions with a phenotypic trait within families, as is typical for highly penetrant Mendelian disorders. For most common autoimmune diseases, familial aggregation is rather modest, and therefore linkage analysis has quite low statistical power to detect chromosomal regions with shared genetic risk within families. Nevertheless, linkage approaches have occasionally contributed signiﬁcantly to the identiﬁcation of new risk genes, for example NOD2 (nucleotide binding and oligomerization domain 2) in Crohn’s disease (CD) (5, 6), and more recently STAT4 (signal transducer and activator of transcription 4) in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) (7). In the last two years, GWA scans have dominated efforts in gene mapping for autoimmune diseases, and these studies have led to the majority of the new genetic associations discussed in this review. Like candidate gene studies, the analysis is based on association, but in the case of GWA scanning, no particular hypothesis is being addressed. Rather, hundreds of thousands of hypotheses are being addressed simultaneously, without regard to biologic plausibility. This is a purely discovery-driven approach to gene identiﬁcation, free of the limitations imposed by a priori assumptions about which genes and pathways are likely to be involved in the disease un-

der study. Despite early skepticism, GWA scanning has proved to be a remarkably effective method of gene discovery.

Genome-Wide Association Scans: Design and Interpretation The GWA scanning approach is critically dependent on knowledge of the extent and patterns of variation in the human genome. Although the initial sequence of the human genome was an important ﬁrst step, it is really the International HapMap Project that has provided the basis for a rational approach to GWA studies (1). When considering single nucleotide polymorphisms (SNPs), any two unrelated individuals in the population differ by approximately 0.1% across the 3.2 billion base pairs of the genome, or approximately 3 million SNPs. By studying 90 individuals in families from three major racial groups (Caucasian, Asian, and African), the HapMap Project has cataloged the majority of the common SNPs (e.g., SNPs with minor allele frequencies of 5% or greater) in these populations. This has provided a library of millions of SNP markers for use in GWA scans (1). An important result of the HapMap Project has been the realization that to deﬁne most of the common variations among individuals, it is not necessary to genotype all 3 million SNP differences among them, but only a subset of these, on the order of 300,000 to 500,000 SNPs. This is because SNP alleles are distributed nonrandomly among individuals, forming blocks of linkage disequilibrium (LD) that may extend from thousands to many hundreds of thousands of base pairs. This results in a kind of bar code that can be used to deﬁne the common genetic variation across the genome of a given individual. An illustration of this pattern for the genetic region around the PTPN22 gene is shown in Figure 1. This pattern of common variation across genomes has led to the concept of tagging SNPs (8). This involves the use of a single SNP to tag a block of LD formed by many other SNPs, thus allowing for the interrogation of a large section www.annualreviews.org • Genetics of Human Autoimmunity

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rs6679677 (WTCCC)

rs2476601 (R620W)

NM_018364

NM_012411 _

RSBN1: round spermatid basic protein 1

PTPN22: protein tyrosine phosphatase, h nonreceptor type

NM_152696

NM_015967 PTPN22: protein tyrosine phosphatase, nonreceptor type

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CEU phased haplotypes

HIPK1: homeodomain-inte

NM_001010922 C1orf178: pro-apoptotic Bcl-2 protein

NM_001010922

NM_19826

C1orf178: pro-apoptotic Bcl-2 protein

HIPK1: h

Haplotype block pattern CEU: lod

Linkage disequilibrium by D'

Figure 1 A map of the region around the PTPN22 locus on chromosome 1p13 covering approximately 200,000 base pairs. The genes in the region are shown at the top of the ﬁgure. The blue and yellow haplotype block pattern was generated by looking at combinations of single nucleotide polymorphisms (SNP) alleles in 90 Caucasian subjects from the HapMap Project. Note that a limited number of patterns are observed, generating a kind of bar code for each subject. The lower portion of the ﬁgure shows a heat map in which the intensity of red color reﬂects the degree of correlation [linkage disequilibrium (LD) measured by D ] among SNPs across the region (indicated by tick marks). Note that widely separated SNPs are highly correlated. Two markers associated with type 1 diabetes (and other autoimmune diseases) are shown at the top. Marker rs2476601 is likely to be the causative variant in this region. Note that another marker (rs6679677) nearly a distance of 100 kb also strongly associates with diabetes. This emphasizes that it is difﬁcult to assign the causative locus on the basis of associations alone, as discussed in the text.

of the genome with a single marker. SNP tagging may be applied in a pairwise fashion, or may also involve using several SNPs to predict the presence of a third. This ability to impute the likely presence of untyped SNPs is based on the information provided by the HapMap on the patterns of LD in the population under study. Although computationally intensive, this approach is applied across the entire genome to generate data on markers that have not been 366

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actually typed in the original study (9), which facilitates combining data generated on different typing platforms. Of course, the extent of correlation among SNP alleles in LD is often less than complete, and therefore statistical methods must be applied. Two commonly used measures are a standard correlation coefﬁcient (r2) and D . D is a measure of LD among markers that is normalized to the maximum possible LD, given

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the allele frequencies, with a D = 1 reﬂecting maximally possible LD. The lower section of Figure 1 illustrates a typical representation of the extent of LD using the D measure across a large region around the PTPN22 locus. Note that a large block of high LD encompasses several genes over a ∼200,000 base pair region. Thus, many SNPs in this region are likely to provide evidence of association for diseases that are associated with PTPN22, and indeed, this was observed in a recent GWA screen (10) for marker rs6679677 (location shown on Figure 1). Conversely, any association observed with such a SNP may be due to causal variants in any of the genes in this LD block. In the case of PTPN22, there is additional functional and biologic evidence that this gene is involved in autoimmunity owing to the effects of a nonsynonymous SNP rs2476601 indicated in the Figure. However, without this additional evidence, it is nearly impossible to prove that PTPN22 is the relevant risk gene in this region or that the rs2476601 marker used to ﬁrst detect the association is causative. Once a SNP association is observed and conﬁrmed, much work remains to be done to establish which genetic variants in the region are actually responsible (e.g., causative) for the association. Furthermore, because many GWA studies employ 500,000 or more SNPs across the genome, each addressing a separate hypothesis, the statistical signiﬁcance levels must be adjusted for multiple testing. An overall p value of <5 × 10−7 is now widely accepted as compelling evidence of true association, although it is quite clear that lower degrees of statistical signiﬁcance often reﬂect real associations. In any case, truly convincing association always requires multiple replications in independent data sets. A major consideration for GWA scans, as well as any case-control association study, is the issue of proper matching of cases and controls. The availability of genome-wide SNP data across many different populations has now permitted the use of so-called ancestry informative markers (AIMS) to match more precisely cases and controls for their ethnic background.

This is quite straightforward for major racial groups, such as Asian, Caucasian, and African, but is more challenging within these groups (11). The pattern of allelic variation observed for the PTPN22 risk allele in European Caucasian populations nicely illustrates this problem. As shown in Figure 2, the T1858C SNP displays a wide range of allele frequencies in the normal population, generally increasing in frequency going from southern to northern Europe. Therefore, if the cases and controls are taken from different European subpopulations, there is considerable risk of false positive (or negative) results. This phenomenon is generally referred to as population stratiﬁcation. In the experience of many investigators, selfreport by study participants is an unreliable indicator of ancestry, but in the context of GWA studies, it is possible to correct for unknown population stratiﬁcation using the entire set of SNP markers. This is generally done using a principal components approach (12) or by measures of multidimensional geometric distance among groups of subjects based on allele frequency distributions across the genome (13). More recently, matching of European populations has also been done using selected AIMS (14). One of the advantages of this approach is that it allows for the use of publicly available control data sets in GWA studies, even when the details of ancestry are not known for these control subjects. Finally, this brief discussion of GWA studies would be incomplete without some mention of statistical power and sample size requirements. Most of the associations with autoimmunity involve the detection of odds ratios between 1 and 2, with many associations on the lower end of this range. The sample sizes required to generate statistical signiﬁcance in the setting of GWA scan (p < 5 × 10−7 ) can be very large, depending on the allele frequency in the population and the odds ratios to be detected. For risk ratios on the order of 2 or more, samples sizes of 1000 are generally adequate. However, for risk ratios in the range of 1.2–1.3, even sample sizes of three or four thousand may have low statistical power depending on marker www.annualreviews.org • Genetics of Human Autoimmunity

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11.6 %

10.4 % 10.3 %

7.8 %

15.5 %

12.6 %

7.8 %

2.5 % 7.4 %

2.1 %

Figure 2 The allele frequencies of the PTPN22 risk allele (T) across Europe. Note that there is a gradient of increasing frequency of this allele moving from southern to northern geographic regions. This gradient emphasizes the importance of carefully matching case and control subjects for association studies, even within European populations.

allele frequency (2). This magnitude of a population sample is now considered a minimum for a thorough analysis in the setting of a GWA scan, and truly comprehensive genetic studies will require considerably larger sample sizes to be studied in the future.

GENETIC ASSOCIATIONS WITH AUTOIMMUNITY We are currently in a period of rapid data accumulation on the variable and complex genetic underpinnings of human autoimmune diseases. Nevertheless, several themes are emerging from the ﬁrst wave of results. First, some genetic variants clearly predispose to multiple autoimmune diseases, thus providing a gratifying conﬁrmation that many of these diseases share common pathways of pathogene368

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sis, despite their highly heterogeneous clinical manifestations. At the same time, the lack of such overlap for some diseases provides evidence that distinct mechanisms also exist. Second, multiple genes are involved in predisposition to each disease, and typically the genetic associations are quite modest. Third, with a few exceptions, the actual causative genetic variations that explain the associations have not been deﬁnitively established. Finally, even the partial data now available suggest that an extraordinarily wide range of different pathways and considerable genetic heterogeneity underlies autoimmunity—some expected, and some entirely unexpected—which offers a rich source of new hypotheses and experimental directions to pursue. For the purposes of this review, we focus on the most convincing associations reported to

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date, and our discussion emphasizes the major themes and pathways that are emerging from the new data. Many associations likely to be real but that do not (yet) reach widely accepted levels of statistical signiﬁcance have not been included because the complete delineation of these genes is a moving target, and in any case a truly comprehensive discussion is impractical given the potentially large number of genes involved. Table 1 provides a list of leading genetic associations that are proven or highly likely to hold up after further study and that have also provided clues to the underlying mechanisms and pathogenesis of autoimmunity. Most of these ﬁndings have been generated from GWA scans, but many also were discovered using candidate gene approaches or linkage analysis. The genes listed in Table 1 are grouped according to their presumed major cellular functions, although it is fair to say that in all cases the exact mechanism by which these genes contribute to risk for autoimmunity is still unknown. The MHC associations with autoimmunity are not included in the Table but are discussed brieﬂy in a separate section. Finally, the list of genes in Table 1 is clearly just the ﬁrst wave of results that are going to emerge over the next few years. For example, as we were completing this review, convincing evidence for 30 genetic associations with CD was reported (15).

Intracellular Tyrosine Phosphatases As listed in Table 1, a number of intracellular signaling molecules have been associated with autoimmune disorders. Perhaps the most replicated and broadly relevant of these associations is with the intracellular tyrosine phosphatase PTPN22. The initial association of PTPN22 with type 1 diabetes (T1D) was reported by Bottini et al. (16), who took a candidate gene approach and focused on a nonsynonymous amino acid polymorphism (R620W) that was judged likely to have functional correlates. In an independent effort, Begovich et al. (17) selected PTPN22 as part of a limited genomewide screen of likely functional variants in sev-

eral thousand candidate genes, informed in part by previous linkage results. This led to the association of PTPN22 with RA. Both associations have now been widely replicated, and the PTPN22 associations with these and several other autoimmune diseases are among the most robust in the literature. For RA and T1D the PTPN22 620W allele confers a nearly two-fold risk for disease, with odds ratios in the range of 3–4 for homozygous individuals. Thus, in terms of strength of association, PTPN22 is second in importance only to the MHC for these two diseases. The patterns of association between the PTPN22 620W allele and autoimmunity are instructive on many levels. First, PTPN22 was among the ﬁrst and most convincing demonstrations that common susceptibility genes underlie diverse autoimmune phenotypes. In addition to T1D and RA, PTPN22 is associated with Graves’ disease (GD) (18–20), Hashimoto thyroiditis (21), myasthenia gravis (22), systemic sclerosis (23), generalized vitiligo (24), Addison’s disease (25), and alopecia areata (26). Associations with juvenile idiopathic arthritis (27–29) and SLE (30, 31) have generally been weaker than for RA and T1D. Strikingly, there is no evidence of association with multiple sclerosis (MS) (32, 33), and the 620W allele actually appears to be protective for CD (15). These contrasting patterns of association are likely to reﬂect fundamental similarities and differences in the mechanisms underlying the pathogenesis of these disorders. In general, it appears that an important feature of the PTPN22-associated diseases is that they all have a prominent component of humoral autoimmunity. Knockout animals for Lyp (also known as PEP, the mouse ortholog of PTPN22) exhibit enhanced T cell activation in combination with an increased production of antibodies (34). This is consistent with the ability of PTPN22 to dephosphorylate Lck at the activating phosphotyrosine 394, leading to persistent phosphorylation and Lck activation in knockout animals. Yet somewhat surprisingly, the consequence of the 620W risk allele in humans is apparently a lower degree of T cell activation [an increased www.annualreviews.org • Genetics of Human Autoimmunity

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Genetic loci with confirmed associations with human autoimmune disorders

Gene

Location

Function

Diseasesa

Selected references

Intracellular signaling molecules and receptors PTPN22

1p13.3

TCR and BCR signaling and other?

RA, SLE, AITD, T1D

16, 17, 21, 167, 193, 194

BANK1

4q22

B cell activation/BCR signaling

SLE

80

TNFAIP3

6q23

Ubiquitin editing enzyme; inhibitor of TNFR signaling/NF-κB pathway

RA, SLE, CD

45–48

BLK

8p23

B cell activation

SLE

31

PTPN2

8p11.3

Negative regulator of T cell activation

CD, T1D

10, 15

TRAF1

9q33

Regulates TNFR signaling/NF-κB pathway

RA

51, 195

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Intracellular pattern-recognition receptors IFIH1

2q24

Receptor for viral dsRNA

T1D, GD

10, 42, 87, 88

NOD2/ CARD15

16q12

Intracellular receptor for bacteria, signals via NF-κB

CD

5, 6

Transcription factors REL

2p13

Member of NF-κB

RA

P.K. Gregersen & K. Siminovitch, unpublished data

STAT4

2q32.2

Regulates IFN-γ pathway

RA, SLE

7, 108

IRF5

7q32

Regulates type 1 IFN pathway

SLE

89, 93

NKX2-3

10q24.2

Regulates development of intestinal and secondary lymphoid organs and B and T cell homing

CD

10, 15, 159

Cytokines and cytokine receptors IL2/Il21

4q26

T cell regulation

T1D, RA, Celiac disease

133, 134

IL23R

1p31.1

Th17 homeostasis

PSA, PSO, CD, AS

10, 15, 123–125

IL7RA

5p13

Memory T cell homeostasis

MS

140, 141, 145

IL2RA

10p15.1

T cell/Treg homeostasis

MS, T1D, GD

137–141, 196

IL12B

15q31.1

Development of T cell subsets, Th1 and Th17

PSO, CD

15, 124, 135

Membrane receptors and costimulatory molecules CTLA4

2q33

T cell costimulation inhibitory

T1D, RA

10, 147, 167

ITGAM

16p11.2

Immune complex clearance/leukocyte adhesion

SLE

30, 31, 150

CD40

20q12

B/T cell costimulation

RA

65

Production of IgM, TNF-α, IL-2 via NF-κB pathway Autophagy related ATG16L1

2q37.1

Autophagy

CD

15, 157, 158

IRGM

5q33.1

Autophagy

CD

10, 15, 159, 197

ARTS1

5q15

Peptide trimming for MHC I

AS

125

PADI4

1p36.13

Enzymatic peptide citrullination

RA

165, 167

INS

11p15.5

Target autoantigen

T1D

170, 171

TSHR

14q31

Target autoantigen

AITD

125, 176, 177

Enzymes

Autoantigens

a Abbreviations: AITD, autoimmune thyroid disease; AS, ankylosing spondylitis; CD, Crohn’s disease; GD, Graves’ disease; MS, multiple sclerosis; PSA, psoriatic arthritis; PSO; psoriasis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; T1D, type 1 diabetes.

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threshold for T cell receptor (TCR) signaling] (35, 36). One clear biochemical consequence of the 620W polymorphism is to reduce the binding of PTPN22 with the intracellular kinase Csk (16, 17). Indeed, amino acid position 620 of PTPN22 is located within one of several SH3 binding sites in the PTPN22 molecule. An important role of Csk is to inhibit Lck activity by phosphorylation of amino acid 505 of the Lck molecule (37). Whether this particular activity is affected by the 620W polymorphism in PTPN22 is unclear. Bottini and coworkers have proposed a model for interactions among Lck, PTPN22, and Csk that may explain the elevation of thresholds for TCR signaling (N. Bottini, personal communication), with the overall implication that reduced, rather than elevated, T cell triggering may be part of the phenotypic predisposition to autoimmunity. A similar tendency to increased thresholds for receptor triggering has also been reported in B cells (36). PTPN22 is widely expressed in many hematopoietic cell types, and several different substrates of PTPN22 have been described (37). However, the overall functions of PTPN22 in non-T cells are largely unknown. PTPN22 is also involved in the activation of endogenous cannabinoids (38). Thus, the exact mechanism for this genetic association is still unresolved, and indeed there could be multiple mechanisms. In this respect, the PTPN22 is an excellent example of how gene discovery is hypothesis generating, and the number of hypotheses can be quite large, even for a single genetic association in which the likely causative variant has been identiﬁed. The disease-associated DNA sequence polymorphism (rs2476601, 1885C>T) resides in a rather large haplotype block encompassing the entire PTPN22 gene as well as several ﬂanking loci shown in Figure 1. As mentioned in the introduction, in the recent GWA studies reported by the Wellcome Trust, the PTPN22 association was actually picked up by a marker that is outside of the PTPN22 gene itself (10). Thus, as with all association studies, the question is whether the polymorphism used to identify the association is actually the causative

variant. Resequencing of the PTPN22 locus by Carlton et al. (39) showed that the 620W allele was the only variant that distinguished the risk haplotype from a second nonassociated haplotype. Modest evidence for associations with additional PTPN22 haplotypes have not been replicated. The importance of the 620W allele is further supported by the fact that there is no association with PTPN22 in the Asian populations, and indeed Asian populations rarely carry the 620W variant. Attempts to identify additional PTPN22 variants that may associate with RA in Asian populations have not been successful (40, 41). Thus, although the genetic data are not totally comprehensive across the entire risk haplotype, it is highly likely that the 620W allele is directly responsible for the associations with T1D, RA, and many other autoimmune diseases. But again, proof of causation depends on combining such genetic evidence with biochemical function and integration of functional differences into a larger pathway of pathogenesis. This has certainly not been fully accomplished for PTPN22 for any disease, but it is further along toward this goal than most of the other genetic associations discussed in this review. A second intracellular tyrosine phosphatase, PTPN2, encoded on chromosome 18p11 has also been associated with human autoimmunity; convincing associations have been reported for CD (10, 15) and T1D (42), with odds ratios in the range of 1.3. Weaker PTPN2 associations with other autoimmune phenotypes such as RA and GD (42) have not yet been conﬁrmed. PTPN2 is thought to be the relevant gene on chromosome 18p11 because it is the only known gene located in the associated region over 100 kb, but no clear causative variants have been identiﬁed. PTPN2 is an appealing candidate gene. It is ubiquitously expressed and is clearly involved in immune function. PTPN2-knockout animals exhibit a fatal inﬂammatory wasting syndrome (43), with accompanying abnormalities in multiple cell types. PTPN2 appears to have a negative regulatory role on IL-2R signaling in T cells, consistent with the fact that Jak1 and Jak3 are among www.annualreviews.org • Genetics of Human Autoimmunity

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its substrates (44). In addition, PTPN2−/− animals have enhanced macrophage Jak1 phosphorylation in response to IFN-γ and increased sensitivity to LPS. Thus, it is reasonable to propose that PTPN2 variants in humans may alter the thresholds for these, or other, signaling pathways (43).

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Susceptibility Genes in Tumor Necrosis Factor Receptor and NF-κB Signaling Pathways Recently, a number of genetic associations have emerged that implicate molecules in the tumor necrosis factor (TNF) family of molecules as well as NF-κB signaling pathways. Although it is still far from clear if these various observations are related to a common pathway of pathogenesis, we discuss them here together, in part to illustrate how the rapid accumulation of genetic data can generate new hypotheses for exploration. In late 2007, Plenge et al. (45) reported the association of SNP markers near the TNFAIP3 locus on chromosome 6q23 with RA. This association has been conﬁrmed (46) and extended to SLE (47, 48). The TNFAIP3 gene encodes a cytoplasmic zinc ﬁnger protein known as A20 in the mouse, and A20 is a major negative regulator of TNF-induced NF-κB signaling pathways. Knockout animals for A20 die prematurely from widespread inﬂammation and cachexia and are hypersensitive to TNF (but not IL-1) with associated inability to downregulate NF-κB (49). The mechanisms by which A20 regulates NF-κB signaling are complex and incompletely deﬁned. A20 possesses dual properties of ubiquitination and deubiquitination, and may act by regulating TNF receptor– associated factors (TRAF)2 and IKKγ, with more recent evidence of regulatory effects on receptor-interacting protein (50). Interestingly, A20 expression is generally low in most cells, but it is induced by NF-κB, consistent with a role in feedback regulation on NF-κB signaling. Several different polymorphisms have been associated with autoimmunity, including 372

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a nonsynonymous coding SNP (Phe127Cys) with some evidence of reduced negative regulatory ability for TNF-induced NF-κB signaling by the susceptibility allele (47). A GWA screen for RA reported in late 2007 also revealed an association with a region on chromosome 9q that contains the TRAF1 as well as the C5 locus (51). This has been replicated, and although both TRAF1 and C5 are compelling candidate genes, the most recent data strongly suggest that TRAF1 is likely to be the causative locus (52). TRAFs are a family of cytoplasmic adapter molecules that mediate signaling by a broad array of TNF receptor family members (53). TRAF1 is distinct from all other TRAF family members in that it lacks zinc ﬁnger and RING domains that are responsible for mediating downstream signaling directly. Thus, an important function of TRAF1 appears to be the regulation of receptor signaling mediated by other TRAFs. TRAF1 is of interest in the context of this discussion because it interacts with TRAF2 (a molecule regulated by A20) to regulate signaling through CD40, a TNF receptor family member (TNFRSF5). Both negative and positive effects on CD40 signaling have been ascribed to TRAF1 (54, 55), but a recent model proposes that TRAF1 cooperates with TRAF2 to regulate the efﬁciency of CD40 signaling in B cells, particularly through the NF-κB pathway (56, 57). Recently, it has become apparent that CD40 is also an important costimulator in T cells, with predominant activation through NF-κB pathways, again with evidence of dependence on TRAFs for signaling (58). Unlike other TRAFs, the level of expression of TRAF1 is tightly regulated and increases on cell activation, including activation through CD40 (59). Although A20 and TRAF1 are involved in a variety of signaling pathways, a renewed focus on their role in CD40 signaling is prompted by the fact that CD40 itself has been associated with risk for autoimmunity. CD40 associations were ﬁrst reported in GD (60), and with few exceptions this ﬁnding has been replicated (61, 62). A likely risk allele is located in a Kozak sequence (marker rs1883832), and there is

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evidence for an effect on expression levels of CD40 (63). The possibility that CD40 may also associate with risk for SLE was raised by ﬁne mapping studies on chromosome 20 (64), but even more compelling evidence comes from a recent large meta-analysis in RA, in which genome-wide levels of signiﬁcance were achieved using a marker (rs4810485) that is a near perfect proxy for the rs1883832 risk allele associated with GD (65). Finally, very recent data on a second North American GWA scan in RA (P.K. Gregersen & K. Siminovitch, unpublished data) have provided deﬁnitive evidence for an association with the NF-κB family molecule c-Rel. The relevant causative alleles have not been identiﬁed, but this association is potentially relevant to the current discussion because CD40 interacts with crel in several ways. First, CD40 transcriptional regulation is inﬂuenced by c-rel (66, 67). In addition, recent reports suggest that, the CD40 and c-Rel proteins can physically interact and form a heterodimer that is translocated to the nucleus and has transcriptional regulatory activity for known c-rel target genes, including CD154, BLyS/BAFF, and Bﬂ-1/A1 (68). The fact that a membrane protein such as CD40 has a nuclear transcriptional function is surprising, but not entirely novel; similar functions have been reported for other cell surface receptors (67). These genetic data raise the possibility that the genetic associations of A20, TRAF1, CD40, and c-Rel may reﬂect the involvement of a common cell signaling pathway in autoimmunity (Figure 3). The costimulatory role of CD40 on the APC–T cell interaction has traditionally been viewed as one of its main functions, and indeed a wide range of cells can express CD40 and can be triggered by CD40 ligand expressed on activated T cells. For example, in the synovium of RA patients CD40 is expressed on CD14+ synovial cells, and CD40-CD154 ligation leads to production of TNF-α and IL-1α and -β, thereby contributing to chronic inﬂammation (69). The CD40 association with GD has likewise been hypothesized to result from overactivity of CD40 on thyrocytes (63).

In contrast to this traditional view, recent studies have emphasized the importance of CD40 in the maintenance of effector T cell populations in autoimmunity. Beginning with early studies in the autoimmunity-prone nonobese diabetic mouse strain (70), evidence has accumulated that a T cell subset expressing low levels of CD4 but high levels of CD40 (designated Th40 cells) is responsible for driving T1D in experimental animals (71). Extending this hypothesis, Waid and coworkers (72) have shown that in the nonobese diabetic mouse the ratio of Treg/Th40 cells is shifted in favor of the autoaggressive Th40 cells. The Th40 cells have a higher resistance to Fas-induced cell death and in fact are protected against Fas-induced cell death if stimulated with CD40. This ﬁnding was recently supported by Vaitaitis & Wagner (73), who also showed that Th40 cells not only are protected against Fas-induced cell death but also have increased levels of the antiapoptotic proteins Bcl-XL and cFLIP43 . Combined with the fact that CD40 costimulation increases proliferation of Th40 cells, the upregulation of these antiapoptotic factors might explain the expanded Th40 population in autoimmunity. The relevance of these ﬁndings to human autoimmunity is supported by ﬁndings that patients with T1D have dramatically increased frequency of CD4lo CD40+ T cells in peripheral blood compared with T2D patients or healthy controls (74), and these cells show evidence of reactivity with candidate autoantigens. Another study in humans has recently documented a polymorphism of CD40 that enhances CD40 signaling and is commonly found in subjects of Mexican and South American heritage (75). These ethnic groups are known to be at risk for increased severity of SLE. The relationship of this CD40 polymorphism to clinical outcome in lupus has not been adequately studied. Clearly, these data mandate further experiments and, particularly, the need to integrate functional studies with the genetic data in humans. They also suggest that it will be fruitful to look at other genes that may be involved in the regulation of TNFR and NF-κB pathways, as www.annualreviews.org • Genetics of Human Autoimmunity

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CD154 Genes implicated in human autoimmune disease

CD40

CD40

Lipid raft TRAF1

TRAF2 TRAF2

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BAFF CD154 Bﬂ/A1

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Figure 3 Genes associated with autoimmunity regulate the CD40/NF-κB signaling pathway. The CD40/NF-κB pathway regulates numerous immune-related functions, such as T and B cell proliferation and activation. After association with its ligand, CD40 trimerizes and translocates to lipid rafts, where it interacts with intracellular mediators, including TNF receptor–associated factors (TRAF)1 and 2, which mediate activation of the IκB kinase (IKK) complex. Through phosphorylation, IKK targets the IκB molecule for destruction, which releases NF-κB to translocate into the nucleus and bind to promoter regions of target genes. The NF-κB pathway is regulated by the inhibitor A20 (encoded by TNFAIP3) and by TRAF1, both found to be associated with autoimmune disorders. Interestingly, CD40 has also been detected in the nucleus of B cells, where it interacts with c-rel and initiates transcription of target genes. Both CD40 and c-rel are recently discovered candidate genes for rheumatoid arthritis (RA) (see text).

well as other facets of immune regulation mediated by CD40.

B Cell–Associated Signaling Molecules Despite the prominent abnormalities in autoantibody production, a direct role for primary B cell defects has been difﬁcult to prove for the common human autoimmune diseases. This is 374

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largely because studies in humans must be conducted after disease has developed, and any B cell abnormalities observed may be secondary to the inﬂammatory environment accompanying active disease. Although biologic therapies directed at B cells can be effective in some autoimmune disorders such as RA (76) and MS (77), this does not prove a primary B cell defect. Therefore, it should be gratifying to B cell

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immunologists that genetic associations with B cell–speciﬁc genes are emerging from the current studies. B cell scaffold protein with ankyrin repeats (BANK1) is an adapter protein that was originally identiﬁed in 2002 by Yokoyama et al. (78) These investigators also showed that BANK1 is tyrosine phosphorylated as a result of BCR signaling and appears to regulate calcium mobilization mediated by IP3R. More recently, BANK1 has been implicated in negative regulation of CD40 signaling in B cells. BANK1-knockout animals exhibit normal B cell development but have increased germinal center formation and enhanced responses to T cell–dependent antigens (79). Starting with a GWA study in the Swedish population, Marta Alarcon-Riquelme and colleagues (80) provided convincing evidence for an association of BANK1 with SLE, including several nonsynonymous coding variants and polymorphisms potentially affecting mRNA splicing. However, at this early stage no deﬁnitive causative variants have been clearly identiﬁed, and so far BANK1 has not been implicated in any other autoimmune diseases. A second B cell–speciﬁc gene, B lymphocyte kinase (Blk), has also been associated with risk for SLE (31). This molecule is a member of the src tyrosine kinase family, and little is known about its speciﬁc role in B cell biology. Knockout animals do not display any phenotype, which suggests a redundant function, at least in mice. Such redundancy is not necessarily consistent across species, as demonstrated by the fact that deﬁciency of another B cell tyrosine kinase, Btk, leads to dramatically different phenotypic severity in mice and humans. The genetic associations with Blk have been replicated in a second GWA study in SLE (30), and currently unpublished data suggest that an association with Blk is likely to be present in RA as well (P.K. Gregersen, unpublished data). Blk is one of two possible candidate genes in the region of association, the other being an open reading frame (C8orf13) of unknown function (31). However, the relevant genetic markers are associated with difference in levels of expression

of both of these genes, where the risk haplotype confers lower levels of Blk expression (31). The association of BANK1 and Blk with lupus and possibly other autoimmune disorders raises a number of different mechanistic possibilities. In the case of BANK1, it appears that genetic alterations may affect B-T interactions by virtue of changes in CD40 signaling thresholds. Interestingly, the BANK1 effects on CD40 signaling appear to be primarily mediated through Akt (79), rather than the NF-κB pathways discussed above in the context of TNFR family signaling pathways. In contrast, and consistent with the quantitative changes in expression conferred by the risk alleles, Blk may subtly alter threshold events during early B cell selection. After more genetic studies are carried out to conﬁrm and reﬁne the likely causative variants, the pursuit of these and other hypotheses is clearly in order and may ﬁnally permit the detection of intrinsic B cell functional phenotypes that confer risk for human autoimmunity.

Intracellular Pattern-Recognition Receptors One of the ﬁrst successes to come out of genetic mapping efforts in complex disease was the identiﬁcation of CARD15 (NOD2) as a risk gene for CD in 2001 (5, 6). Following traditional linkage analysis indicating a risk gene on chromosome 16q12 (81), two groups took complementary approaches to gene identiﬁcation, one pursuing traditional ﬁne mapping (5) and the other focusing on CARD15 as a compelling candidate gene in the region (6). The associations with CARD15 are distinct from many of the other genes listed in Table 1 in that the CARD15 risk alleles are relatively uncommon in the normal population (1–5%), and the risk ratios for CD in heterozygotes are in the range of 2–3, with much higher risk ratios (approaching 20) for homozygotes. The low frequency of these alleles suggests that these risk variants arose fairly recently in the European population, and not surprisingly, they are extremely rare in other major ethnic groups. www.annualreviews.org • Genetics of Human Autoimmunity

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CARD15 functions as an intracellular receptor for bacterial-derived peptidoglycans and activates NF-κB pathways in response to these ligands (82). It is expressed in intestinal epithelial cells, as well as in endothelial cells, neutrophils, and monocyte-derived cells such as macrophages and dendritic cells (DCs). Thus, there are potentially both local intestinal and systemic effects of CARD15 genetic variants. The CARD15 risk alleles involve nonsynonymous changes and frameshift mutations involving the leucine-rich repeat in the C-terminal end of the molecule, a region that is required for signaling responses to peptidoglycans (83). Disease-associated mutations alter the response to peptidoglycan stimulation in terms of cytokine production and gene expression patterns (84). However, despite such functional studies, it is not clear exactly what mechanism(s) explains the CARD15-mediated susceptibility to CD. CARD15 alleles also appear to inﬂuence intestinal location of disease (82), and interestingly, there is also evidence that these alleles inﬂuence the severity of graft-versus-host disease in bone marrow transplantation (85). Furthermore, different mutations in the CARD domain of CARD15 are responsible for another inﬂammatory disorder, Blau syndrome, with a Mendelian dominant pattern of inheritance (86). Thus, similar to PTPN22, although the likely causative alleles in CARD15 have been identiﬁed, there is much work to be done to identify the mechanisms by which these alleles cause the various disease phenotypes (82). Interferon-induced with helicase C domain protein (IFIH1) is another example of an intracellular pattern-recognition receptor that appears likely to associate with autoimmunity. This association was ﬁrst identiﬁed in the context of a GWA study in T1D (87) and has been conﬁrmed in this disease (10, 42), with perhaps stronger contributions to risk for GD as well (88). Although there are several other genes in the region of association on chromosome 2q24, a nonsynonymous SNP in IFIH1 is likely to be a causative allele in the region. IFIH1 is one of two well-deﬁned intracellular receptors (the other being RIG-1) for viral dsRNA,

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and these two intracellular receptors recognize distinct but overlapping forms of dsRNA. In addition, deﬁciency of IFIH1 or RIG-1 in vivo leads to susceptibility to infection by different sets of viruses. Thus, on the one hand, this association is consistent with the proposed involvement of viral infection as an environmental risk factor for T1D. On the other hand, this hypothesis has not been advanced for GD, raising the possibility of additional inﬂuences of IFIH1 on the innate immune response.

Transcription Factors: Interferon Regulatory Factors 5 (IRF5) and Signal Transducer and Activator of Transcription 4 (STAT4) The association of SLE with interferon regulatory factor 5 (IRF5) was ﬁrst reported by Sigurdson et al. (89) in the context of a candidate gene study based on evidence that IFN pathways are involved in the pathogenesis of the disease (90). IRF5 is one of nine IRFs that participate in signaling through Toll-like receptors (TLRs) as well as intracellular patternrecognition receptors such as RIG-1 and IFIH1 (91). IRF5 is required for induction of inﬂammatory responses upon triggering through TLR4, 7, and 9 (92). In the context of lupus, TLR7 and TLR9 are of particular interest because their ligands are composed of nucleic acids that are found in the (ribo)nucleoprotein autoantigens that are likely to be driving the disease. The association of lupus with IRF5 has now been widely replicated and reﬁned (30, 31, 93– 95), with several causative alleles identiﬁed that regulate both splicing and levels of expression of the IRF5 gene (94). The IRF5 associations with lupus also hold up in populations of nonEuropean ancestry (96, 97). Complementing these genetic results, multiple studies have now shown that increased expression of type 1 IFN– regulated genes are characteristic of the lupus phenotype (98), driven at least in part by self-antigens triggering DCs or other antigenpresenting cells through TLR and Fc receptors (90, 99, 100). Furthermore, IFN-α levels are

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heritable (101), and the IRF5 risk haplotype is associated with high serum IFN activity (102). Thus, IRF5 is an example of a genetic discovery that has substantially accelerated progress in an area of disease investigation that was already ongoing, in particular providing support that IFN dysregulation is not simply a secondary abnormality caused by disease activity. Reports of IRF5 associations with other autoimmune diseases, such as ulcerative colitis (103) and perhaps RA in some populations (104), have raised the possibility that this gene may be a general risk factor for autoimmunity. However, these associations need further conﬁrmation in additional populations. Signal transducer and activator of transcription 4 (STAT4) was ﬁrst shown to be associated with both RA and SLE by Remmers et al. (7). This ﬁnding was driven by prior data in RA showing evidence of linkage to a region on chromosome 2q (105). Gene identiﬁcation was carried out using a positional ﬁne mapping approach to the region. Interestingly, the GWA scans in RA have not pointed strongly to STAT4 as a risk gene, which undoubtedly relates to the very modest odds ratios for this association [odds ratio (OR) ∼1.25]. In contrast, the odds ratios for STAT4 associations with lupus are considerably stronger, and thus STAT4 emerges as a prominent association signal in GWA scans in SLE (30, 31). Intriguingly, STAT4 is particularly strongly associated with certain phenotypic subsets of SLE, especially the presence of high titer anti-dsDNA antibody and renal disease (106). The STAT4 associations with both RA and SLE are also observed in Asian populations (107, 108), thus conﬁrming STAT4 as an important common risk gene for these two diseases. STAT4 is also associated ¨ with Sjogren’s syndrome (109); it seems likely that STAT4 is involved in other autoimmune disorders as well, although deﬁnitive studies have not yet been published. Fine mapping and resequencing of the STAT4 risk haplotype continue to support the view that the causative alleles are likely to be located within the third intron of this gene (E.F. Remmers, unpublished data). The risk haplo-

type is common in the Caucasian populations, with a frequency of ∼0.22, and this background population frequency is quite stable across various European subpopulations. The intronic location of the associated SNPs is consistent with functional changes in either the splice patterns or levels of expression. STAT4 is a member of a family of transcription factors of which there are six main members, each with distinct roles in cytokine receptor signaling (110). STAT4 is a key molecule for IL-12 signaling in T cells and NK cells, leading to the production of IFNγ and differentiation of CD4 T cells into a Th1 phenotype (111). Upon IL-12R binding by IL12, STAT4 is phosphorylated and forms homodimers. These homodimers are translocated to the nucleus, where they initiate transcription of STAT4 target genes, including IFN-γ (112). Thus, STAT4−/− mice do not respond to IL-12, lack Th1 responses, and have a predominantly Th2 immune response phenotype (113). Relatively little is known about how the expression of STAT4 itself is regulated at the transcriptional level. STAT4 is expressed in resting CD4+ T cells and NK cells and in Jurkat cells. STAT4 transcription is regulated in part by Ikaros, a zinc ﬁnger transcription factor involved in hematopoietic cell differentiation (114). In contrast, STAT4 is not highly expressed by monocytes or immature DCs but can be induced upon activation and maturation (115). In the case of DCs, NF-κB/Rel proteins upregulate STAT4 transcription during the differentiation into mature human DC in response to LPS, CD40 stimulation, or other activators (116). In this case, the induction of STAT4 transcription in these cells is dependent on the combination of TNF-α and IL-1β. The published work on STAT4 transcriptional regulation is focused on the promoter region 5 to the gene (116), and there is no information concerning the potential role of intronic regions in the regulation of STAT4. Speciﬁcally, the SNPs that are associated with RA and SLE are over 50 kb distant from the 5 promoter region and show no evidence of LD with SNPs in the promoter region. It is nevertheless intriguing that a recent www.annualreviews.org • Genetics of Human Autoimmunity

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study reports that a different level of expression of STAT4 in osteoblasts, but not in T cells, is correlated with the STAT4 risk haplotype (95). Thus, intronic variation in STAT4 may inﬂuence cell type–speciﬁc gene expression through mechanisms that are yet to be deﬁned. Interestingly, Balb/c mice demonstrate signiﬁcant differences in STAT4 expression from other strains (117), but this difference appears to be restricted to macrophages, again emphasizing the importance of examining the relevant cell type when attempting to correlate changes in expression with genotype. Targeting STAT4 by inhibitory oligodeoxynucleotides or antisense oligonucleotides results in suppression of the disease in arthritis models (118), and STAT4-knockout mice are highly resistant to the induction of proteoglycan-induced arthritis. In RA patients, the high expression of STAT4 in DCs in the synovium disappears after treatment with disease-modifying antirheumatic drugs (119, 120). These studies and the association of STAT4 with RA suggest that STAT4 might be a potential therapeutic target. Several other transcription factors are emerging as important susceptibility genes for autoimmunity. The NKX2-3 is a member of a family of homeodomain containing transcription factors, with a conﬁrmed association with CD (10, 15). Mice lacking NKX2-3 are either asplenic or have reduced spleen size, along with a block in formation of Peyer’s patches (121); NKX2-3 appears to regulate the differentiation of a ﬁbroblast component of the splenic stromal architecture (122). The mechanism for the CD association is unknown. STAT3 is also associated with CD (15), which is consistent with the likely involvement of cytokine pathways such as IL-23 in this disease, as discussed below.

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Cytokines and Cytokine Receptors One of the ﬁrst major associations to result from a GWA study of autoimmunity is the association of CD with IL-23 receptor (123). This association has now been conﬁrmed (10, 15) 378

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and extended to psoriasis (124) and ankylosing spondylitis (AS) (125). These three disorders exhibit familial aggregation with one another, and thus a common genetic association among them is not unexpected. The IL-23 receptor is composed of two subunits (p19 and p40), one of which, p40 (IL-12Rb1), is shared with the IL-12 receptor (composed of a p35 and p40 heterodimer). The IL-23 receptor–speciﬁc p19 subunit contains a nonsynonymous amino acid change, Arg381Gln, in which the Gln residue provides signiﬁcant protection against CD. The functional signiﬁcance of this change has not been elucidated, and it is likely that additional variants in the p19 molecule also contribute to disease risk, perhaps as a result of splicing or regulatory effects on p19 expression. Furthermore, IL-12RB1 (p40) is located just 3 to the IL-23R (p19) locus. Although there is no direct evidence for genetic associations with p40, regulatory inﬂuences on p40 may result from polymorphisms in the IL-23R (p19) gene. The additional association of both CD (15) and psoriasis (124, 126) with the β subunit of the IL-12 cytokine itself (IL-12B) adds to evidence that the balance in activity of IL-23 and IL-12 cytokine pathways is an important component of disease pathogenesis in these disorders. This genetic evidence for the importance of IL-12 and IL-23 pathways in CD is supported by experimental studies in animal disease models, as well as by recent data on the role of these cytokines in Th17 cell differentiation (127). Both IL-12 and IL-23 are upregulated in the disease, and biologic therapies that block IL-12/IL-23 pathways are emerging as effective therapies (128). In addition to inducing widespread inﬂammation and colitis (129), IL-23 injection into skin produces psoriaticlike lesions in experimental animals (130), and blockade of IL-12/IL-23 is also showing promise in the clinic as a treatment modality for psoriasis (131). Thus, the biology and genetics of IL-12 and IL-23 pathways are coming together in a way that is likely to alter profoundly the understanding of disease pathogenesis as well as the treatment of these disorders.

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However, much work needs to be done to fully understand the complexity of the genetic factors, and it remains to be seen whether the identiﬁcation of genetic subgroups of disease will actually impact the selection of therapy and management of individual patients. It will also be of great interest to see how the genetic ﬁndings will improve understanding of AS, a disease whose pathogenesis has an intriguing but still obscure relationship to intestinal abnormalities (132). A role for IL-2 and IL-2R in susceptibility to autoimmunity has also been reported, although in general the evidence is less clear than for IL-12 and IL-23 pathways. A region on chromosome 4q27 has been unequivocally associated with Celiac disease (133), and there is evidence that this association extends to other autoimmune disorders including T1D (10, 42, 134), GD (42), and RA (10, 134). Associations with psoriasis have also been suggested (135). However, this region contains four genes in strong LD: KIAA1109-Tenr-IL-2-IL-21. The ﬁrst two of these genes have no known relationship to immune function, although both IL-2 and IL-21 are clearly of interest, especially because IL-2 is a risk gene for T1D in the nonobese diabetic mouse (136). Despite resequencing efforts at the IL-2 and IL-21 locus, no obviously functional variants have been identiﬁed (42). Additional support for a role for IL-2 comes from the fact that allelic variation in the genetic region encoding the IL-2 receptor is also clearly associated with T1D (10, 137, 138), again with some evidence for association with GD (139), RA (10), and MS (140, 141). Resequencing efforts have failed to identify clearly the causative variants in the IL-2RA gene (138), despite being guided by information on the regulatory regions that control the expression of this gene (142). However, risk haplotypes have been correlated with circulating levels of soluble IL-2R (138). Given the obvious functional importance for IL-2 in the growth and differentiation of T cells (142), including regulatory T cells (143, 144), it seems likely that IL-2 and/or IL-2R receptor polymorphisms will ultimately be shown to have a direct causative role in

disease susceptibility for multiple autoimmune disorders. Finally, recent genetic studies in MS have focused attention on the gene encoding the IL-7 receptor as a new risk locus for this disease (140, 145). A functional SNP that inﬂuences splicing and expression has been identiﬁed (145), although this ﬁnding has not yet been translated into a clear causal relationship to disease pathogenesis. IL-7 appears to play a complex role in T cell development and peripheral T cell homeostasis (146). Interestingly, some evidence for an association of IL-7R with T1D has also been reported (42).

Costimulatory Molecules and Other Cell Surface Receptors The cytotoxic T lymphocyte antigen 4 (CTLA4) associations with T1D and autoimmune thyroid disease provided one of the ﬁrst convincing demonstrations that genetic variation in costimulatory molecules is involved in disease susceptibility (147). These associations with T1D have been conﬁrmed in recent GWA scans (10), and a role for CTLA4 in human autoimmunity is widely accepted (148). As with many other associations, the strength of these associations is modest, ranging from OR ∼1.15 in T1D to OR ∼1.5 in GD. Although the exact mechanisms and causative alleles remain to be proven, these genetic advances have been accompanied by the development of biological therapies targeting CTLA4, and their efﬁcacy provides further conﬁrmation of the importance of this pathway. Other cell surface molecules with negative regulatory effects on T cells, such as PDCD1, have also been associated with autoimmunity, in particular SLE (149). However, the strength and reproducibility of these ﬁndings across different populations is still being deﬁned. One of the more intriguing and robust associations to emerge from the recent GWA scans in SLE is with integrin αM (ITGAM or CD11b) (30, 31). ITGAM has also been the focus of extensive candidate gene analyses, driven by the results of previous linkage www.annualreviews.org • Genetics of Human Autoimmunity

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studies (150). Thus, like STAT4 and CARD15, this demonstrates that linkage information can lead to gene identiﬁcation even in a genetically complex disease. It is highly likely that the major causative allele in ITGAM is a nonsynonymous amino acid change in the extracellular portion of the molecule. ITGAM encodes the α chain of the αM β2 integrin known variously as Mac1, CR3, or CD11b/CD18. This molecule binds a range of different ligands including ICAM1 and ICAM2, certain complement components (C3bi), ﬁbrinogen, and GPIbα (150). It is unclear which of these functions explains the associations with lupus, although changes in neutrophil expression of ITGAM (151) have been described, and abnormalities of immune complex clearance are characteristic of the disease. Indeed, Fc receptor polymorphisms have long been associated with SLE (152, 153), and genetic evidence for involvement of other genes in this family have recently been reported (154). These new genetic ﬁndings will surely catalyze a renewed effort to understand the details of these pathways in SLE (155).

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Genes Involved in Autophagy One of the more unexpected ﬁndings to emerge from the recent GWA scans has been the identiﬁcation of genes that function in autophagy as risk factors for CD—an example of how genetic ﬁndings can catalyze new perspectives on disease pathogenesis. Autophagy is a phylogenetically ancient mechanism by which the cell can degrade and dispose of intercellular constituents in a regulated manner, and also provides a way of disposing of intracellular infectious agents without destroying the cell itself (156). Two genes in the autophagy pathway, ATG16L1 (157, 158) and IRGM (10, 159), have been associated with CD. In the case of ATG16L1, a potentially causative nonsynonymous variant (A197T) has been associated with disease, and an upstream insertion deletion polymorphism in IRGM that is associated with disease has recently been shown to affect expression (160). It was further shown that expression levels of IRGM have an inﬂuence on 380

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the efﬁciency of antibacterial autophagy (160). Because the host response to intestinal bacteria is important for the pathogenesis of CD, a role for autophagy in this process is clearly a leading hypothesis to explain these genetic associations. However, autophagy may also play a role in other aspects of the immune response, particularly with regard to the antigen presentation (161), including within thymic epithelium, where high levels of autophagy have been observed (162).

Genes with Novel Enzymatic Functions Two novel associations of genes with enzymatic functions have emerged from recent genetic data. One of these, PADI4, is a member of a family of peptidyl arginine deiminases encoded on chromosome 1p35-36 (163). The function of these enzymes is the conversion of arginine residues to citrulline in mature proteins, and such posttranslational modiﬁcation appears to be important for a variety of structural molecules, such as keratin, histones, and myelin basic protein. Citrullination also accompanies inﬂammation, and in the case of RA, local citrullination of ﬁbrin and other proteins is observed in the inﬂamed synovium (164). Over the past decade, it has become apparent that antibodies to citrullinated peptides are quite speciﬁc to RA, and therefore the discovery in 2003 of an association between PADI4 and RA emphasized the importance of citrullinated antigens in this disease (165), and also contributed to the current view that anticitrulline antibodies deﬁne a distinct subset of RA. Interestingly, the associations with RA of PADI4 have only been convincingly replicated in Asian populations (166), and the causative alleles have not been deﬁned. Although there are hints of association in non-Asian populations, a role for PADI4 in disease risk in Caucasians has been difﬁcult to demonstrate (167). This difﬁculty may relate to population differences in environmental exposures, such as smoking, that are risk factors for the development of anticitrulline antibodies (168). Thus, the PADI4 associations

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with RA offer an opportunity to explore geneenvironment interactions in the setting of an autoimmune disease. A second enzyme of interest is the aminopeptidase regulator of TNFR1 shedding 1 (ARTS1). As implied by the name, ARTS1 has a role in the cleavage of cell surface receptors for proinﬂammatory cytokines including IL-1R2 and IL-6Ra, as well as TNFR1. Genetic alterations that affect cell surface receptor shedding are clearly important for susceptibility to inﬂammatory disease, as demonstrated by the TNF receptor–associated periodic syndrome (TRAPS) (169), a Mendelian disorder associated with defects in receptor shedding [see review by Masters et al. (3), this volume]. Thus, defects in ARTS1 cleavage functions could have proinﬂammatory effects. Interestingly, ARTS1 is also active in the endoplasmic reticulum and is involved in trimming peptides for MHC class I presentation. Given the strong associations of AS with HLA-B27, this is another potential explanation for the involvement of ARTS1 in risk for AS.

Autoantigens as Susceptibility Genes The discovery over a decade ago that regulatory polymorphisms in the insulin gene are associated with T1D (170, 171) has fostered the view that alterations in tolerance for speciﬁc autoantigens may underlie susceptibility to autoimmune disease. In the case of T1D, this is further supported by the demonstration of insulin-speciﬁc T cells in the pancreas of affected individuals (172), and by the fact that autoantigens under the control of the Aire gene are presented to developing thymocytes during thymic selection (173, 174). Furthermore, there is considerable variation of thymic expression of potential tissue autoantigens among individuals (175), obviously raising the possibility of genetic regulation of these phenotypes. This has led to the exploration of autoantigens as candidate genes for genetic association. Other than insulin, the only compelling genetic support for this hypothesis in humans is the association of the TSH receptor (TSHR) with GD

(125, 176, 177). Interestingly, even comprehensive genome-wide SNP screens may miss such associations for technical reasons, as was the case for insulin in the Wellcome Trust study (10). Thus, it seems likely that further examples of risk alleles in autoantigens will emerge in the future.

The Major Histocompatibility Complex The MHC is the predominant genetic region of importance for many autoimmune disorders; a basic unresolved issue is still the precise role of the various associated HLA alleles in disease pathogenesis. However, the recent availability of dense SNP marker sets that span the MHC has also revealed evidence that loci in addition to the well-established HLA class II associations are relevant in several autoimmune diseases. For example, in a recent detailed analysis in T1D, risk is conferred by the HLA-B locus (178), in addition to the known class II risk alleles. By performing analyses that condition upon the known risk alleles, similar evidence of genetic complexity has emerged for other disorders such as RA (179, 180), MS (181), and myasthenia gravis (182). Because of the extensive LD of some common autoimmune risk haplotypes, such as A1-B8-DR3, it will be challenging to fully dissect these issues using a purely genetic approach. Deep resequencing will likely be required, along with functional studies on newly deﬁned candidate polymorphisms. Thus, the current data suggest that the MHC still contains valuable genetic insights that have yet to be fully mined.

Copy Number Variation and Rare Genetic Variants: The Next Frontier This review has focused on the recent genetic associations of autoimmunity with common genetic variants, generally SNPs. However, it is now apparent that variation in copy number of genes or DNA sequences is an extremely common form of genetic difference among individuals in the population (183, 184). Some of www.annualreviews.org • Genetics of Human Autoimmunity

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these have been associated with autoimmunity (154), but we are still in the early stages of establishing the overall extent of this variability. In addition, it is now clear that copy number change can arise de novo (185), and this may explain the sporadic appearance of complex disorders such as autism (186) and schizophrenia (187, 188). The extent to which this type of somatic genetic change contributes to autoimmunity is unknown, but it is striking that even twins commonly exhibit copy number differences between them (189). Could this contribute to the relatively high rate of discordance for autoimmunity among monozygotic twin pairs? Finally, the contribution of multiple rare variants to autoimmune phenotypes must be considered. A compelling recent example involves the association of multiple rare alleles in TREX1 with lupus (190). TREX1 is a DNA exonuclease that was originally implicated in a Mendelian disorder, Aicardi-Goutieres syndrome (AGS1) that is accompanied by some clinical manifestations reminiscent of lupus (191). Multiple rare variants in TREX1 are associated with sporadic lupus, several with distinct functional consequences for cellular localization of this enzyme (190). Beyond the fas-

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cinating biology of this ﬁnding, TREX1 is a beautiful example of how multiple rare alleles in unexpected places can contribute to autoimmunity. It is highly likely that more such associations will be discovered as the technology for resequencing large sections of the genome becomes more ﬁnancially accessible.

CONCLUSION In this review, we have attempted to give a broad overview of the fast moving ﬁeld of human genetics as it applies to the problem of human autoimmunity. It is virtually certain that by the time of publication numerous additional risk genes will be identiﬁed and that some genetic associations that we have chosen to pass over in discussion will be revealed as critically important. For example, we have not discussed various genes of unknown function, some of which have compelling associations with disease (192). However, we think it unlikely that any of the genes discussed here will be shown to be false positives. Thus, although the details will change, it should be quite obvious that the recent outpouring of genetic data is producing a rich harvest of new avenues for investigation.

DISCLOSURE STATEMENT P.K.G. reports stock ownership in Genentech, Illumina, and Amgen. P.K.G. received consulting fees of less than $10,000 from Roche Pharmaceuticals in 2008. L.O. is not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS This work was supported by grants to P.K.G. from the National Institutes of Health (AI068759, AR44422, AR12256, AR72232), the National Arthritis Foundation, and the American College of Rheumatology. L.O. and P.K.G. are also supported by the Eileen Ludwig Greenland Center for Rheumatoid Arthritis and the Laurie Strauss Leukemia Foundation. LITERATURE CITED 1. Frazer KA, Ballinger DG, Cox DR, Hinds DA, Stuve LL, et al. 2007. A second generation human haplotype map of over 3.1 million SNPs. Nature 449:851–61 2. Iles MM. 2008. What can genome-wide association studies tell us about the genetics of common disease? PLoS Genet. 4:e33 382

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Cell-Mediated Immune Responses in Tuberculosis Andrea M. Cooper Trudeau Institute, Saranac Lake, New York 12983; email: [email protected]

Annu. Rev. Immunol. 2009. 27:393–422

Key Words

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

T cell, B cell, phagocyte, regulation, memory, inﬂammation

This article’s doi: 10.1146/annurev.immunol.021908.132703

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0066-4154/09/0707-0393$20.00

Tuberculosis is primarily a disease of the lung, and dissemination of the disease depends on productive infection of this critical organ. Upon aerosol infection with Mycobacterium tuberculosis (Mtb), the acquired cellular immune response is slow to be induced and to be expressed within the lung. This slowness allows infection to become well established; thus, the acquired response is expressed in an inﬂammatory site that has been initiated and modulated by the bacterium. Mtb has a variety of surface molecules that interact with the innate response, and this interaction along with the autoregulation of the immune response by several mechanisms results in less-than-optimal control of bacterial growth. To improve current vaccine strategies, we must understand the factors that mediate induction, expression, and regulation of the immune response in the lung. We must also determine how to induce both known and novel immunoprotective responses without inducing immunopathologic consequences.

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INTRODUCTION

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Immunity: the ability to stop or signiﬁcantly limit a pathogen from causing disease Acquired cellular immune response: the adaptive response of the vertebrate immune system that results in the development of antigen-speciﬁc lymphocytes and thereby in the enhanced speciﬁcity of the effector mechanisms of the system

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The pathogenesis of tuberculosis is the product of the interaction between bacterial virulence and host resistance, which are two distinct and independent variables. Although we have long understood this aspect of tuberculosis pathogenesis (1), modern tools have allowed us to deﬁne the role of speciﬁc bacterial and host components to an unprecedented degree. The desired end point of these efforts is to identify virulence factors and drug targets within the bacterium as well identify components of the host’s immune system that can be augmented and indeed altered by vaccination. In this review, I touch only lightly on the classic literature of the early modern period, as this work has been eloquently reviewed in two Annual Review of Immunology articles (2, 3). I focus on the relatively recent literature, which has beneﬁted from the maturation of available tools that has taken place since publication of the mouse and bacterial genome (4, 5). The use of these tools and the ability to genetically alter the bacterium (6, 7) have resulted in an increased ability to manipulate the disease model in a directed manner. Tuberculosis continues to have a detrimental impact on public health worldwide. Indeed, with approximately one-third of the world’s population exposed, the 5% of those exposed who eventually develop disease translates into 8 million new cases per year (8). When we consider the ability of HIV infection to reduce immunity to Mycobacterium tuberculosis (Mtb) infection, the consequences for spread of both drug-sensitive and drug-resistant tuberculosis are daunting (9, 10). Furthermore, while the disease’s public health impact is enormous and warrants the high level of interest, the disease and the immunopathologic lesions it evokes have also fascinated immunologists since the birth of the discipline. Perhaps the key reason for immunologist’s interest is that both immunity and pathogenesis are mediated by the lymphocyte response to mycobacterial infection. Thus, while the absence of an acquired cellular immune response leads to limited or no immunity, the ab-

Cooper

sence of this response also limits the classical caseation associated with pathogen transmission. This statement is perhaps best supported by considering the consequences of HIV infection on the development of tuberculosis. Tuberculosis is an index disease for HIVinfected individuals and develops when CD4 numbers are still much higher than numbers indicating a predisposition to other opportunistic infections (11). However, when we assess the immunopathologic consequences of Mtb infection in AIDS patients, there is a much altered disease state (11) and an altered inﬂammatory response. Speciﬁcally, there is a dominant granulocytic inﬁltrate and necrosis but not the typical caseous necrosis seen in non-HIV-infected tuberculosis granulomas (12). We also see this strong tendency to granulocytic involvement in the mouse model, in which the CD4 molecule is genetically disrupted (13). The acquired cellular response, as represented largely by CD4 T cells, therefore provides protective immunity while also promoting the development of mononuclear lesions and the caseous necrosis required for transmission. The duality of the role of the acquired cellular response leads to the apparently contradictory presence of a strong cellular immune response at the site of unresolving disease. The most important aspect of the acquired cellular response is the rapidity with which it is expressed. If the response is too slow, bacteria grow and reach a point where although a potentially protective response is being expressed, the environment is such that it is not effective. In this same vein, it is clear that dose plays a role in the ability of the host to control bacteria. Speciﬁcally, if one is infected by too high a dose, then the local bacterial burden may reach a level that interferes with the efﬁcient expression of protective immunity. These ideas were brought together eloquently by Rich (1) using the lung histopathology from patients in the predrug era to describe the natural history of the disease. He suggested that the acquired cellular response could control bacterial growth but that it failed to do so in the face of high numbers of bacteria. To support this idea, he observed that within

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the same patient large lesions tend to progress while small ones are restrained in their growth. Furthermore, the nature of metastatic lesions was different from the primary lesion in that they are generally circumscribed and bacterial growth is controlled. Finally, he reported that the large number of bacteria that arrive at a new site as a result of aspirating large primary lesions usually results in a sizeable progressive lesion. This interpretation predates our understanding of much of the acquired cellular immune response but supports the importance of the kinetics of the response, the importance of the environment within which the response must occur, and the potential for regulation of the response by either the bacteria or the acquired response itself. The importance of lymphocytes in controlling tuberculosis was underappreciated in early work as, although these cells were clearly present in lesions, their function was unknown. It was early mouse model work that demonstrated that T cells were required for antituberculous immunity in systemic (14, 15) and aerosol (16) challenge models. That CD4 T cells were the primary mediators of antituberculous immunity was shown as a result of transfer models and then later by the use of gene-deﬁcient mice (17, 18). Furthermore, gene-deﬁcient mice demonstrated the importance of cytokine-mediated macrophage activation in the control of bacterial growth (19, 20). The relevance of these studies to the human condition was demonstrated by the observation that HIV-mediated loss of CD4 T cells rendered patients susceptible to tuberculosis (11) and that people genetically deﬁcient for the cytokine-mediated macrophage activation pathway were also susceptible to tuberculosis (21). Although we have made signiﬁcant progress in understanding what lymphocytes do during both primary and recall responses to mycobacterial infection, the vaccine currently in use, a derivative of Mycobacterium bovis called bacille Calmette-Gu´erin (BCG), was ﬁrst developed in 1921 when we had no idea what lymphocytes do. This early vaccine is clearly effective

for limiting disseminated childhood tuberculosis (22), but its efﬁcacy is less obvious for pulmonary disease (23), and we are still limited in the rational design of vaccines by our lack of understanding of either the primary or the recall acquired cellular response to tuberculosis. To make signiﬁcant progress in vaccine development, we need to understand the interaction between the acquired cellular immune response and the pathogen from the beginning of infection throughout the disease process. Several important issues must be addressed: Is the acquired cellular response that is expressed upon infection truly optimal? To what extent is it modulated by the pathogen? What types of effector cells are activated by infection? Is each subtype required for immunity, or does it act to mediate pathologic or regulatory consequences? Furthermore, to what extent do lymphocytes induced by infection modulate the function of other cells and to what extent does the pathogen modulate the function of host cells? Indeed, we need to know how much the inﬂammatory site itself affects the ability of effector cells to mediate their protective function. Finally, we need to identify antibacterial activities that are not induced during natural infection but that can be augmented by vaccination and that do not lead to adverse pathologic consequences. This list of objectives is somewhat daunting, but the ﬁeld has made dramatic progress recently, and in light of new tools and approaches further progress should result in signiﬁcant breakthroughs (24).

INITIATION OF THE T CELL RESPONSE The initiation of the T cell response following mycobacterial infection has been studied indirectly for many years. The ﬁrst modern analysis (17) demonstrated that lymphocytes induced by systemic infection were capable of mediating protection upon transfer by day 5 post primary infection. This measure of protective function was seen ﬁrst in L3T4 (CD4) T cells and later in Ly2 (CD8) T cells, and both populations could protect against aerosol challenge. The www.annualreviews.org • Cellular Immunity to Tuberculosis

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Inflammation: the physiological process by which the body responds (in this case) to an invading and persistent pathogen, including acute inﬂammation associated with activation of infected tissue macrophages and chronic inﬂammation associated with mononuclear cell accumulation

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protective ability of cells produced in the ﬁrst 25 days post challenge was sensitive to the effects of cyclophosphamide, indicating that these were dividing cells and were likely what is now termed effector T cells (17). Later studies used the effector function of antigen-speciﬁc cells as an indicator of the initiation of the cellular response and demonstrated that dissemination of bacteria to the draining lymph node occurred following aerosol infection (25). Chackerian and colleagues (25) showed that the draining lymph node was the ﬁrst site of expression of effector function, followed by the spleen and the lung. They took the temporal correlation between lymphocyte activation and bacterial arrival in the lymph node to mean that acquired cellular immunity was initiated in the draining lymph node by bacteria disseminating from the lungs via the lymphatic drainage and that further dissemination occurred systemically thereafter (25). This paper also highlighted the importance of accelerated kinetics in the induction of protective acquired cellular immune responses. Speciﬁcally, the dissemination of the bacteria from the lung and the induction of effector cell responses for the resistant B6 mouse were compared with the susceptible C3H mouse. The resistant strain had equivalent bacterial growth in the lung compared with the susceptible strain, but the dissemination and induction of cellular responses were accelerated in this strain compared with the susceptible strain (25). Thus, the slow induction of the immune response was clearly detrimental to the ultimate success of the response. Although Chackerian and colleagues (25) demonstrated that induction of effector function required dissemination of bacteria from the lung to the draining lymph node, they did not directly address the issue of naive T cell activation. This is an important issue, as the mechanism resulting in the slow expression of effector function in the antigen-speciﬁc lymphocyte population was not deﬁned. One hypothesis to explain this slow induction is that Mtb exerts a regulatory activity via either inhibition of migratory activity or by modulation of antigenpresenting cell (APC) function. Alternatively, Cooper

slow induction could simply be a consequence of the low number of bacteria delivered to an organ that has a limited ability to initiate cellular responses and that the delay is due to lack of inﬂammation and antigen. To address this issue, researchers needed to determine when and where sufﬁcient antigen is presented to initiate activation of naive T cells. For this, one needs T cell receptor (TCR) transgenic mice, in which naive T cells express a TCR speciﬁc for Mtb antigens that are expressed early in infection. To this end, two groups have recently reported data that strongly support the conclusion of Chackerian and colleagues that antigen-speciﬁc T cells are ﬁrst activated within the draining lymph node of the lung and that this activation occurs only after a delay. In one study, proliferation of antigen-speciﬁc naive cells was examined and found to occur ﬁrst in the draining lymph node and after the dissemination of bacteria to this organ (26). Proliferation is a downstream effect of naive T cell activation, however, and to detect the ﬁrst events in activation, a second paper examined the induction of CD69 on naive antigen-speciﬁc T cells. In this latter study, CD69 expression was seen on T cells in the draining node ﬁrst and after the arrival of bacteria in this organ (27). Thus, by any measure of naive T cell activation, it is clear that no T cell activation occurs within the host before day 9 following aerosol infection and that the lung draining lymph node is the ﬁrst place where it takes place. In all studies, the response was coincident with the arrival of viable bacteria in the node, demonstrating that dissemination is likely essential to priming of the response (Figure 1). Why then is the dissemination so slow? In all three of the above models, the bacteria was delivered to the lung via an aerosol cloud containing particles of a size that can pass through the lower bronchioles and be deposited in the alveolar space. Once in this location, the cells most likely infect alveolar macrophages, although this is impossible to test owing to the small number of events. The deposition of bacteria in the alveolar space places it in the lung parenchyma rather than the mucosal tissue of the airways, and there are distinct differences in

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8 – 9 days post-infection

Once bacteria arrive in draining lymph node, naive T cells are activated, proliferate, and become eﬀector cells

Bacteria DC Airway mucosa

Dendritic cells sampling the mucosal airway respond rapidly to pathogen signals and migrate to draining lymph node

Lymph node Eﬀector T cells

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DC Dendritic cells sampling the alveolar tissue do not respond rapidly to pathogen signals; mycobacteria are not rapidly transported to the draining lymph node

Lung

Availability of speciﬁc cytokines will deﬁne the phenotype of the eﬀector T cells

DC

Alveolar tissue

Eﬀector T cells migrate from the lymph node through circulation and are attracted to the lung tissue by inﬂammation

Infected phagocyte Antigen-speciﬁc eﬀector T cells remain in inﬂammatory lesion and mediate protection

18 – 20 days post-infection Figure 1 Low-dose aerosol infection, which approximates the natural delivery route for induction of tuberculosis, results in low numbers of Mtb (red ) being deposited in the lower airways and the alveolar tissue. Bacteria do not disseminate from the lung until 9 days post infection, when they can be detected in the draining lymph nodes. This dissemination coincides with the ﬁrst activation of naive T cells ( purple). The fact that bacteria do not disseminate rapidly suggests that they either inhibit migration of dendritic cells or that the cells that they infect cannot migrate readily to the lymph node. Activation of naive T cells occurs in the presence of live bacteria, and effector cells develop with expected kinetics. The effector cell phenotype will depend on the availability of speciﬁc cytokines. These effector cells migrate to the lung in response to inﬂammation and mediate protection by activating infected phagocytes ( pale red ). The response takes 18–20 days to reach an effective level and thereby to stop bacterial growth.

the phenotype and function of the cells encountered in these two compartments of the lung (28). The events that occur following initial infection with a low dose of bacteria have not been deﬁned; however, after nine days and substantial expansion of the population, the bacteria

enter the lymphatic drainage, when they are ﬁrst detected in the draining lymph node (25– 27). Before this, even the delivery of an external inﬂammatory mediator, which should mobilize any infected cells to the lymph node, fails to accelerate the dissemination of bacteria or the www.annualreviews.org • Cellular Immunity to Tuberculosis

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initiation of immunity (26). Although a more than tenfold increase in dose can accelerate dissemination, it will accelerate by only one to two days, suggesting that where the bacteria are deposited is a key contributor to the slowness of the response (27) (Figure 1). The involvement of the dendritic cell (DC) in the migration of bacteria to the lymph node is assumed to be essential to initiation of the response. Therefore, the different functionality of these cells in the airways compared with the alveolar areas may deﬁne the kinetics of the response. Mtb-infected DCs delivered to the lung via the intratracheal route are capable of migrating to the draining lymph node and initiating cellular responses (29–31). It is unlikely, however, that these cells are deposited in the alveolar space, and indeed we do not know if they represent the functional aspects of the DCs that occupy this unique environment. Conducting airway DCs are considered to be active samplers of the mucosal environment and to migrate readily to the draining node (32, 33). In contrast, DCs within the alveolar tissue are in a regulated environment with surfactant protein and alveolar macrophages that may limit their ability to respond to infection and to migrate to the lymph node as rapidly as would DCs in the airway tissue (28) (Figure 1). The delay in dissemination of bacteria and the initiation of the cellular response may therefore reﬂect the infection of alveolar phagocytes (both macrophages and DCs) that are resistant to migration and, indeed, to activation. This resistance to migration and activation is likely crucial to the maintenance of the alveolar space as an air exchange surface as opposed to an inﬂammatory site. The slow initiation of the acquired immune response by Mtb may therefore be a product of the slow growth of this bacteria and of the relatively immunoprivileged nature of the alveolar tissue.

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DEVELOPMENT OF CELLULAR RESPONSES DCs are currently considered to be the most efﬁcient inducers of activation in naive T cells. 398

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This efﬁciency stems from the fact that they provide not only the antigen-speciﬁc stimulus but also secondary and tertiary signals that promote efﬁcient development of effector T cells. The effect of mycobacterial infection on DC function has been studied extensively. Indeed, the classic demonstration that immature DCs can phagocytose particles and become efﬁcient APCs used BCG as the maturation agent (34). More recently, however, the ability of Mtb to interfere with T cell stimulation has been suggested by the fact that DCs infected in vivo are less efﬁcient at stimulating antigen-speciﬁc T cells than are equivalent uninfected DCs (35). As discussed above, once bacteria reach the draining lymph node, initiation of naive T cell activation occurs. Whether this is as a result of direct interaction between lung-derived bacteria-infected DCs has not been deﬁnitively proven. However, in a model system in which antigen-pulsed DCs are delivered to the lung intratracheally, those DCs exposed to Mtb before delivery are clearly better at migrating to the draining node and initiating T cell activation (29–31). Furthermore, whereas IL-12p40 promotes this migration, IL-10 may limit it (29, 31, 36). These data suggest that simple exposure to Mtb is not sufﬁcient to limit the ability of an infected DC to initiate T cell activation. Infected lung DCs migrate from the lung to the draining lymph node, but this migration is difﬁcult to see before day 14 of infection, and this is after the initial migration and initiation of T cell activation (35). Despite our inability to deﬁne the exact population delivering the bacteria to the lymph node, bacteria clearly are in the lymph node when activation of naive T cells occurs, and therefore the bacteria could inhibit the process of activation once initiated. Importantly, however, as the naive T cells become activated, they proliferate, upregulate CD44, downregulate CD62L, and begin to accumulate in the lung with the expected kinetics (27) (Figure 1). That this activation occurs with the expected kinetics indicates that the simple presence of the bacteria in the lymph node does not affect initial T cell activation; we do not know, however, whether the expansion

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and survival of the newly activated T cells is optimal. Indeed, the observations (a) that viral or bacterial infection of the lymph node results in altered chemokine expression in the T cell area and (b) that this affects generation of new antigen-speciﬁc responses (37, 38) both suggest that mycobacterial infection of the draining lymph node may affect naive T cell activation. Once cells are activated, they must migrate to the primary site of infection, and this migration takes place 15–18 days post infection (27) (Figure 1). Whether this recruitment is delayed and, indeed, whether it depends on antigen or inﬂammation have not been deﬁnitively determined. However, new real-time imaging techniques are providing improved understanding of the factors modulating the generation of the inﬂammatory site. In particular, a recent paper has begun to determine how T cells and macrophages interact within the granuloma: Following systemic infection with BCG, tissue macrophages of the liver take up the bacteria, and a granuloma forms as a result of migrating local uninfected macrophages and monocytes from the blood. T cells then enter the granuloma and remain within it, using the myeloid cells as a framework for moving throughout the granuloma (39). Live imaging using the zebraﬁsh model is also providing information about the kinetics and development of the cellular response to mycobacterial infection (40, 41). In particular, the ability to genetically modify both bacterium and host in this model will allow for novel insight into the factors affecting the development of granulomata (40–42).

NEW SUBSETS OF EFFECTOR T CELL As discussed above, although we know that lymphocytes accumulate in the inﬂammatory lesion and that they probably mediate protective immunity by activating infected phagocytes, we have not yet fully deﬁned their function in vivo. Indeed, although it is widely assumed that CD4 T cells making IFN-γ are required for protective immunity, this assumption is based largely on correlative data. In particular, it is

clear that cessation of bacterial growth correlates with arrival of IFN-γ-producing CD4 T cells (43–46) and that loss of CD4 T cells increases the likelihood of succumbing to tuberculosis (11). However, although we understand the role that CD4 T cells play in protecting against bacterial growth, we have not yet fully deﬁned either their capacity to mediate protection or the mechanisms by which they mediate immunity. Clearly, various CD4 T cell effector subtypes exist, ranging from early activated cells making only IL-2, to cells making IFN-γ, to multifunctional cells expressing IL-2, IFN, and TNF, and the presence of these multifunctional cells is associated with protection (47). Furthermore, cytolytic CD4 T cells have been identiﬁed in mice (43) and humans (48). Multifunctional cells are seen at high frequency in tuberculosis patients (49) and also in those people in high incidence areas (50) and in vaccinated infants (51). Development and function of all effector cells depend on the ability of DCs within the draining node to prime and promote survival of these cells efﬁciently. This priming requires expression of antigen in the context of MHC, costimulatory molecules, and the necessary cytokines that promote T cell polarization. The role of IL-12p70 in IFN-γproducing cells has been demonstrated extensively (52); however, the conditions required to generate and maintain the multifunctional and cytolytic antigen-speciﬁc lymphocytes have not been fully deﬁned (Figure 1). Investigators have recently identiﬁed other subsets of functional T cells, such as those producing IL-17 and IL-22, and these cells have been seen in the mouse model and in humans exposed to tuberculosis (53). We do not yet know their protective role in tuberculosis; however, their recent discovery demonstrates that we are still learning about the cellular response to tuberculosis even in the well-deﬁned mouse model. IL-17-producing antigen-speciﬁc cells are induced in mice following aerosol infection; these cells and indeed most of the IL-17 response in the lung depends on the presence of IL-23 (54). γδ T cells are a source of IL-17 and produce this cytokine very early following a www.annualreviews.org • Cellular Immunity to Tuberculosis

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high-dose intranasal challenge with BCG (55). A large portion of the IL-17 response in the mouse model is within the γδ T cell population (56). When IL-17 is blocked during a high-dose challenge, neutrophil recruitment is hindered, and this may alter subsequent development of inﬂammation (55). In the absence of IL-23 and, therefore, in the majority of the IL-17 response in the mouse model, there is a modest alteration in the early inﬂammatory response (54). Whether these cells are protective or damaging we do not yet know, but when Mtb-infected animals are repeatedly challenged with mycobacterial antigen, the lesions become necrotic and contain a higher frequency of granulocytes (57, 58). Recently, we investigated the role of IL-17 in this enhanced pathology and found that increased lesion size and increased granulocyte presence in the lesions was dependent on IL-23 and could be ablated by the delivery of anti-IL-17 antibody (A. Cruz, personal communication). These data suggest that the nature of the inﬂammation that develops in response to chronic antigen exposure depends on IL-23 and IL-17. In addition, there is the potential for a dual role for these two cytokines in the response to Mtb infection in the mouse. Whether this response is equally important in humans is as yet unknown; however, CD4+ antigen-speciﬁc IL-17- and IL-22-producing cells can clearly be detected in humans exposed to Mtb, although only IL-22 is detected in the lung (50).

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EFFECTOR FUNCTIONS Determining the lymphocyte response to mycobacterial infection is essential to rational vaccine development; however, the response of the phagocyte to both infection and activation is also crucial in this regard. Speciﬁcally, if infected phagocytes are resistant to activation, despite the presence of acquired cellular immune responses, then this is an important limitation of any vaccine-induced response. Infected phagocytes also limit cellular responses as a result of feedback pathways involving products of phagocyte activation (59). 400

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In most vertebrate infections, bacterial growth occurs logarithmically until acquired immunity is expressed, whereupon control of bacterial growth occurs (60). This pattern is true for the B6 mouse model (61), in which there is a clear inability to control bacterial growth if phagocyte-activating cytokines such as IFN-γ (19, 20) and TNF (62) are missing and if the phagocyte cannot elaborate large amounts of nitric oxide (63). However, there are other mouse strains that either fail to stop bacterial growth or cannot maintain control of the infection. The cause of increased susceptibility varies depending on mouse strain (25, 64–68). This variability illustrates how complex the control of this Mtb infection is, but also provides tools for investigating the mechanisms involved. Speciﬁcally, a potentially novel function of infected phagocytes has recently been suggested by the use of linkage analysis. By crossing C3HeB/J mice, which are susceptible to Mtb, with resistant C57BL/6J mice, investigators have identiﬁed a locus (sst1) that is associated with decreased survival and increased development of necrotic lesions (68). The candidate gene within the sst1 locus, Ipr1, is induced in phagocytes upon infection with Mtb and is associated with ability to control bacteria and limit necrotic death of infected macrophages (68). Importantly, the impact of the locus is highest in the lung, and resistance is associated with reduced necrosis and increased apoptosis in the lung lesions. There is no difference in development of T cell responses in resistant and susceptible mice; however, vaccination is less effective in the susceptible mice, which suggests that defective phagocyte responses can limit the protective effect of vaccination (69). Unlike the data from most animal models, the data from humans regarding the role of speciﬁc cellular pathways in controlling Mtb are less clear. Although most humans who are exposed do not become diseased, exposure to the pathogen does result in the development of an acquired delayed-type hypersensitivity (8). The mechanism mediating control of Mtb in people exposed but not developing disease is currently unknown. However, the prolonged expression

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of an antigen-speciﬁc T cell response and the increased risk of disease in AIDS patients and in those lacking elements of the IFN-γ/IL-12 pathway suggest that acquired cellular immunity is induced upon infection and provides protection in the majority of those exposed. Recent studies have focused on the unique abilities of human phagocytes to control Mtb. In particular, mechanistic analysis of antimycobacterial activity in humans provides potential explanations for the increased susceptibility to disease exhibited by those with vitamin D deﬁciency. Early work implicated vitamin D in the ability of human macrophages to control Mtb proliferation (70, 71). More recently, investigators demonstrated that addition of 1α,25 dihydroxyvitamin D3 (the biologically active metabolite of vitamin D) reduces growth of mycobacteria in human peripheral blood mononuclear cells in a dose-dependent manner. This action is mediated via the nuclear vitamin D receptors and is associated with induction of the cathelicidin hCAP-18 gene. Addition of the active peptide LL-37, which is cleaved from cathelicidin, reduces growth of Mtb in culture (72). Toll-like receptor (TLR) activation of human macrophages and monocytes (but not DCs) results in upregulation of the vitamin D receptor and of the vitamin D1-hydroxylase genes and leads to induction of the antimicrobial peptide cathelicidin and killing of Mtb (73). The link between vitamin D–triggered antimycobacterial activity in monocytes and cathelicidin has been conﬁrmed using siRNA inhibition of 1α,25 dihydroxyvitamin D3 –induced cathelicidin protein production, which resulted in increased mycobacterial growth (74). A second macrophage function important in antimycobacterial activity is autophagy. Cells perform autophagy to “clean house” by sequestering their own cytoplasm into an autophagosome that is then delivered to the lysosome (75). IFN-γ induces autophagy, and inhibition of autophagy increases the viability of intracellular mycobacteria in mice and humans (76, 77). Interestingly, this activity has been linked to immunity-related p47 guanosine triphosphatases, one of which, lrgm1 (LRG-47), is

essential for IFN-γ-mediated control of mycobacterial growth in mice (78); the human ortholog of this gene, IRGM, also plays a role in autophagy and the control of mycobacterial burden (77). The autophagic response to Mtb is abrogated by IL-4 and IL-13 (79) and enhanced by TLR4 ligation (80). Careful analysis and identiﬁcation of novel effector functions capable of limiting bacterial growth in both animal models and humans will provide increased insight into potential pathways for augmenting immunity both in intact and in immunocompromised individuals.

TOLL-LIKE RECEPTORS AND PATTERN-RECOGNITION RECEPTORS Both the innate and acquired response to Mtb infection depends to a large degree on recognition of Mtb as a pathogen by the patternrecognition receptors. Of particular interest to researchers are the TLRs and the common adapter molecule MyD88, which mediates many of the intracellular signaling events after pattern-recognition receptor ligation. MyD88 is required for survival upon mycobacterial infection (81–84), and the disease phenotype exhibited as a result of this deﬁciency is interesting. MyD88-deﬁcient mice can generate high levels of cytokine in the lung, and ex vivo stimulation of cells with mycobacterial antigen results in an IFN-γ response; however, bacterial growth is not regulated, and the lung develops an acute necrotic pneumonia. Analysis of the phagocytes in these mice reveals that they exhibit reduced responsiveness to the activating signals produced by the acquired response (82– 84). In MyD88-deﬁcient mice, therefore, the induction of some aspects of acquired immunity remain intact, whereas the effector function of the phagocytes is lost. Investigators initially thought that loss of TLR2-, TLR4-, and/or TLR9-induced phagocyte activation was the deﬁning element of the MyD88 deﬁciency phenotype, and indeed deﬁciency of TLR2 and TLR9 results in increased susceptibility to Mtb infection (85). However, direct www.annualreviews.org • Cellular Immunity to Tuberculosis

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comparison between TLR2/4/9 deﬁciency and MyD88 deﬁciency clearly shows that deﬁciency in these TLRs does not account for the susceptibility seen in the MyD88 mice (84). Other work investigated the function of MyD88 as an adapter of IL-1 and IL-18 receptor signaling, revealing that the absence of IL-1R but not the absence of IL-18R or the Toll-IL-1R domaincontaining adapter protein (TIRAP) recapitulated the MyD88 phenotype; speciﬁcally, absence of IL-1R led to increased susceptibility to disease, induction of cellular responses with the absence of phagocyte activation, and the development of necrotic pneumonia (86). Although MyD88 and IL-1R appear essential for induction of phagocyte activation and survival of acute infection, deﬁciencies in TLR are associated with reduced induction of acquired immunity and increased susceptibility to chronic or high-dose infection. For example, whereas the absence of TLR4 or TLR6 results in little impact on infection or disease in mice, the absence of TLR2 results in slightly increased bacterial growth in low-dose aerosol and greater susceptibility to high-dose aerosol (85, 87–89). TLR2 has been implicated in recognition of mycobacterial antigens and modulation of phagocyte function. In particular, lipoproteins from Mtb that are recognized by TLR2 can limit the ability of macrophages to upregulate MHC class II in response to IFN-γ; this effect is seen after prolonged exposure and is associated with reduced antigen presentation (90–95). Mycobacterial peptidoglycan can also inhibit IFN-γ-mediated induction of the class II transactivator (CIITA) by a TLR2-, MyD88independent pathway (96, 97). Despite the strong in vitro data, addressing the effect of the bacteria on phagocyte function in vivo has been difﬁcult. Such an experiment was attempted recently, however. By infecting a chimeric mouse containing congenically marked phagocytes that were either TLR2 sufﬁcient or deﬁcient, investigators assessed the relative importance of TLR2-dependent and -independent pathways on MHC class II expression in vivo in response to Mtb. Although they demonstrated that MHC class II regulation in infected lungs

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depends on the presence of the IFN-γ receptor, the presence of TLR2 does not affect the level of MHC class II on either infected or uninfected phagocytes within the infected lung (98). This could mean either that both TLR2-dependent and TLR2-independent pathways are involved in regulating MHC class II in vivo or that Mtb does not regulate MHC class II in vivo. Some evidence suggests that Mtb can alter T cell activation without altering class II expression (35), but this still needs to be demonstrated in vivo. Other studies show that when macrophages enter infected lung tissue they upregulate MHC class II in an IFN-γ-dependent manner (99). The fact that reduced acquired immunity and increased immunopathologic consequences seen in TLR2 and TLR9 mice (85, 88) only modestly affect the control of bacterial growth indicates that the low-dose aerosol model may not stress the immune response to a sufﬁcient degree. This may explain why the profound differences in cellular responses of TLR-deﬁcient cells seen in vitro are not recapitulated in vivo. With regard to induction of the acquired response, we can conclude, on the basis of speciﬁc TLR expression, that DCs and macrophages have different responses to Mtb (85, 100). The level and activity of TLR-mediated signaling by DCs encountering the bacteria will inﬂuence the outcome of the cellular response, but in low-dose aerosol infection any differences may result in subtle outcomes early in infection and may only be seen by careful analysis. For example, the relative levels of IFN-γ- and IL-17-producing cells during mycobacterial infection in both mouse and human will depend on the level of speciﬁc cytokines present during and after T cell activation (101). A recent analysis of the response of human DCs to Mtb found that IL-23 was preferentially expressed, likely in a TLR2-dependent manner, compared with IL-12p70 in the absence of IFN-γ; IL-10 was also induced. Following IFN-γ activation, DCs responded to Mtb by producing both IL23 and IL-12p70 and by reducing the level of IL-10 (102). These data suggest that in humans,

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before expression of IFN-γ, the preferential T cell effector type would likely be an IL-17 producer but that thereafter both the IFN-γ and IL-17 response would be promoted. In the mouse model, Mtb-infected DCs generate IL-12 and IL-23 and promote both IFN-γ and IL-17 populations in CD4 T cells (54) (Figure 1). The C-type lectins are also implicated in modulation of phagocyte function in mycobacteria response (103, 104). Mycobacteria can affect DC function via ligation of the C-type lectin DC-SIGN on immature human DCs, and this interferes with LPS-induced maturation, while promoting IL-10 production (105). This interference appears to be mediated by the acetylation of the p65 subunit of NF-κB that prolongs the transcription rate of the IL10 gene (106). The C-type lectin Dectin-1, in conjunction with TLR2, is involved in the cytokine response of macrophages to Mtb (107) and in Mtb induction of IL-12p40 in splenic DCs (108). Our understanding of the role of speciﬁc cell types and receptors in the development of tuberculosis is still being developed. Thus, although we may not consider a speciﬁc ligand and receptor interaction as essential to survival, it could be crucial for the development of balanced acquired cellular immune responses. We should therefore continue to determine the role of these molecules in both protection and development of pathologic consequences.

PHAGOCYTE PHENOTYPES IN THE INFECTED LUNG To understand the dynamic environment within which the acquired cellular immune response must act to mediate protection, we must deﬁne the cellular components of the inﬂammatory response to Mtb infection. It has long been assumed that the phagocytic mononuclear cells that accumulate at the inﬂammatory site in the Mtb-infected lung are monocytederived macrophages. Recent data make clear, however, that even in the naive lung the myeloid cell population is a complex mixture of phenotypes (28). Indeed, ﬂow cytometry and

immunohistochemistry to study the cell phenotypes accumulating in the lesion following infection have challenged the conventional view that macrophages are the principal host cell for Mtb. Speciﬁcally, early observations that mycobacteria could infect cells of DC phenotype in vivo (109) have been followed by demonstrations that Mtb infects a variety of cells within the lung as the lesion develops (35, 110). The potential importance of the DC in the lung lesion was suggested when the DEC205 (CD205) DC marker was used to determine that the vacuolated mononuclear cells usually called “foamy macrophages,” which comprise a large portion of the inﬂammatory lesion, are in fact positive for this DC marker (111) and are CD11bhigh and CD11chigh . A similar subset was recently determined to be a primary host for Mtb within the infected lung (35). The origin of these cells and their function in controlling infection are still under investigation. However, a recent study using cell transfer techniques has shown that circulating monocytes can become one of ﬁve different types of cell when entering the Mtb-infected lung. This study demonstrates that when circulating monocytes are recruited to the lung in response to mycobacterial infection, they express high levels of CD11c, MAC-3, and MHC class II (112). In an important analysis demonstrating the phenotype of the protective effector cell, a correlation between CD205 and iNOS expression occurred in the CD11b+ CD11c+ monocyte– derived cells that accumulated in the lung (112). The cellular environment within the lung clearly becomes more complex as infection develops into disease. Determining the nature of the infected cells and indeed the overall environment within the lesion is crucial to rational design of vaccines, as we need to know whether cells induced by vaccination are capable of functioning within the inﬂammatory environment generated by the bacteria. In this regard, it has recently been demonstrated that the inﬂammatory lesions in several animal models [but not in the intact mouse (113)] are locally hypoxic environments (114). Whether this environment could affect cellular immunity is www.annualreviews.org • Cellular Immunity to Tuberculosis

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unclear, as these hypoxic sites are highly disrupted; the possibility should be considered, however. One ﬁnal consideration of an indirect impact of mycobacterial infection on the T cell response is the fact that several mycobacterial species infect the thymus, even when they have entered the body though the lung. Although this infection does not result in granulomatous responses, the normal function of the thymus is likely affected by this infection (115).

EFFECTOR B LYMPHOCYTES The ability of acquired immune responses to limit bacterial growth is key to survival upon Mtb infection; however, the fact that this immunity is insufﬁcient to eliminate the bacteria suggests several potential failures in the response. The ﬁrst is that the phagocyte is not capable of eliminating bacteria regardless of activated state. The second is that macrophage activation is optimal but that it is limited by the bacterium. The third is that activation of the infected phagocyte by T cells is compromised within the inﬂammatory environment. This latter failure could be addressed and potentially remedied either by generating T cells more able to function in the inﬂammatory site or by modulating the inﬂammatory site. The importance of CD4 and CD8 T lymphocytes in controlling Mtb in the lung has been reviewed extensively elsewhere (2, 3), and these cells are the focus of most current vaccine development. In this section, I propose the potential for B cells to affect the cellular control of Mtb infection. Although the location of antigen-speciﬁc T cells within the granuloma has not been deﬁned, it is clear that CD3+ , CD4+ , and CD8+ T cells are present within the inﬂammatory lesion. The largest population of lymphocytes within the lesions, however, are the B220+ cells, which are likely B cells (116). These cells accumulate in follicles that look like nascent lymphoid tissue, are dependent on speciﬁc chemokines, and may play a role in regulating immunity within the lesional site (117) (Figure 2). Furthermore, although low-dose aerosol infection 404

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of B cell–deﬁcient mice does not result in increased susceptibility to bacterial growth, it does alter lesion development (118–120). And when B cell–deﬁcient mice are challenged intravenously with a large dose of bacteria, they are more susceptible to disease (121). The role of B cells in immunity to tuberculosis may therefore need reassessment. Recently, higher dose aerosol infection has supported a role for B cells in immunity against lung disease (122, 123). These recent papers propose that the nascent follicles in the lung are germinal centers and that they contain PNA- and GL7-positive cells as well as T helper follicular cells; the latter express CXCR5 and respond to CXCL13. In the absence of B cells, mice exposed to these higher dose aerosols have exacerbated immunopathologic consequences with increased neutrophil accumulation; these changes in the cellular response correlate with an increase in bacterial burden (122). An extension of this work has shown that the nature of the Fcγ receptor expressed in the lung correlates with susceptibility; thus, whereas activating receptors aid protection, inhibiting receptors limit protection (123). These data suggest that either the B cells and their products or the follicles themselves can modulate both the inﬂammatory and the cytokine response. In support of this assumption, a postexposure vaccine model demonstrated the ability of B cells to affect the inﬂammatory site; in the model, the absence of B cells resulted in a severe increase in pathologic consequences (120). The high frequency and potential of B cells to affect both immunity and immunopathologic consequences at the site make the potential of these cells difﬁcult to ignore. Whether it will be possible to harness the power of B cells by vaccination will depend on our understanding of the degree of antigen speciﬁcity and of the speciﬁc functions expressed by these cells. I raise an element of caution here, however, as guinea pigs, which are highly susceptible to death from Mtb infection, elaborate an early T cell response in lung lesions that is subsequently replaced by a predominantly B cell and granulocyte response (124). Whether the B cells are causative of the susceptibility of guinea

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Mtb modulates surface molecule (TDM) to alter the response of mononuclear cells

Antigen-speciﬁc eﬀector T cell function is limited by regulatory T cells

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B cells accumulate within lesion, and nascent lymphoid follicles form; B cells aﬀect protective and pathologic functions

Regulatory cytokines (IL-10, IL-27) and molecules (lipoxins, DAP12) regulate the lymphocyte response

Infected phagocytes are activated by antigen-speciﬁc eﬀector T cells; T cells limit the accumulation of granulocytes

Figure 2 The inﬂammatory lesion within the lung is a dynamic environment containing a variety of protective and regulatory cells. Effector T lymphocytes ( purple) mediate control of bacterial growth and the mononuclear composition of the granuloma. Regulatory T lymphocytes (orange) also accumulate in the lesion and limit the ability of the acquired response to stop bacterial growth. Infected phagocytes elaborate cytokines and effector molecules that limit the activity of the lymphocyte response. B cells (blue) accumulate within the lesion in the form of nascent lymphoid follicles; these cells can affect bacterial control and the immunopathologic consequences of infection. Mtb (red ) can modulate the inﬂammatory response via the modiﬁcation of trehalose dimycolate (TDM) expressed at the bacterial surface.

pigs will have to be determined, and the function of B cells within protected and susceptible species may provide insight into what represents a useful B cell.

CONTROL OF T CELL RESPONSES Despite the potential of B cells, it is the T cell response that must mediate protection once Mtb is within host cells. In mice, the lung, liver, and spleen are infected following aerosol infection; however, signiﬁcant disease, as measured by the level of inﬂammatory involvement of parenchymal tissue, occurs primarily in the lung (125, 126). In the spleen and liver, inﬂammatory lesions are small and contained, whereas in the lung, these lesions continue to develop and become destructive at rates that depend on the strain of mouse or species of host (60). To control tuberculosis, we need to understand

what is regulating the cellular response in the lung and also whether removal of this regulation will allow for efﬁcient clearance of the bacteria or result in overwhelming inﬂammation, which could kill the host. There is mounting evidence that the immune response to Mtb infection is regulated. With the increased interest in regulatory T cells, the potential of these cells to modulate the immune response to tuberculosis has been the focus of several investigations. When a CD4 T cell population is depleted of regulatory T cells (based on surface phenotype) and transferred into Rag mice, this population can mediate efﬁcient control of bacteria; the regulatory activity is not dependent on IL-10 (127). Furthermore, in a direct aerosol infection, T cells with a regulatory cell phenotype accumulate both in the draining lymph nodes of the lung and within the lung itself at a rate similar to effector T cells (128). These regulatory T cells are also www.annualreviews.org • Cellular Immunity to Tuberculosis

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located within the inﬂammatory lesion (Figure 2). Using a chimera model in which cells sufﬁcient for the transcription factor required for regulatory T cell development were congenically marked, it was possible to deplete regulatory T cells from infected mice and demonstrate that depletion of regulatory T cells results in a modest reduction in bacterial number (128). Together, these data suggest that regulatory T cells do affect the ability of the effector T cell response to mediate immunity; however, it is unclear what the long-term consequences of depletion of this regulatory activity are. The potential role of IL-10 in regulating the protective cellular response to Mtb has been investigated extensively in recent years. The human data are of interest, as there are reports of an association between the risk of developing tuberculosis and the presence of speciﬁc IL-10 polymorphisms. One example is the increased tendency toward development of primary progressive tuberculosis in humans with increased innate IL-10 responses (129). This association is not seen in all populations, and a recent meta-analysis suggests that, although we see a trend toward an association between increased susceptibility and IL-10 polymorphisms, it is not strong (130). In the mouse model, CBA mice have a strong IL-10 response to Mtb as expressed in the lung phagocytes, and this strain develops progressive tuberculosis, which is poorly controlled by the cellular response (65). Modulating this excess IL-10 by anti-IL-10 treatment reduces the susceptibility of this strain (131). If IL-10 is overexpressed in the lungs of Mtb-infected B6 mice, then bacterial growth is poorly controlled compared with wild-type mice (65). In the B6 model, IL-10 is expressed at modest levels in lung phagocytes (65), and in its absence there is a modest impact on control of bacterial growth (132) (Figure 2). These data suggest that IL-10 can affect cellular immunity but that its involvement depends on genetic factors affecting its expression in response to infection; this may explain the variable nature of the impact of IL-10 polymorphisms on the progression of human disease.

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In several other mouse models, the loss of one gene can result in improved bacterial control (Figure 2). For example, in the absence of IL-27R activity, bacterial burden is decreased, whereas there is increased inﬂammation within the parenchyma (133, 134). Despite this improved bacterial control, the genedeﬁcient mice succumb to infection earlier than do wild-type mice (134). In a different model, the enzyme required for the generation of the immunoregulatory lipoxins, 5-lipoxygenase, is detrimental to reduction of bacterial numbers, as mice deﬁcient for this enzyme elaborate greater Type 1 immunity to mycobacterial infection (135). Although the effect of this deﬁciency on survival has not been reported, we know that the absence of this enzyme is detrimental to the survival of mice infected with Toxoplasma gondii owing to excessive Type 1– induced damage (136). In another model, the absence of the chemokine receptor CXCR3 also results in improved control of bacterial burden in the chronic phase of disease, the extent of which is mouse strain dependent. In this model, there is no apparent impact on the inﬂammatory response, and it appears that CXCR3 expression regulates the size of the antigenspeciﬁc T cell population (137). Finally, comparison of the phagocyte response to infection between DAP12-deﬁcient and wild-type mice demonstrated that the DAP12-deﬁcient cells had a much greater NF-κB activation and an increased cytokine response to infection than did wild-type cells. This resulted in an enhanced inﬂammatory response and increased numbers of IFN-γ-producing antigen-speciﬁc cells in the tissue, which further resulted in reduced bacterial burden within 25 days of infection and an accelerated and increased granulomatous response within the lung (138). These data suggest that DAP12, which is an immunoreceptor tyrosine-based activation motif (ITAM)containing coreceptor, regulates the cellular response following mycobacterial infection, likely through its ability to regulate the phagocyte response to the pathogen. The regulatory nature of the inﬂammatory lesion that occurs in response to Mtb means that

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acquired cellular immunity should be expressed as rapidly as possible to control bacterial growth before any regulatory activity is expressed. One way to do this is via vaccine-induced generation of immunological memory.

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MEMORY T CELL RESPONSES As discussed above, following infection via the aerosol route there is a signiﬁcant delay in the induction of acquired cellular immune responses. This delay likely contributes to the establishment of a productive infection and progression to disease in susceptible individuals. The best way to accelerate the kinetics of acquired cellular responses is to expose the adaptive cells to speciﬁc antigens of the pathogen in an environment that will generate long-lived antigen-speciﬁc cells capable of remembering the pathogens upon subsequent challenge; this is, of course, the basis of vaccination. While we know that vaccination is an excellent way to protect against some diseases (primarily those controlled by antibody), vaccination against tuberculosis is not generally protective against pulmonary disease in adults (23) (Figure 3). In contrast, vaccination can signiﬁcantly limit the dissemination of disease to other organs and does protect children from disease sequellae such as tuberculous meningitis (22). To improve vaccination strategy, we must understand why the current strategy is ineffective. One potential issue with current models is that, although sizeable populations of circulating antigen-speciﬁc memory cells are induced, the kinetics of the protective cellular memory response to aerosol challenge is only accelerated by a few days (45, 46, 139, 140). This modest acceleration allows for earlier expression of antimycobacterial activity within the lung, but as there is still a substantial delay a signiﬁcant bacterial burden becomes established before the memory response. The memory cells then have to act within an environment that the Mtb has had an opportunity to modulate (Figure 3). Because dissemination occurs at the same time as activation of T cell responses, disseminated bacteria likely encounter the acquired response

shortly after arriving at a site and are therefore less able to modulate the environment. This may be why BCG is only modestly protective against lung disease but is very effective against disseminated disease (22). It also explains why measuring the circulating population of antigen-speciﬁc IFN-γ-producing cells does not provide a good correlate of protection (141). Speciﬁcally, although memory cells may be present in the circulation, it is the ability of these cells either to populate the lung or to get to the lungs quickly upon infection that is important; this ability is not measured by determining frequency of antigen-speciﬁc effector cells within the peripheral blood. To overcome the low frequency of antigenspeciﬁc cells within the lung, investigators have employed in situ boosting regimens. BCG priming in the lung followed by boosting with either BCG or modiﬁed vaccinia virus Ankara (MVA)-expressing antigen85 (Ag85) demonstrated that extremely good protection against aerosol challenge could be obtained and that this correlated with induction of Ag85-speciﬁc cells within the lung draining lymph node (142). A separate study using intramuscular DNA vaccination found that very few Mtb-reactive CD8 T cells are in the airway, despite a substantial systemic population. Unlike BCG vaccination, in which a predominantly systemic population of cells can mediate a modest level of protection in the murine model, this systemic CD8 population was not protective. However, if the cells were recruited to the lung lumen by low-dose antigen exposure, these cells could mediate protection from challenge (143) (Figure 3). The authors of this work took these data to demonstrate that the protective activity of antigen-speciﬁc memory T cells is deﬁned by the location of the cells. In an effort to induce mucosally located antigen-speciﬁc T cells, these authors have delivered DCs transduced to express Ag85 intranasally to mice. These DCs migrate to the lung mucosa and the draining lymph nodes in an IL-12p40-dependent manner and generate antigen-speciﬁc effector cells in the airway lumen in an IL-12-independent manner. This route of vaccination resulted in a www.annualreviews.org • Cellular Immunity to Tuberculosis

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a 8 – 9 days post-infection

Circulating memory T cells recognize antigen in lymph node and are activated

DC

Lung

Alveolar tissue

Bacteria arrive in alveolar tissue and take 9 days to get to the lymph node

Resident memory T cells see antigen quickly and respond to activated infected phagocytes or recruit other eﬀector memory T cells

Eﬀector T cells

Lymph node DC

Eﬀector memory T cells migrate from lymph node through circulation and are attracted to the lung tissue by inﬂammation

Infected phagocyte Bacteria still have 15 days to grow and modulate environment before eﬀectory memory T cells arrive in lung

12 – 15 days post-infection

b Bacterial burden in lung

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Bacteria

Eﬀector cells arrive at day 20 in naive mice

Eﬀector cells arrive at day 15 in vaccinated mice Eﬀector cells arrive earlier and can stop bacterial growth and reduce the impact of the bacteria on inﬂammatory site

Time in days post-infection

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modest level of protection (36). Further data on isolate it from the protective response. In this the longevity and any potential immunopatho- regard, we have determined that IL-23 and logic role of lung-resident effector cells should IL-17 appear to be required for the enhanced be obtained prior to using pulmonary vaccina- pathologic responses seen in repeatedly exposed Mtb-infected mice (A. Cruz, personal tion as a tool to control tuberculosis. Investigators have proposed prime-boost communication). Although IL-17-producing cells may be regimens for augmenting the routine BCG vaccination given either to newborns or to ado- detrimental during chronic infection, we have lescents, under the rationale that the response identiﬁed a function for these cells during to BCG wanes; this has been demonstrated re- the recall of antigen-speciﬁc IFN-γ-producing cently in a study from the UK (144). The MVA- memory CD4 T cells. In a model of vaccineexpressing Ag85, shown to be protective in mice induced protection using a subunit vaccine con(142), has been used to boost BCG-induced re- taining a peptide from an early expressed Mtb sponses in people and has been found to be antigen (ESAT-6) delivered in an adjuvant of highly immunogenic (145). Boosting with this monophosphoryl lipid A, trehalose dicorynovaccine results in populations of polyfunctional mycolate, and dimethyldioctadecylammonium, cells that retain proliferative potential (146). Al- we have shown that a population of antigenthough it is difﬁcult to test the protective efﬁ- dependent cells responsible for IL-17 produccacy of these prime-boost regimens in humans, tion is induced in the lung (46). These cells rethe data support the use of these regimens to spond more rapidly to aerosol challenge and are required for early expression of chemokines maintain cellular immune responses. One concern with repeated boosting is the within the lung. In their absence, neither an acpotential to induce pathologic consequences. celerated IFN-γ response nor vaccine-induced As discussed above, the repeated delivery of protection is seen in the lung (46). These data mycobacterial antigen to already infected mice suggest that surveillance cells can be induced can result in augmentation of the pathologic in the lung by subcutaneous vaccination and response in the lung (57, 58, 120). This is that vaccination may be rationally targeted to reminiscent of the work by Koch in which he these cells. The caveat to this is, of course, demonstrated that delivery of live mycobacteria that these cells could also mediate destructive or mycobacterial antigen to tuberculous guinea immunopathologic consequences if not sufﬁpigs could result in necrosis and sloughing of ciently regulated. The increase in our ability to modulate mythe skin (1). This effect depended on antigen dose; at lower doses, hypersensitivity is seen cobacteria genetically has allowed the design without necrosis (1). Although there has been and testing of genetically modiﬁed mycobaclittle evidence of this “Koch phenomenon” teria as vaccine candidates. This is a ratioduring the currently performed prime-boost nal method of generating candidates as long models, we may be able to boost the protective as we can augment immunogenicity while reresponse to a greater degree if we can identify ducing virulence. Two recent studies highlight what mediates any damaging response and the practicality of this approach. In one, the ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 3 (a) The host with previous antigenic experience has an increased frequency of memory lymphocytes either in the circulation ( purple) or within the lung ( pale red ). Cells within the lung can respond to infection before bacteria migrate to the lymph node and can therefore activate infected phagocytes and stop bacterial growth. Cells within the lung can also recognize infection and act rapidly to recruit circulating memory cells that may require dissemination of bacteria to become activated. As effector cells arrive in the lung, they activate phagocytes and stop bacterial growth. (b) The cessation of bacterial growth by memory cells can occur 5 days sooner than is seen for naive cells. This acceleration, though signiﬁcant, does not stop substantial bacterial growth, and thus effector cells must act within a site that has been initiated and modulated by Mtb. If the memory response can be improved such that the response occurs within 10 days as opposed to 15, then the T cell response may be expressed more efﬁciently and thereby limit the development of disease (red line). www.annualreviews.org • Cellular Immunity to Tuberculosis

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expression of two genes in BCG that promote antigen translocation to the cytoplasm of infected cells results in improved protection compared with the parental BCG, likely because of cross-priming (147). In a more recent study, vaccination with an Mtb mutant lacking the ability to inhibit apoptosis of infected macrophages resulted in enhanced CD8 T cell responses and early protection in both mice and guinea pigs (148). By increasing our understanding of the nature and functionality of vaccine-induced cells, we will provide a rational basis for new vaccine design. Key elements of this understanding include the location of the vaccine-induced cells and their ability to respond quickly and efﬁciently within a highly regulated environment. We can achieve this using basic animal models, but we also need to improve our analysis of human vaccination.

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HUMAN CELLULAR RESPONSES Recent technological advances have allowed for improved analysis of the cellular responses occurring in vaccinated, Mtb-exposed, and tuberculosis patients. The most dramatic improvement in data generation has been in analyzing antigen-speciﬁc responses and identifying several parameters of responding cells using ﬂow cytometry. Analysis of cellular responses has shown that infected and diseased individuals express a high frequency of multifunctional cells (49). Recent studies of a European population reported a limited Mtb-speciﬁc IL-17speciﬁc response but a strong IL-17 response to fungal antigens (149). In contrast, when South African populations are examined, Mtb-speciﬁc IL-17- and IL-22-producing T cells can clearly be identiﬁed both in exposed and diseased individuals, although the frequency of such cells in the periphery is lower in the diseased patients (50). Despite the low frequency of these T cells peripherally, IL-22 can be detected in the bronchoalveolar lavage of patients, suggesting that IL-22-producing cells are active in the lung of patients (50). This compartmentalization of the

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response has also been seen in a recent study using an Ag85/HLA-A∗ 0201 pentamer to assess frequency of antigen-speciﬁc cells in tuberculous children before and after drug treatment. These patients had a low frequency of antigenspeciﬁc CD8 T cells in the blood, but this frequency increased after treatment (150), possibly because the treatment reduced inhibition of T cell activation by the bacteria or because lung lesions were resolving and thus recruitment of antigen-speciﬁc cells from the blood to the lesional site was reduced. Interestingly, the circulating antigen-speciﬁc cells had low cytokine and cytolytic activity before treatment, whereas more activated cells were present in the lung before treatment (150). A separate study found that at the beginning of treatment the frequency of antigen-speciﬁc IFN-γ-producing cells is higher than that for IL-2-producing antigen-speciﬁc cells but that as treatment progressed over 28 months the dominance of the IFN-γ response is lost and most responding cells are of the IL-2-producing phenotype (151). Further detailed studies will allow for greater understanding of the kinetics and nature of the antigen-speciﬁc cellular response in humans. The high level of BCG vaccination occurring in newborns in South Africa has allowed for an unprecedented screen of the ability of BCG to induce speciﬁc cell types in this population. In a recent study, cells from BCG-vaccinated newborns were restimulated in vitro with BCG, and the cellular response was analyzed by ﬂow cytometry. Importantly, whereas IL-4- and IL-10-producing CD4 T cells occurred at low frequency, IFN-γ, IL-2, and TNF were all produced either singly or in combination by activated CD4 T cells. Activated CD8 T cells were less frequent and when present were predominantly IL-2 and/or IFN-γ positive. In a telling detail, many of the responding T cells were not positive for IFN-γ; thus, the effector phenotype of many of the responding cells is still to be determined. The majority of the responding IFN-γ-producing cells had an effector cell surface phenotype, whereas those expressing IL-2

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alone were of the central memory phenotype (51, 152). One aspect of the human immune response that has received increased attention recently is the identiﬁcation of antigens recognized by exposed individuals. In a study using synthetic peptide arrays of known immunodominant antigens, the antigen speciﬁcity of a large number of CD8 T cells clones was obtained and new epitopes identiﬁed (153). In a separate study, the regions of difference (RD) that have been identiﬁed between Mtb and BCG were used to compare the peptide-speciﬁc responses of patients with tuberculosis and healthy individuals vaccinated with BCG. In this study, both groups responded to peptides from the RD1 region (perhaps reﬂecting a high degree of exposure to Mtb). However, RD12, RD13, and RD15 peptides elicited an IL-10 response from peripheral blood mononuclear cells, suggesting that antigens within the different RD are associated with distinct cellular responses (154). These kinds of studies are being used to ﬁll the gaps in our understanding of the speciﬁcity of the T cell response to Mtb in humans (155). Although acquired cellular immunity is the focus of many studies, the study of the innate response of humans to infection has been gaining momentum recently. Neutrophils have been implicated in antimycobacterial immunity owing to their ability to provide antibacterial activity to infected macrophages when phagocytosed (156), as ﬁrst shown in the mouse model with M. avium and M. microti experiments (157) and conﬁrmed using human cells and virulent Mtb (158). Both apoptotic neutrophils and puriﬁed neutrophil granules can reduce the viability of extracellular bacteria and augment the ability of infected macrophages to reduce bacterial growth. The granules trafﬁc to the early endosomes and colocalize with the bacteria (158). In an exciting extension of this work, the potential importance of neutrophils in protection against infection was assessed in 187 contacts of newly diagnosed Mtb patients (159). In this study, a whole blood assay was used to demonstrate that when neutrophils were depleted from whole

blood the release of antimicrobial peptides in response to mycobacterial exposure was dramatically reduced; the same peptides were also antimycobacterial. Also, when neutrophils were depleted from the whole blood, the ability of the blood to limit mycobacterial viability was reduced. Importantly, an inverse relationship between the number of peripheral neutrophils and the risk of Mtb infection in contacts of pulmonary tuberculosis patients was observed, suggesting that neutrophils may play a protective role very early in infection (159). Human natural killer (NK) cells also mediate antimycobacterial activity. When macrophages are infected with Mtb, they upregulate vimentin, and NK cells can lyse infected mycobacteria via ligation of the vimentin by the NKp46 molecule on NK cells (160–162). This ability of NK cells to lyse infected cells is seen with infected human alveolar macrophages, suggesting that this interaction could play a role in the earliest responses to infection (160). An extension of this work demonstrated that human NK cells can lyse expanded T cells that express a regulatory phenotype (CD25+ FoxP3+ ) (163). How this ability of NK cells to regulate the acquired response affects disease will be intriguing to determine. Indeed, although the effect of NK activity on disease is not clear, NK-mediated cell lysis is reduced in patients with advanced disease (162). In the mouse model, IL-12p70-dependent NK cell activity can control bacterial growth in the absence of acquired immunity and also limits accumulation of granulocytes at lesional sites (164). The study of human immunity is being improved by tools for visualizing the cellular response both immediately ex vivo and even in vivo (24). Despite these advances, experimenting on humans directly is still difﬁcult, and it is therefore imperative that we maintain an active dialog between those studying mycobacterial infection in various animal models and those studying the disease in humans. Comparative pathology is an excellent tool to improve our knowledge of this complex disease.

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MTB MODULATES ITS ENVIRONMENT

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The chronicity of Mtb infection results in an inﬂammatory and dynamic environment within which the acquired cellular immune response must act. In this regard, the effect of the mycobacterium on cellular inﬂammation is a key element. The different mycobacterial species such as M. leprae and M. ulcerans induce profoundly different consequences upon infection compared to Mtb. However, it is only recently that the genetic variability and strain differences within Mtb have been appreciated to be extensive and to contribute to the pathogenesis of disease (165). The effect of this genetic variability is now well established. As we identify more strains and their ability to modulate immunity, it is clear that Mtb has a variety of tools with which to modulate the inﬂammatory response to infection. This is not surprising because the bacterium depends on the inﬂammatory response to mediate dissemination of disease. It is also not surprising because the surface of the bacteria contains a variety of highly stimulatory molecules the structures of which are ﬁnely tuned to interact with the immune response (Figure 2). The effect of very small changes in one of the major inﬂammatory molecules that comprise the cell wall of mycobacteria can be dramatic. For example, an Mtb mutant that lacks the pcaA gene (Rv0470c) and thus is unable to cyclopropanate alpha mycolates cannot persist in the mouse model despite reaching initially equivalent levels of infection in the lung (166). The reduced virulence of this mutant is not apparent in TNF-deﬁcient mice, suggesting that it is the induction of TNF by the altered molecule that is associated with the decreased virulence of this mutant. The primary molecule affected by this change is likely the trehalose dimycolate (TDM), as systemic delivery of the mutant TDM is much less able to induce either TNF or a prolonged inﬂammatory response compared with the TDM from the parental strain (167). While inability to cyclopropanate alpha mycolates reduces the inﬂammatory activity of

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TDM, an inability to trans-cycloproponate methoxy and ketomycolates increases the inﬂammatory activity of TDM. Thus, in a mutant lacking cmaA2, a gene that is required to transcycloproponate methoxy and ketomycolates, normal bacterial growth occurs in vivo, but the mutant is more virulent than the parental strain. This virulence correlates with increased inﬂammatory involvement in infected organs, where larger but lymphopenic lesions are seen. The mutant is therefore hyperinﬂammatory, and this is associated with altered TDM structure (168). Modiﬁcation of the TDM is also involved in induction of the IL-12p40 gene. An Mtb mutant lacking the mmaA4 gene, which is required for oxygen-containing modiﬁcations of cell wall mycolic acids, is less virulent in vivo, and this reduced virulence is not observed in IL-12p40deﬁcient mice. Both the mutant bacteria and its TDM induce more IL-12p40 than do the wildtype Mtb, and the wild-type TDM is inhibitory (169). In a further example of the role of TNF induction in the virulence of mycobacteria, some isolates of the highly successful W-Beijing strains can modulate their induction of TNF. These strains are present throughout the world and are associated with acute outbreaks (170). Several virulent isolates of the W-Beijing strains apparently do not have the frameshift mutation, present in many strains of Mtb, that prevents generation of phenolic glycolipid (PGL), and thus they produce this lipid. In the mouse model, there is little effect on the bacterial burden as a result of the generation of PGL; however, infected mice are subject to greater mortality. When the ability to make this lipid is disrupted by mutation, the hypervirulence is lost, and there is an increase in induction of TNF, IL-6, and IL-12. Overexpression of the PGL by mycobacteria or treatment with free PGL results in decreased induction of these cytokines (171). Virulence of the W-Beijing strains in a rabbit model of tuberculosis meningitis also depends on the production of PGL (172). Further analysis of the role of the

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PGL suggests that its ability to affect virulence may depend on other as yet undeﬁned aspects of the bacteria, as simple expression of the PGL in the laboratory strain H37Rv does not result in hypervirulence but does inhibit cytokine induction (173). Furthermore, when the acquired cellular response to one of the hypervirulent W-Beijing strains is examined, a signiﬁcant Th1 type immune response is induced but is lost as a regulatory T cell population expands (174). Thus, although we know that the W-Beijing strains are clearly altered in their ability to modulate the immune response to infection and that the expression of PGL is essential to this process, we still have not deﬁnitively determined the exact mechanism by which this hypervirulence occurs. Mtb devotes considerable energy to directing the induction of the cellular response to infection, and the ability of strains and mutants to modulate the kinetics and extent of cytokine induction plays an important role not in the control of bacteria but in the nature of the inﬂammatory response induced.

CONCLUSIONS Our knowledge of the cellular response to Mtb infection has improved dramatically over the years. We have not as yet deﬁned the type of acquired cellular response that will mediate immunity and that should therefore be induced by vaccination; this severely limits our ability to develop novel vaccines. We are also at a loss to test new vaccines in humans, as we do not have a deﬁnitive correlate of protection. Improvements in locally available technology at sites of high disease incidence are improving our ability to deﬁne the human cellular response to both infection and disease. These improvements combined with greater communication between groups facilitated by funding of large consortia should dramatically improve our understanding of human disease. Combining this greater understanding with deﬁnitive experimentation within the animal models will allow investigators to determine the protective and pathologic function of speciﬁc cellular responses as well as how best to induce each type of response.

SUMMARY POINTS 1. The induction of naive T cell activation occurs more than a week after delivery of bacteria to the lung. Bacterial dissemination to the draining lymph node correlates with initiation of T cell responses in the draining lymph node. 2. A variety of effector lymphocyte subsets are induced by infection, including multifunctional and cytolytic cells. Not all responding cells make IFN-γ. 3. Phagocyte effector function is key to the ability of the host to express immunity. 4. The MyD88-mediated phagocyte response required for control of Mtb is not a TLRdependent event but likely a result of IL-1R activity. TLR ligation is involved in efﬁcient induction of lymphocyte effector subsets and may be important in the balance between protection and pathologic consequences. 5. The phagocytes that accumulate in the lung in response to Mtb infection represent a complex population that includes cells that express both macrophage and dendritic cell markers. 6. The inﬂammatory lesion is a dynamic environment that is regulated both by the bacteria and by the host. TDM is a key bacterial modulator of the immune response, whereas B lymphocytes, T lymphocytes, and phagocytes are key host regulators of the inﬂammatory site.

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7. Vaccine-induced memory cells need to be either in the lung or capable of being rapidly recruited to the lung in order to be effective in limiting bacterial growth following aerosol infection.

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8. Detailed investigation of the human response to infection is highlighting the complexity of the response. Demonstration of novel lymphocyte subsets as well as novel phagocyte functions highlights the need to maintain an open view as to what constitutes a protective response in humans.

FUTURE ISSUES To improve current vaccine strategies, the ﬁeld should: 1. Determine where bacteria are deposited in the lung during primary infection and why it takes 8–9 days for the acquired immune response to be initiated. 2. Determine the factors, both bacterial and host, that regulate expression of immunity in the lung. 3. Identify effector lymphocyte subsets that can mediate protection within the context of an Mtb-induced inﬂammatory site. 4. Identify novel phagocyte functions that can be activated by vaccine-inducible antigenspeciﬁc lymphocytes. 5. Determine how to augment the protective activity of the acquired cellular response without increasing immune-mediated damage. 6. Determine the potential of B cells, which are plentiful at the lesional site, to modulate immunity and/or immunopathologic consequences. Can we (should we) harness B cells by vaccination? 7. Determine how to generate antigen-speciﬁc lymphocytes capable of persisting in the lung and of responding rapidly to aerosol infection. 8. Continue to enhance the consortium arrangements that promote communication between bacteriologists, animal modelers, and those investigating the human disease.

DISCLOSURE STATEMENT The author is not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS Trudeau Institute librarian, Kelly Stanyon, provided excellent library assistance. I apologize in advance to all the investigators who could not be cited owing to space limitations. I thank my colleagues who kindly provided preprints and informative discussions. A.M.C. is supported by the Trudeau Institute, Inc., and by grants from the National Institutes of Health (AI067723, AI46530, AI069121, AG028878) and the American Lung Association (DeSouza award). 414

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154. Al-Attiyah R, Mustafa A. 2008. Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG. Infect. Immun. 76(9):4190–98 155. Blythe M, Zhang Q, Vaughan K, de Castro RJ, Salimi N, et al. 2007. An analysis of the epitope knowledge related to mycobacteria. Immunome Res. 3:10 156. Appelberg R. 2007. Neutrophils and intracellular pathogens: beyond phagocytosis and killing. Trends Microbiol. 15:87–92 157. Silva M, Silva M, Appelberg R. 1989. Neutrophil-macrophage cooperation in the host defence against mycobacterial infections. Microb. Pathogen. 6:369–80 158. Tan BH, Meinken C, Bastian M, Bruns H, Legaspi A, et al. 2006. Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens. J. Immunol. 177:1864–71 159. Martineau A, Newton S, Wilkinson K, Kampmann B, Hall B, et al. 2007. Neutrophil-mediated innate immune resistance to mycobacteria. J. Clin. Invest. 117:1988–94 160. Garg A, Barnes PF, Porgador A, Roy S, Wu S, et al. 2006. Vimentin expressed on Mycobacterium tuberculosis-infected human monocytes is involved in binding to the NKp46 receptor. J. Immunol. 177:6192–98 161. Vankayalapati R, Garg A, Porgador A, Grifﬁth D, Klucar P, et al. 2005. Role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium. J. Immunol. 175:4611–17 162. Vankayalapati R, Wizel B, Weis S, Saﬁ H, Lakey D, et al. 2002. The NKp46 receptor contributes to NK cell lysis of mononuclear phagocytes infected with an intracellular bacterium. J. Immunol. 168:3451–57 163. Roy S, Barnes P, Garg A, Wu S, Cosman D, Vankayalapati R. 2008. NK cells lyse T regulatory cells that expand in response to an intracellular pathogen. J. Immunol. 180:1729–36 164. Feng CG, Kaviratne M, Rothfuchs AG, Cheever A, Hieny S, et al. 2006. NK cell-derived IFN-γ differentially regulates innate resistance and neutrophil response in T cell-deﬁcient hosts infected with Mycobacterium tuberculosis. J. Immunol. 177:7086–93 165. Fleischmann R, Alland D, Eisen J, Carpenter L, White O, et al. 2002. Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J. Bacteriol. 184:5479–90 166. Glickman M, Cox J, Jacobs W. 2000. A novel mycolic acid synthetase is required for cording, persistence and virulence of Mycobacterium tuberculosis. Mol. Cell 5:717–27 167. Rao V, Fujiwara N, Porcelli S, Glickman M. 2005. Mycobacterium tuberculosis controls host innate immune activation through cyclopropane modiﬁcation of a glycolipid effector molecule. J. Exp. Med. 201:535–43 168. Rao V, Gao F, Chen B, Jacobs WJ, Glickman M. 2006. Trans-cyclopropanation of mycolic acids on trehalose dimycolate suppresses Mycobacterium tuberculosis-induced inﬂammation and virulence. J. Clin. Invest. 116:1660–67 169. Dao D, Sweeney K, Hsu T, Gurcha S, Nascimento I, et al. 2008. Mycolic acid modiﬁcation by the mmaA4 gene of M. tuberculosis modulates IL-12 production. PLoS Pathog. 4:e1000081 170. Kremer K, Glynn J, Lillebaek T, Niemann S, Kurepina N, et al. 2004. Deﬁnition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers. J. Clin. Microbiol. 42:4040–49 171. Reed M, Domenech P, Manca C, Su H, Barczak A, et al. 2004. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 431:84–87 172. Tsenova L, Ellison E, Harbacheuski R, Moreira A, Kurepina N, et al. 2005. Virulence of selected Mycobacterium tuberculosis clinical isolates in the rabbit model of meningitis is dependent on phenolic glycolipid produced by the bacilli. J. Infect. Dis. 192:98–106 173. Sinsimer D, Huet G, Manca C, Tsenova L, Koo M, et al. 2008. The phenolic glycolipid of Mycobacterium tuberculosis differentially modulates the early host cytokine response but does not in itself confer hypervirulence. Infect. Immun. 76:3027–36 174. Ordway D, Henao-Tamayo M, Harton M, Palanisamy G, Troudt J, et al. 2007. The hypervirulent Mycobacterium tuberculosis strain HN878 induces a potent TH1 response followed by rapid downregulation. J. Immunol. 179:522–31

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Enhancing Immunity Through Autophagy ¨ Christian Munz Viral Immunobiology, Institute of Experimental Immunology, University Hospital ¨ ¨ of Zurich, CH-8057 Zurich, Switzerland; email: [email protected]

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Key Words

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

innate immunity, immune escape, MHC class II, T cell survival, tolerance

This article’s doi: 10.1146/annurev.immunol.021908.132537 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0423$20.00

Abstract Next to the proteasome, autophagy is the main catabolic pathway for the degradation of cytoplasmic constituents. The immune system uses it both as an effector mechanism to clear intracellular pathogens and as a mechanism to monitor its products for evidence of pathogen invasion and cellular transformation. Because autophagy delivers intracellular material for lysosomal degradation, its products are primarily loaded onto MHC class II molecules and are able to stimulate CD4+ T cells. This process might shape the self-tolerance of the CD4+ T cell repertoire and stimulate CD4+ T cell responses against pathogens and tumors. Beyond antigen processing, autophagy’s role in cell survival is to assist the clonal expansion of B and T cells for efﬁcient adaptive immune responses. These immune-enhancing functions make autophagy an attractive target for therapeutic manipulation in human disease.

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INTRODUCTION

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Microautophagy: budding of cytoplasmic material into the lysosomal lumen for degradation Macroautophagy: engulfment of cytoplasmic constituents into an autophagosome for delivery to degradation in late endosomes and lysosomes Chaperone-mediated autophagy (CMA): signal peptidedependent protein imported into lysosomes via the LAMP-2A transporter

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The eukaryotic cell uses two main degradation systems to recycle macromolecular building blocks: the proteasome and the lysosome (1). In both systems hydrolytic activity is compartmentalized from the surrounding cytoplasm. The proteasome degrades protein substrates in a catalytic chamber of a barrel-shaped polyprotein complex, whereas in the lysosome lipases, DNAses, RNAses, glycosidases, and proteases are conﬁned by a membrane. Access to the degradative interior of the catabolic structures is tightly regulated through protein adaptors for the proteasome (2) and vesicular transport, as well as through membrane channels for the lysosome (3). In addition to the turnover of malfolded proteins, proteasomal degradation is used for the regulation of short-lived proteins, including transcription factors and cell-cycle controls (4). In contrast, lysosomal proteolysis targets long-lived proteins (5), damaged cell organelles (6), and endocytosed material (3) in the steady state but also breaks down cytoplasmic constituents more indiscriminatingly in times of metabolic stress such as starvation (7). Interestingly, recent evidence is emerging that the same post-translational modiﬁcation, namely ubiquitinylation, might be involved in biodegradative targeting to both systems (8), and we discuss below the possible involvement of this protein signal in the intracellular routes to lysosomes, namely autophagic pathways. The proteasomal and lysosomal degradation pathways have developed to maintain, together with biosynthesis, macromolecular homeostasis in the cell. They are used by the immune system to discard foreign substances, including pathogens. Degradation products or components of pathogens are then sensed by receptors for activation of the innate immune system and presentation to the adaptive immune system. This coordinated usage of catabolic products leads to the initiation of comprehensive and protective immune responses. I discuss in the following section how a group of intracellular pathways, described by the term autophagy, contribute to the restric-

Munz ¨

tion of pathogens, activation of the immune system, antigen presentation to T cells, and the maintenance of T and B cell function. These recently realized functions of a long-known metabolic process, autophagy, offer the possibility of manipulating immune responses for enhanced immunity or tolerance during disease processes.

AUTOPHAGIC PATHWAYS: MICROAUTOPHAGY, MACROAUTOPHAGY, AND CHAPERONE-MEDIATED AUTOPHAGY As mentioned above, the molecular basis of cytoplasmic constituent transport to lysosomes for degradation, called autophagy, has only recently been elucidated. Christian de Duve, one of the founding fathers of modern cell biology, coined this term in 1963 to explain earlier electron microscopic observations of mitochondrial degradation in double-membrane vesicles, a process that had been named autolysis (9). Today, three separate pathways have been identiﬁed that transport cytoplasmic content into lysosomes for degradation (Figure 1). These pathways are called microautophagy, macroautophagy, and chaperone-mediated autophagy (CMA) (10). During microautophagy, whose conservation in higher eukaryotes has not been clearly documented, the lysosomal membrane, called the vacuolar membrane in yeast, buds into the lysosomal lumen, carrying with it cytoplasmic material in an autophagic body. This autophagic body is then degraded with its content by lysosomal hydrolysis. In contrast to direct budding into lysosomes, macroautophagy assembles a vesicle around the cargo, which is destined for degradation. This vesicle, called the autophagosome, envelops its substrate with two membranes. These are assembled from poorly understood membrane sources by the fusion of small transport vesicles into a cup-shaped isolation membrane, which then closes over the autophagic cargo, and fuses with late endosomes and lysosomes to

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PI3K/Beclin-1

Atg12

Autophagosome

Atg5

Atg7 (E1-like) Atg10 (E2-like) Atg12

HSP40

Atg16L1 Bag-1 HSC

70 KFER Q

G140

Hip Hop

HSP90

HSC70

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Chaperone-mediated autophagy Figure 1 Autophagic pathways delivering cytoplasmic constituents for lysosomal degradation. Microautophagy involves budding of cytoplasmic content into the lysosome. Macroautophagy assembles a separate transport vesicle, the autophagosome, using the lipid kinase signaling complex of VPS34 and Atg6/Beclin-1 and two protein conjugation systems with the ubiquitin-like molecules Atg8 and Atg12. Autophagosomes then fuse with endosomes and lysosomes. Chaperone-mediated autophagy imports signal peptide–containing proteins via LAMP-2A into lysosomes.

amphisomes and autolysosomes, respectively. In these fusion vesicles, the inner autophagosome membrane and the autophagosome cargo are degraded by lysosomal hydrolysis. More than 30 autophagy-related genes (atg) have been identiﬁed as crucial for this process in yeast. Although mammalian homologs and functions are still being deﬁned for most of these genes, two ubiquitin-like systems and one lipid kinase signaling complex involved in autophagosome formation have been characterized and are now frequently targeted by immunologists for their studies on innate and adaptive immunity (10). One of these systems couples Atg8 to phosphatidylethanolamine (PE) on the outside and inside of the assembling au-

tophagosome membrane. Six Atg8 homologs exist in higher eukaryotes such as humans: MAP1LC3A, MAP1LC3B, MAP1LC3C, GABARAP, GABARAPL1/GEC1/Apg8L, and GABARAPL2/GATE-16 (11). For PE coupling, Atg8 is processed by the cytosolic protease Atg4 to cleave off ﬁve C-terminal amino acids and to make a C-terminal glycine residue (G120) accessible. Atg8 is then activated by the E1-like enzyme Atg7 and conjugated by the E2-like enzyme Atg3 to PE. Whereas Atg8 gets recycled from the outer membrane after completion of the autophagosome by Atg4, Atg8 that is coupled to the inner membrane stays associated with the autophagic vesicle and is degraded with this membrane by lysosomal hydrolysis. Therefore, ﬂuorescent

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Autophagosome: double-membrane vesicle delivering macroautophagy substrates for lysosomal degradation Atg: autophagyrelated gene Adaptive immunity: immune responses imprinting immunological memory by expansion and preservation of cell clones with somatically recombined receptors

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protein–tagged Atg8 and its mammalian homolog LC3 have been used to visualize autophagosomes (12, 13). In addition, lipidated Atg8/LC3 (LC3-II) runs with a smaller apparent molecular weight of 16 kD in SDS gel electrophoresis than uncoupled Atg8/LC3 (LC3-I), and its turnover can be used as a quantitative measure of macroautophagy (14). Therefore, Atg8 is not only an essential protein for autophagosome formation, but also for experimental monitoring of macroautophagy. Its function in autophagosome formation is, however, much less clear. Two functions have been suggested for Atg8. First, Atg8 may facilitate the fusion of membrane vesicles for the assembly of the isolation membrane and then the completion of autophagosomes. Along these lines, liposomes containing PE-coupled Atg8 can perform hemifusion, and this hemifusion activity is required for autophagosome extension in yeast (15). The Atg content also governs isolation membrane expansion and autophagosome size (16). Second, Atg8 may tether the forming isolation membrane to the autophagic cargo. Support for this suggestion comes from the identiﬁcation of a protein called p62 (or sequestosome 1) that binds to Atg8/LC3 and also to polyubiquitinylated protein aggregates. However, that this protein is required by autophagosomes in substrate engulfment has not been documented (17, 18). For Atg8 to perform these potentially important functions during autophagosome generation, another ubiquitinlike conjugation system is required. This system involves ligation of Atg12 to Atg5, which then forms a complex with Atg16L1 at the outer isolation membrane. During this conjugation reaction, Atg12 is coupled via its Cterminal glycine residue (G140) to a lysine residue in the Atg5 protein (K149) with the assistance of the E1-activating Atg7 and the E2-conjugating Atg10 enzymes (19). The complex of Atg16L1 and the Atg5-Atg12 conjugate stays with the outer isolation membrane during elongation but leaves it after autophagosome completion. Similar to lipidated Atg8, the function of the Atg5-Atg12/Atg16L1 complex Munz ¨

is not entirely understood. However, because of its requirement in and facilitation of Atg8 conjugation, investigators have proposed that the Atg5-Atg12/Atg16L1 complex functions as an E3-like ligase for Atg8 lipidation, catalyzing this reaction and determining the site of Atg8 attachment (20, 21). All three components of this complex have been targeted by small interfering RNA (siRNA)-mediated silencing or gene knockout to speciﬁcally inhibit macroautophagy (14). For the Atg5-Atg12/Atg16L1 complex to catalyze Atg8 lipidation, autophagosome nucleation needs to be initiated. The complex involved in this contains type III phosphatidylinositol 3-kinase (PI3K or VPS34) and Atg6/Beclin-1, among other components, which modulate the activity of these two proteins (22). VPS34 and Atg6/Beclin-1 have also been targeted to modulate macroautophagy. 3-methyladenine, wortmannin, and LY294002 (which are all PI3K inhibitors) and lithium (which lowers IP3 levels) have been frequently used as pharmacological inhibitors of this pathway (23–25). Atg6/Beclin-1 can be targeted for macroautophagy inhibition by siRNAmediated silencing (14) and haploinsufﬁciency (26), or overexpression leads to macroautophagy stimulation (14). The VPS34/Beclin1, Atg5-Atg12/Atg16L1, and Atg8-PE complexes are all involved in generating a transport vesicle for cytoplasmic constituents to the lysosome, whereas in the third autophagosomal pathway, this translocation across the lysosomal membrane is solved by a transport channel. CMA imports proteins with a signal peptide into lysosomes. A pentapeptide of RNAse A, KFERQ, was initially described as the prototypic signal peptide to target substrate to CMA, but investigators then realized that a more degenerate sequence of a glutamine preceded or followed by one or two basic, one or two hydrophobic, and one acidic residue in a pentapeptide can also fulﬁll this function (27). This signal peptide is recognized by cytosolic HSC70 chaperones, which unfold the CMA substrate and dock to the lysosomal membrane with the help of the cochaperones HSP40, Hip, Hop, Bag-1, and HSP90 (28). The docking

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of the chaperone complex, which guides CMA substrates, is dependent on the C-terminal cytosolic domain of lysosome-associated membrane protein (LAMP)-2A. And, despite this protein’s single transmembrane domain, investigators have suggested that it multimerizes to form the translocation channel for CMA substrates (29). LAMP-2A is assisted in the translocation of CMA substrates into the lysosome by lysosomal HSC70 (30). Regulation of cytosolic or lysosomal HSC70 and LAMP-2A can inﬂuence CMA translocation rates, but CMA is usually only upregulated during prolonged metabolic stress conditions, such as starvation, whereas macroautophagy is the earlier response to these stressors (28). Therefore, at least three pathways exist that deliver cytoplasmic constituents for lysosomal degradation, and for two of them (macroautophagy and CMA), substrates and their characteristics have been identiﬁed in mammals with innate and adaptive immune systems.

AUTOPHAGIC SUBSTRATES To appreciate how the different autophagic pathways might contribute to innate and adaptive immunity, one needs to understand what substrates can be targeted by them. In contrast to the nonselective uptake of cytoplasmic material under starvation conditions for the primary goal of energy generation, ensuring cellular survival, a more speciﬁc substrate selection seems to occur under steady-state conditions and during immune activation. Early metabolic labeling studies showed that short-lived proteins were primarily turned over by proteasomal degradation, whereas long-lived protein levels were primarily inﬂuenced by lysosomal proteolysis (5). Such a long-lived protein is GAPDH (glyceraldehyde-3-phosphate dehydrogenase) with the extraordinarily long halflife of 130 h (31), which has been described as a substrate of CMA (32) and has been found in puriﬁed autophagosomes (33). The structures in which these primarily long-lived proteins are compartmentalized and in which they become substrates for macroautophagy are cell

organelles and protein aggregates (7). This suggests that this degradation pathway is accessed by substrates that are too large for proteasomal degradation and/or cannot be kept soluble by chaperones for delivery to the proteasomal catalytic chamber. Along these lines, a variety of studies have demonstrated the macroautophagic degradation of depolarized mitochondria (6, 34–36), expanded endoplasmic reticulum (37), mature ribosomes (34, 38), excess peroxisomes (39), and protein aggregates (40). For most of these cytoplasmic structures, however, it remains unclear how they are recruited to isolation membranes for macroautophagy. Only the peroxisomal protein Pex14p has been implicated in the selective macroautophagy of peroxisomes via recognition of the N terminus of this protein (41), and it has been suggested that the Cvt19 receptor recognizes substrates for the macroautophagy-related cytoplasm to vacuole pathway (Cvt), by which yeast imports hydrolases into its lysosomal vacuole (42, 43). Similar to Cvt19-mediated recruitment of the isolation membrane to hydrolase precursor aggregates, mammalian autophagosomal membranes likely anchor substrates to their inner isolation membrane. Both PIP3 , a product of the VPS34/Beclin-1 complex, and Atg8/LC3 have been suggested as such anchors for macroautophagy substrates (17, 44). With Alfy and p62/SQSTM1, two proteins were identiﬁed that have PIP3 (FYVE) and Atg8/LC3 binding domains, respectively, and that localize to protein aggregates (18, 44). Interestingly, p62/SQSTM1 also has a binding domain for ubiquitin and promotes polyubiquitinylated protein aggregate formation (18, 45). Polyubiquitinylated protein aggregates accumulate in macroautophagy-deﬁcient hepatocytes and neurons (46–48) and in autophagosomes upon inhibition of lysosomal proteolysis ¨ (D. Schmid, M. Gannag´e & C. Munz, unpublished observations). In addition, ubiquitination regulation inﬂuences the macroautophagy of mature ribosomes (38). Such polyubiquitinylated protein aggregates are formed in neurodegenerative proteinopathies, such as Huntington’s

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and Alzheimer’s disease (40). Indeed, polyglutamine-expansion mutations, such as those seen in pathogenic huntingtin variants, promote protein aggregation, and both huntingtin and Alzheimer precursor protein have been documented as macroautophagy substrates (49–51). Encouragingly, macroautophagy stimulation was able to ameliorate neurodegeneration in mouse and ﬂy models of Huntington’s disease (52). These studies suggest that polyubiquitinylation may serve as a general signal for degradation, directing soluble proteins for proteasomal degradation and protein aggregates for macroautophagy. Such a speciﬁcity in autophagic cargo selection broadens the role of this catabolic pathway from its essential role for survival during starvation (53) to an essential housekeeping function in the removal of damaged and

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excess organelles, as well as pathogenic protein aggregates. This function should not be underestimated, as it might signiﬁcantly contribute to the longevity of an organism (7, 22). In addition, selectivity to macroautophagy also enables the immune system to use it for innate immunity by targeting pathogens for degradation (see Figure 2).

RESTRICTION OF INTRACELLULAR PATHOGENS BY MACROAUTOPHAGY The ability of macroautophagy to remove large cytoplasmic structures such as organelles and protein aggregates with selectivity enables this pathway to be used to clear intracellular bacteria, parasites, and viruses. Because macroautophagy also seems to be the only pathway by

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Figure 2 Innate immunity by and immune escape from macroautophagy. Both free cytosolic pathogens such as group A Streptococcus and microbial phagosomes containing, for example, Mycobacterium tuberculosis are delivered by autophagosomes for lysosomal degradation. Some pathogens interfere with macroautophagy at two steps. Shigella ﬂexneri prevents its engulfment by macroautophagy, and herpesviruses inhibit autophagosome formation. Several RNA viruses (e.g., picornaviruses) and bacteria (e.g., Brucella abortus) seem to stabilize autophagosomes in order to replicate at their surface or within them. This autophagosome stabilization is achieved presumably by inhibition of autophagosome fusion with lysosomes. 428

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which large structures can be delivered for hydrolytic destruction, it might be involved in the cell autonomous clearance of all intracellular pathogens. In case this mechanism fails, infected cells are left with the only other option of progression to cell death and clearance of the pathogen, as well as cellular debris by phagocytes. With respect to bacterial and parasitic pathogens, macroautophagy is able to target both free microbes in the cytosol and pathogencontaining phagosomes (54). An excellent example for the former is the clearance of group A Streptococci (GAS), which escape from endosomes via streptolysin O, a hemolytic toxin that they encode (55). Interestingly, Atg5, and therefore macroautophagy-dependent clearance of GAS, was associated with the formation of large Atg8/LC3-positive autophagic vacuoles 10 μm or larger in diameter that had engulfed GAS chains, whereas conventional mammalian autophagosomes for metabolic turnover are usually 0.5–1.5 μm in diameter (7). This indicates quite some ﬂexibility in the system and suggests that macroautophagy cargo can determine the size and form of the isolation membrane. It is tempting to speculate that the large GAS-containing autophagosomes are generated by isolation membrane recruitment to the pathogen surface, possibly in a receptormediated fashion. A similar uptake of cytosolic replicating bacteria has been suggested for Rickettsia conorii (56) as well as metabolically inhibited Listeria monocytogenes (57). The other, more common mechanism by which macroautophagy clears intracellular bacteria and parasites seems to be targeting the pathogen-containing phagosome. The most extensively studied pathogen in this respect is Mycobacterium tuberculosis (Mtb), which conditions the phagocytic vesicle after uptake primarily by macrophages to avoid fusion with lysosomes (58). Macrophage activation by interferon (IFN)-γ, for example, leads to enhanced fusion of bacterial phagosomes with autophagosomes, delivering the pathogencontaining vacuoles for lysosomal degradation.

Interestingly, IFN-γ-induced members of the immunity-related p47 GTPases (IRGs) in mice, and overexpression of LRG-47, one of the 23 IRGs in mice, enhances autophagosome formation and macroautophagy-dependent clearance of Mtb-containing phagosomes in mice (58). In contrast to mice, the three human IRGs are not IFN-γ inducible, but overexpression of one member, IRGM, also leads to enhanced macroautophagic clearance of vacuolar Mtb in human myeloid cells (59). It is now obviously of interest how human IRGM levels are regulated and which stimuli could lead to Mtb clearance via this mechanism in human cells. In addition to the delivery of the bacterial pathogen to lysosomes, macroautophagy seems also to deliver bactericidal substances to the Mtb degradation compartment, which is probably an amphisome or autolysosome. Interestingly, ubiquitin, which in the form of polyubiqitinylated protein aggregates becomes part of macroautophagy substrates, is broken down to bactericidal peptides and gives rise to part of the Mtb-destroying activity delivered by autophagosomes (60). Therefore, macroautophagy delivers not only pathogens for lysosomal degradation, but also additional bactericidal substances for the elimination of bacteria. Similar to the Mtb-containing vesicles, conditioned phagosomes are also used by the parasite Toxoplasma gondii for its replication and can be targeted by both mouse IRGs and macroautophagy (61–63). However, in contrast to the macroautophagy upregulating role suggested for IRGs during Mtb infection, the mouse IRGs IIGP1 and IGTP seem to disrupt the T. gondii–conditioned parasitophorous vacuole membrane and even the parasite’s membrane, exposing it for degradation by macroautophagy (62, 63). This mechanism of T. gondii destruction seems to be efﬁciently induced by CD40-mediated stimulation of infected macrophages (61). These studies suggest that pathogen-conditioned phagosomes might need processing for efﬁcient clearance of the contained bacteria and parasites. Conﬁrming this notion, Salmonella enterica induces

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macroautophagy and replicates in conditioned vacuoles (Salmonella-containing vacuoles), which were only efﬁciently targeted to autophagic delivery for lysosomal destruction when the Salmonella-containing vacuole was damaged by the type III secretion apparatus of this pathogen (64). Interestingly, some Salmonella bacteria escape from the Salmonella-containing vacuole and accumulate in polyubiquitinylated proteins on their surface (65), which could mark them for destruction. In addition to the cell autonomous innate immunity for the clearance of pathogens by macroautophagy, this process has also been shown to contribute to the survival of cells targeted by bacterial exotoxins such as Vibrio cholerae cytolysin (66). Exposure to this cytolysin induces macroautophagy, and Atg5-deﬁcient cells are more susceptible to cytolysin-induced cell death. Therefore, macroautophagy might not only protect against pathogens by targeting them for destruction, but it might also protect cells from their exotoxins. These mechanisms could also contribute to the restriction of gut commensals to mucosal surfaces. Evidence of this comes from genetic association studies in Crohn’s disease, a form of idiopathic inﬂammatory bowel disease (67–69). The gut inﬂammation in this disease is thought to come from the uncontrolled invasion of commensals into the mucosa. This inﬂammation was found to be associated with mutations in atg16L1 (T300A) and IRGM (T313C), and these two genes are, as outlined above, involved in autophagosome generation and the targeting of bacterial vacuoles for lysosomal degradation. Thus, macroautophagy conﬁrms resistance to bacterial and parasitic pathogens by different mechanisms and might be crucial to maintaining a peaceful coexistence between host and commensals at mucosal surfaces. In contrast to macroautophagy’s role in protection against bacterial and parasitic pathogens, relatively little is known about its protective function against viruses during innate immunity. Although autophagosome accumulation has also been observed during several Munz ¨

viral infections, such as human parvovirus B19 (70) and hepatitis C virus (71), the contribution of macroautophagy regulation during innate immunity against these pathogens remains poorly deﬁned. Indeed, only herpesvirus particles of herpes simplex virus 1 (HSV1) have been observed in autophagosomes of infected cells (72). In vivo, Atg6/Beclin-1 overexpression protected against encephalitis by Sindbis virus infection (73) and was required for innate immune responses against tobacco mosaic virus in plants (74). However, how macroautophagy contributes to protection against these viruses remains unclear. One interesting protection mechanism was suggested by studies on vesicular stomatitis virus (VSV) and Sendai virus infection of plasmacytoid dendritic cells (DCs) (75). This study demonstrated that recognition of these RNA viruses by Toll-like receptor 7 (TLR7), one pathogen-associated molecular pattern (PAMP) recognition receptor, was dependent on the delivery of cytosolic viral replication intermediates to endosomes via macroautophagy. Atg5-dependent TLR7 recognition elicited then secretion of antiviral type I IFNs. These ﬁndings, however, seem to be cell-type dependent because VSV infection of mouse embryonic ﬁbroblasts (MEFs) elicited less type I IFN in the presence of macroautophagy than in its absence (76). The cytosolic RNA helicase retinoic acid–inducible gene I (RIG-I) seemed primarily to mediate stimulation of type I IFN production in VSVinfected MEFs, and Atg5-Atg12 conjugates directly interacted with RIG-I and the downstream type I IFN effector IFN-β promoter stimulator 1 via their caspase recruitment domains. Therefore, macroautophagy regulation by viruses can inﬂuence pathogen detection by PAMP recognition receptors for the secretion of antiviral type I IFNs during innate immune responses. In summary, macroautophagy seems to mediate innate immunity to bacterial, parasitic, and viral pathogens by different mechanisms, ranging from directly degrading intracellular microbes to assisting host cells to obtain resistance to infection.

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REGULATION OF MACROAUTOPHAGY BY PATHOGENS Most microbes are probably efﬁciently cleared by autophagic and other innate immune mechanisms. However, the small subset of bacteria and viruses that we recognize as pathogens has developed strategies to outsmart our defenses. Therefore, it is not too surprising that many also target macroautophagy. These pathogens target two steps of macroautophagy, autophagosome formation and its fusion with lysosomes. A good example of escape from engulfment by autophagosomes is the bacterium Shigella ﬂexneri (77). S. ﬂexneri secretes the IcsB protein, which blocks S. ﬂexneri recognition by the autophagic machinery. Therefore, IcsB-deﬁcient S. ﬂexneri gets trapped in autophagosomes and becomes restricted in its replication. At a later stage of efﬁcient conversion of autophagosomes into degradative compartments by acidiﬁcation and fusion with lysosomes, macroautophagy is blocked by metabolically active L. monocytogenes (78). These bacteria replicate most efﬁciently in the cytosol but can also be taken up into spacious Listeria-containing phagosomes (SLAPs) in a macroautophagy-dependent manner. They maintain slower replication after damaging these vesicles by listeriolysin O. Listeriolysin O–negative bacteria are degraded after the formation of SLAPs. It is tempting to speculate that listeriolysin O–induced pores abolish autophagosome acidiﬁcation and therefore inhibit lysosomal degradation in SLAPs. Similar to L. monocytogenes, a number of other pathogens seem to establish their replicative niche in autophagosomes by preventing their maturation to degradative compartments. These include Francisella tulatensis, Brucella abortus, Porphyromonas gingivalis, Leishmania mexicana, Chlamydia trachomatis, Coxiella burnetii, Legionella pneumophilia, and Anaplasma phagocytophilum (79–87). In addition to preventing autophagosome fusion with lysosomes or blocking degradation after fusion with lysosomes, some of these bacterial replica-

tive compartments continue to fuse with autophagosomes, presumably beneﬁting from delivered macroautophagy cargo as a source of nutrients for the pathogens. Therefore, bacterial pathogens can both escape from engulfment into autophagosomes and subvert autophagosomes to become their replicative niche. Although the molecular basis of escape from macroautophagy is not well deﬁned for most bacterial pathogens at the level of interaction with the macroautophagic machinery, some viral escape mechanisms have been linked to viral proteins and have been carefully characterized (88). Examples of these mechanisms exist in herpesviruses. For the α-herpesvirus HSV1, ﬁrst the viral gene product ICP34.5 (89) and then a subdomain of this protein (90) have been shown to inhibit macroautophagy. ICP34.5 binds to Atg6/Beclin-1 via its N-terminal domain and prevents autophagosome initiation via this interaction. Although macroautophagy inhibition might not be crucial for efﬁcient HSV1 replication in vitro (91), neurovirulence of HSV1 was signiﬁcantly attenuated by deletion of the Atg6/Beclin-1 interacting domain from ICP34.5. In addition to HSV1, the γherpesviruses Kaposi sarcoma-associated herpesvirus and murine γ-herpesvirus 68 also encode Atg6/Beclin-1-interacting proteins that prevent autophagy (92, 93). The viral Bcl-2 (v-Bcl-2) of Kaposi sarcoma-associated herpesvirus and murine γ-herpesvirus 68 blocks macroautophagy in this fashion at the autophagosome initiation step. Finally the βherpesvirus human cytomegalovirus also inhibits macroautophagy, but the molecular basis for this remains unclear (94). In contrast to these macroautophagy inhibition mechanisms by DNA viruses, some RNA viruses seem to inhibit autophagosome degradation. This mechanism of macroautophagy manipulation has been suggested primarily for poliovirus, a positive-strand picornavirus. Early ultrastructural studies on poliovirus-infected cells have detected the accumulation of doublemembrane vesicles (95). Isolation of these

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vesicles revealed that they apparently contained cytosolic material (96) and could be induced by the expression of the poliovirus proteins 2BC and 3A (97). Atg8/LC3 localizes to these membranes, and their formation can be inhibited by the siRNA-mediated silencing of Atg12 and Atg8/LC3 (98). Furthermore, lipidation of Atg8/LC3 could be induced by transfection of the poliovirus protein 2BC alone (99). The viral replication machinery is assembled on the surface of these double-membrane vesicles, and macroautophagy inhibition decreased virus production, whereas macroautophagy stimulation increased poliovirus yield (98). Thus, it was suggested that poliovirus stabilizes autophagosomes and prevents their fusion with lysosomes to use their membranes as scaffolds for its replication. Autophagosome accumulation was also observed during infection with the ﬂavivirus dengue virus (100). Analogous to poliovirus replication, dengue virus titers increased upon macroautophagy stimulation and decreased in Atg5-deﬁcient MEFs. Similar claims were made for rhinoviruses (98) and the coronavirus mouse hepatitis virus (101). However, at least for the replication of mouse hepatitis virus, macroautophagy does not seem to be required in macrophages and MEFs because loss of Atg5 in these cell types did not decrease the viral yield (102). These data could suggest that the generation of replication compartments for RNA viruses might only require a subset of Atgs, and therefore these vesicles might not be classical autophagosomes. Instead of hijacking autophagic membranes for its replication, human immunodeﬁciency virus (HIV) seems to use macroautophagy for CD4+ T cell depletion. Both inhibition of this prosurvival pathway in infected T cells (103) and induction of cell death, which is dependent on macroautophagy induction, in uninfected bystander T cells (104) might contribute to immunodeﬁciency induced by HIV. Although the molecular basis by which HIV could downregulate macroautophagy in infected cells remains unclear, autophagosome accumulation in bystander T cells seems to be induced by the interaction between the HIV envelope protein and

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the chemokine receptor CXCR4. These ﬁndings demonstrate once more that HIV is not well adapted to the human species because although these multiple T cell depletion mechanisms might initially give the virus the advantage of unchallenged replication, in the end, they efﬁciently deplete the viral host cell. A cleverer usage of the autophagic machinery is demonstrated by the ﬂavivirus bovine viral diarrhea virus, which has incorporated part of the Atg8/LC3 sequence for speciﬁc proteolytic processing of its polyprotein by the Atg4 protease presumably (105). This strategy enables some isolates of the virus to use the conserved Atg4 protease to liberate viral proteins from its polyprotein. Thus, both viruses and bacteria regulate macroautophagy for their beneﬁt primarily by escaping macroautophagy-mediated restriction or utilizing autophagic membranes for replication niches, and for some this adaptation is necessary for their pathogenicity.

TLR AGONISTS, CYTOKINES, AND TNF FAMILY RECEPTORS IN THE MODULATION OF MACROAUTOPHAGY Cytokines and surface receptors involved in both innate and adaptive immune response signaling have evolved to counteract the immune escape mechanisms of pathogens. Among these, TLRs; the tumor necrosis factor (TNF) family receptors CD40L, TNF-R, and TNF-related apoptosis-inducing ligand (TRAIL); and IFNs have been implicated in augmenting macroautophagy via their signaling (Figure 3). Investigators have suggested that, among the signals for innate immune activation, TLR2, -3, -4, -7/8, and -9 inﬂuence macroautophagy (106–108). Most of these studies have been performed in the murine macrophage RAW264.7 cell line and primary mouse or human macrophages. Engagement of TLR2 by its ligand zymosan led to the recruitment of the macroautophagy machinery to late endosomes, which facilitated lysosome fusion with phagosomes (106). The authors suggested, on the basis of ultrastructural analysis, that this was

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Figure 3 Macroautophagy regulation by signaling molecules of the immune system. PAMP receptors, tumor necrosis factor (TNF) family receptors, and cytokines modulate macroautophagy by regulating autophagosome formation and fusion with lysosomes and endosomes. Toll-like receptor (TLR) 4, 7, 8, and 9 (as well as TNF, IFN-γ, and TRAIL receptor) induce the formation of autophagosomes, whereas IL-4, IL-13, and Ipaf signaling inhibits it. CD40 and TLR2 signaling enhances autophagosome fusion with lysosomes and endosomes.

an independent use of the macroautophagy machinery for endosome maturation, because the analysis did not reveal double membranes engulﬁng the phagosome. However, one should keep in mind that the amphisome forms in an intermediate step in macroautophagy, and these vesicles are not surrounded by two membranes, but rather resemble late endosomal multivesicular bodies. Therefore, part of the enhanced phagosome fusion with lysosomes could oc-

cur after autophagosome fusion with late endosomes. In addition to TLR2, stimulation of TLR3, -4, -7, and -8 increased macroautophagy after incubation with polyinosinicpolycytidylic acid, lipopolysaccharide, singlestranded RNA, and imiquimod, respectively (107). Among these, TLR7/8 signaling restricted the growth of bacillus Calmette-Gu´erin mycobacteria in mouse macrophages. A similar mycobacterial clearance assay was used

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to demonstrate the functional relevance of macroautophagy enhancement by TLR4 stimulation with lipopolysaccharide (108). With respect to TLR9 stimulation by CpG-rich DNA sequences, enhanced macroautophagy as measured by LC3-II accumulation was reported in one study (106), but to a lesser extent in another (107). Therefore, there is a hierarchy in the ability of TLR engagement to stimulate macroautophagy, and TLR4 and -7/8 mediate the strongest induction of autophagosome formation. In contrast to macroautophagy regulation by TLRs, little is known so far about cytosolic PAMP recognition receptors, such as the RNA helicases RIG-I and mda5 and the NOD-like proteins or about their inﬂuence on autophagosome formation. Only one NOD-like protein, Ipaf, seems to inhibit macroautophagy induction by Shigella infection (109). Therefore, macroautophagy regulation by PAMP recognition receptors might be ﬁnetuned by activating and inhibitory signals given by different receptors upon recognition of several pathogen constituents. In addition to TLR and NOD-like protein engagement, cytokines of both innate and adaptive immunity have been demonstrated to regulate macroautophagy. These include type I and II IFNs, TNF-α, and the Th2 cytokines IL-4 and IL-13. The type I IFN-inducible double-stranded RNA-dependent protein kinase R (PKR) was required for restriction of HSV1 infection in vitro and in vivo (89, 90). Attenuated neurovirulence of HSV1 deﬁcient in the Atg6/Beclin-1 interacting domain of ICP34.5 was dependent on PKR, and PKRdeﬁcient mice were unable to control mutant HSV1 infection (90). Although we can indirectly deduce type I IFNs’ effects on macroautophagy from the dependence of viral clearance on IFNα/β-inducible factors, the exposure of mouse macrophages to type II IFN has been shown directly to upregulate autophagosome formation and the clearance of mycobacteria via macroautophagy (58). IFN-γ-mediated upregulation of macroautophagy seems to proceed more efﬁciently and with faster kinetics in mouse than in human cells (110), pos-

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sibly because mouse, but not human, IRGs are INF-γ inducible (59). Apart from IRGs, IFN-γ might also induce macroautophagy via deathassociated protein kinases (DAPKs). Human epithelial cells increase macroautophagy after overexpression of DAPK and its close relative DAPK-related protein kinase 1 (111). In addition to type I and II IFNs, TNF-α can stimulate macroautophagy. However, this has only been shown for cells with low levels of NF-κB activation (112). Ewing sarcoma cells were susceptible only to TNF-α-mediated macroautophagy upregulation after compromising their ability to activate NF-κB. Finally, counteracting these macroautophagy-inducing cytokines (which, in part, all support the development of efﬁcient antiviral and antitumor Th1-polarized immune responses), Th2 cytokines such as IL-4 and IL-13 seem to block macroautophagy induction (113). Because Th2-polarized immune responses efﬁciently support humoral immunity against extracellular pathogens, the hallmark cytokines of this T cell polarization might not need to induce macroautophagy for intracellular pathogen restriction. These studies suggest that a number of cytokines of both the innate immune system [IFN-α/β produced by plasmacytoid DCs, TNF-α secreted by myeloid DCs, and natural killer (NK) cells and IFN-γ produced by NK cells] and the adaptive immune system (IFN-γ, IL-4, and IL-13 secreted by T cells) can regulate macroautophagy for the clearance of intracellular pathogens and might also facilitate antigen processing for T cell stimulation via this pathway as discussed below. Apart from these soluble mediators of innate and adaptive immunity, several surface receptors involved in the communication among components of the immune system also reportedly regulate macroautophagy. Among these, TRAIL increased macroautophagy in human epithelial cells (114). Accordingly, macroautophagy induction by TRAIL was compromised by inactivation of Fas-associated death domain, the signaling adaptor protein of the TRAIL receptor. Similar to TRAIL-mediated macroautophagy induction, other cell death– inducing stimuli have been shown to induce

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macroautophagy, and in most settings this seems to be the ﬁnal strategy of cells to rescue themselves from apoptosis. However, the interplay among macroautophagy, apoptosis, and necrosis differs among experimental systems and has been reviewed recently (115). Another TNF family receptor shown to modulate macroautophagy is CD40L. Upon engagement by CD40, T. gondii–containing phagosomes were delivered more efﬁciently to lysosomes in a macroautophagy-dependent manner (61). CD40L-mediated clearance of T. gondii worked efﬁciently in mouse and human macrophages. Finally, and unrelated to TNF family receptors, B cell receptor (BCR) signaling was implicated recently in TLR9 recruitment to autophagosome-like compartments (116), but it remains unclear if macroautophagy is involved in this recruitment, or if TLR9 and BCR colocalize in amphisomes, which also receive input from autophagosomes. Nevertheless, the immune system can enhance macroautophagy after PAMP recognition and through communication between lymphocytes using both surface receptors and cytokines. This macroautophagy modulation assists in the clearance of intracellular pathogens and is used for antigen presentation to the adaptive immune system, which I discuss next.

ANTIGEN PRESENTATION ON MHC MOLECULES AFTER AUTOPHAGY Not only are cellular degradation systems such as the proteasome and lysosome used for the elimination of unwanted intracellular material, such as invading pathogens, but their products also are utilized to alarm the immune system. This is achieved by displaying products of these degradation systems for the activation of innate lymphocytes such as NKT cells and T cells for adaptive immunity. Autophagy is no exception to this rule, and its substrates can be processed for antigen presentation. In this section, I focus primarily on antigen processing for MHC class II presentation because most available data suggest a role for autophagy in delivering intra-

cellular proteins for CD4+ T cell stimulation. However, I speculate at the end of this section on possible roles for autophagy in glycolipid presentation on CD1d and antigen processing onto MHC class I molecules. CD4+ and CD8+ T cells monitor peptides generated by lysosomal and proteasomal degradation, respectively. These are presented on MHC class I molecules to CD8+ and on MHC class II molecules to CD4+ T cells. MHC class I ligands are derived primarily from short-lived cytosolic or nuclear proteins, such as cyclins (117), and defective ribosomal products that are rapidly degraded after faulty protein biosynthesis (118). Peptides, the products of proteasomal hydrolysis, are then transported into the endoplasmic reticulum by the transporter associated with antigen processing, where they can be further trimmed by the aminopeptidase ERAAP before loading onto newly synthesized MHC class I molecules (119, 120). Only stable complexes of high-afﬁnity peptides with MHC class I molecules are then released and transported to the cell surface for presentation to CD8+ T cells. In contrast, MHC class II molecules are loaded in late endosomes, called MHC class II– containing compartments (MIICs) (121). MHC class II molecules reach this compartment under the guidance of a chaperone called invariant chain (Ii). Ii not only prevents premature peptide loading onto MHC class II molecules in the endoplasmic reticulum, but also carries sorting signals in its cytosolic domain that steer the Ii/MHC class II complex to the MIICs. Ii is degraded in the MIICs by lysosomal proteolysis, and its last remnant, the class II associated Ii chain peptide, is then replaced by high-afﬁnity peptides with the help of the chaperone HLADM in humans or H2-M in mice. The complex of high-afﬁnity peptide ligands and MHC class II molecules is then transported to the cell surface for CD4+ T cell immune surveillance. MHC class II ligands are generated by lysosomal proteolysis, and their source proteins can reach the MIICs by endocytosis. However, there is now mounting evidence that a substantial proportion of MHC class II ligands is also derived from intracellular source proteins and

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Figure 4 Antigen processing for major histocompatibility complex (MHC) class II presentation via autophagy. Both macroautophagy and chaperone-mediated autophagy deliver antigens for presentation on MHC class II molecules to MHC class II–containing compartments (MIICs). These antigens are then degraded by lysosomal hydrolysis for loading onto MHC class II molecules with the assistance of the chaperone HLA-DM.

EBV: Epstein Barr virus LCL: lymphoblastoid cell line

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that autophagic pathways contribute to the delivery of these to the MIICs (Figure 4). Indeed, mammalian autophagosomes fuse with late endosomes prior to fusion with lysosomes (122), and the fusion vesicles, called amphisomes, have been characterized ultrastructurally by electron microscopy (123, 124). Isolated amphisomes are multivesicular and multilamellar late endosomes. The same morphology has been described for the MIICs (125), which may be conventional late endosomes equipped with the molecular machinery for loading of MHC class II molecules in antigen-presenting cells. In addition to the morphological similarity between amphisomes and MIICs, autophagosomes fuse frequently with MIICs (13). In these studies, we labeled autophagosomes with GFP-Atg8/LC3 and quantiﬁed their fusion with MIICs by im-

Munz ¨

munoﬂuorescence microscopy. The MIICs were visualized as HLA class II+ HLADM+ LAMP-2+ vesicles. In human epithelial cell lines, primary monocyte-derived DCs, and Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCLs), we documented that more than 50% of MIICs received input from autophagosomes. Ultrastructurally, GFP-Atg8/LC3+ HLA class II+ compartments resembled large multivesicular endosomes in immunogold electron microscopy. Interestingly, GFP-Atg8/LC3 and MHC class II molecules were primarily localized in close proximity to the intravesicular membranes, which have been suggested to primarily support MHC class II loading with antigenic peptides owing to colocalization of HLA-DM and MHC class II molecules (125). In addition, colocalization of Atg8/LC3 with MHC

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class II molecules in F. tularensis–containing vesicles of infected macrophages was observed, providing further evidence for the fusion of autophagosomes with MIICs (126). Therefore, autophagosomes fuse frequently with MIICs, and Atg8/LC3 could deliver substrates to the intravesicular membranes of these late endosomal compartments for MHC class II loading. Evidence that the fusion between autophagosomes and MIICs leads to MHC class II loading comes from the analysis of the natural ligands of MHC class II molecules (Table 1). The analysis of MHC class II–bound peptides revealed that 20–30% originates from cytosolic and nuclear proteins (127–129), and these could be processed for MHC class II presentation at least in part by autophagic pathways. Table 1

As described above, Atg8 is partially degraded with the autophagosome content by lysosomal hydrolysis. Peptides of two mammalian Atg8 homologs, LC3 and GABARAP, have been isolated from human and mouse MHC class II molecules, respectively (129, 130). LC3 (MAP1LC3B93−109 and MAP1LC3B93−110 ) and GABARAP (GABARAP29−45 ) fragments were found to be natural ligands of HLA-DR and H2-Ag7 molecules on LCLs and mouse pancreatic β cell lines. In addition to these components of the molecular machinery for macroautophagy, the known autophagy substrate GAPDH (32, 33) is the most frequent cytosolic source protein for natural MHC class II ligands (117, 129). It has been eluted from ﬁve different MHC class II alleles but not

Antigen processing for major histocompatibilty complex (MHC) class II presentation after autophagy

Antigen

Evidence for autophagy involvement

Presenting cell type

Reference

Self-antigens eluted from MHC class II molecules Atg8/MAP1LC3B

Component of macroautophagy machinery

Human B-LCL

129

Atg8/GABARAP

Component of macroautophagy machinery

Mouse pancreatic β-cell line

130

GAPDH

Autophagy substrate

Human B-LCL

117

Rad23

Enhanced MHC class II presentation after autophagy stimulation

Human B-LCL

129

HSP70

Enhanced MHC class II presentation after autophagy stimulation

Human B-LCL

129

EF-1α

Enhanced MHC class II presentation after autophagy stimulation

Human B-LCL

129

Cathepsin D

Enhanced MHC class II presentation after autophagy stimulation

Human B-LCL

129

Complement C5

3-MA inhibition of CD4+ T cell recognition

Mouse B cells and macrophages

131

Mucin 1

3-MA and wortmannin inhibition of CD4+ T cell recognition

Human dendritic cells

132

NeoR

3-MA and wortmannin inhibition of CD4+ T cell recognition

Human B-LCL and epithelial cell lines

133

Mtb 85B

Atg6/Beclin-1 siRNA inhibiton of CD4+ T cell recognition

Mouse macrophages and dendritic cells

EBNA1

3-MA and Atg12 siRNA inhibition of CD4+ T cell recognition

Human B-LCL and B cell lines

134

GAD65

Enhanced CD4+ T cell recognition after LAMP-2A overexpression

Human B-LCL

137

SMA

Enhanced CD4+ T cell recognition after LAMP-2A overexpression

Human B-LCL

137

CD4+ T cell antigens

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C. Jagannath, personal communication

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from MHC class I molecules. These studies suggest that known autophagy substrates and molecules that are delivered by autophagosomes for degradation are frequently loaded onto MHC class II molecules. Presentation of autophagy substrates by MHC class II molecules is of functional relevance because it can lead to T cell stimulation. This was initially described for complement C5 overexpressed in mouse B cells, macrophages, and ﬁbroblasts (131). Whereas B cells and ﬁbroblasts presented intracellular C5 to CD4+ T cell lines directly, macrophages did so only after partial inhibition of lysosomal proteolysis. The intracellular route, but not processing after extracellular addition for C5 presentation on MHC class II molecules, was sensitive to 3-methyladenine (3-MA), a pharmacological inhibitor of type III PI3 kinases and therefore of macroautophagy (23). However, although 3-MA-mediated inhibition of MHC class II presentation can serve as a ﬁrst indication that macroautophagy is involved in antigen processing, one has to be cautious as additional effects on endocytosis, lysosomal acidiﬁcation, phosphorylation during signal transduction, and mitochondrial permeability have been reported (14). Nevertheless, similar methodology was used to implicate macroautophagy in the antigen processing onto MHC class II for the tumor antigen Mucin 1 (MUC1) and the model antigen neomycin phosphotransferase II (NeoR) (132, 133). MUC1 is processed by a 3-MA-sensitive pathway for CD4+ T cell stimulation after MUC1-encoding RNA electroporation of DCs. NeoR was processed by an intracellular route in LCLs and in renal cell carcinoma cell lines onto MHC class II molecules. This presentation was sensitive to type III PI3K inhibition by 3-MA and wortmannin, whereas tyrosinase presentation on MHC class I molecules to CD8+ T cells and NeoR processing onto MHC class II molecules after extracellular addition were not. The same treatment prevented the access of intracellular NeoR to lysosomal compartments, suggesting that macroautophagy is involved in the delivery of NeoR to MIICs for lysosomal processing

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and MHC class II presentation to CD4+ T cells. Another group of model antigens that can be used to investigate antigen processing onto MHC class II molecules after macroautophagy comprises those targeted to autophagosomes. We demonstrated that macroautophagy targeting of Atg8/LC3 fusion proteins with inﬂuenza matrix protein 1 (MP1) and the tumor antigen NY-ESO-1 enhances MHC class II presentation of these antigens 5- to 20-fold (13; M. ¨ Gannag´e & C. Munz, unpublished data). This targeting strategy was effective in human epithelial cells, LCLs, and monocyte-derived DCs. Enhanced MHC class II presentation of antigen-Atg8/LC3 fusion proteins was dependent on the molecular machinery of macroautophagy. Fusion proteins with a mutant Atg8/LC3, in which the C-terminal glycine residue was mutated to alanine and could therefore no longer be coupled to PE in the autophagosomal membrane, failed to show enhanced processing onto MHC class II molecules. Furthermore, the siRNA-mediated silencing of Atg12 abolished colocalization of the fusion constructs with MIICs. Therefore, access to autophagosomes results in efﬁcient antigen processing onto MHC class II molecules. In contrast to antigen targeting for enhanced MHC class II presentation and CD4+ T cell stimulation, only two pathogen-derived antigens have been found to require macroautophagy for their presentation on MHC class II molecules to CD4+ T cells. One of these is derived from Mtb, whose clearance by macroautophagy is discussed above. Presentation of the 85B antigen of Mtb (a signiﬁcant candidate vaccine antigen against this mycobacterium) on MHC class II molecules could be enhanced by rapamycin-mediated stimulation of macroautophagy (C. Jagannath, personal communication). This process required live Mtb infection and was sensitive to the siRNA-mediated silencing of Atg6/Beclin-1. In line with macroautophagy’s involvement in 85B processing onto MHC class II molecules, rapamycin treatment localized Mtb to autophagosomes in infected

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macrophages. Interestingly, this treatment enhanced in vivo priming of 85B-speciﬁc CD4+ T cells by Mtb-infected DCs and therefore demonstrated a role for MHC class II presentation after macroautophagy for the ﬁrst time in vivo. The second pathogen-derived antigen for which macroautophagy contributes to MHC class II presentation to CD4+ T cells is the nuclear antigen 1 of EBV (EBNA1) (134). EBNA1 is processed for MHC class II presentation by an intracellular route, making it the most consistently recognized CD4+ T cell antigen of latent EBV infection (135). This intracellular processing involves macroautophagy because EBNA1 could be visualized in autophagosomes and developing isolation membranes by electron microscopy (134). Macroautophagy of EBNA1 contributes to MHC class II presentation because EBNA1-speciﬁc CD4+ T cell recognition of LCL could be inhibited by 3MA and siRNA-mediated silencing of Atg12. Therefore, macroautophagy seems to visualize a subset of pathogen-derived antigens to the adaptive immune system by delivering them for MHC class II presentation to CD4+ T cells. EBNA1 is a long-lived protein that protects itself from rapid proteasomal turnover by its glycine-alanine repeat. These degradation characteristics are shared with another nuclear protein, RAD23, which is involved in nucleotide excision repair. RAD23 carries the UBA2 domain that prevents its proteolysis by the proteasome in cis (136). Interestingly, a RAD23-derived peptide was among the four most signiﬁcantly upregulated natural ligands isolated from MHC class II molecules of LCLs, which had been starved to increase autophagy (129). The others were derived from HSP70, elongation factor-1α, and cathepsin D. Interestingly, starvation-induced autophagy primarily increased the presentation of cytosolic and nuclear antigens but did not signiﬁcantly affect the display of membrane and secreted proteins on MHC class II molecules. These ﬁndings again argue that autophagy delivers primarily cytosolic and nuclear antigens, which are not efﬁciently processed by the proteasome, for MHC class II presentation.

Although most evidence for intracellular antigen processing for MHC class II presentation implicates a role for macroautophagy in this process, other autophagic pathways might also contribute. Indeed, two autoantigens, glutamate decarboxylase 65 and the mutant human immunoglobulin κ light chain SMA, are more efﬁciently presented on MHC class II molecules after the overexpression of LAMP2A, the transporter involved in protein import into lysosomes by CMA (137). Glutamate decarboxylase 65 required processing by the cytosolic proteasomes prior to transport by CMA (138). Even though signal peptide sequences mediating CMA for MHC class II presentation have not been identiﬁed in these autoantigens, these studies suggest that multiple pathways, targeting presumably different subsets of intracellular antigens, might contribute to cytosolic and nuclear antigen processing for CD4+ T cell stimulation. Although most studies suggest that autophagy contributes primarily to MHC class II presentation of antigens, autophagy might also assist in antigen processing for classical and nonclassical MHC class I molecules. Levine’s (139) laboratory has recently shown that clearance of cell corpses, a process giving rise to MHC class I and II cross-presentation by phagocytes, is critically dependent on the capacity of the dying cells to perform macroautophagy. Indeed Atg5- and Atg6/Beclin-1deﬁcient cells failed to produce apoptotic bodies that could display phosphatidylserine on their outside to trigger uptake by phagocytes, and they could not release lysophosphatidylcholine to attract them. This led to incomplete cavitation during early embryonic development and the accumulation of apoptotic corpses in atg5-deﬁcient mice. Furthermore, pharmacological inhibition of macroautophagy with 3-MA accumulated cell corpses during retina development in chickens (140). As in mice, macroautophagy seemed to be required to generate ATP for the surface presentation of phophatidylserine and the efﬁcient engulfment of apoptotic bodies by neighboring cells. Thus, macroautophagy could facilitate

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cross-presentation onto both MHC class I and II molecules by rendering dying cells more susceptible to uptake by phagocytes, including professional antigen-presenting cells such as DCs. In addition to assisting antigen presentation by these classical MHC molecules, macroautophagy possibly could deliver, in addition to proteins, other pathogen-derived constituents for lysosomal degradation and detection by the immune system. Indeed, as discussed above, it might do so by facilitating the detection of PAMPs by TLRs (75). Moreover, the nonclassical MHC class I molecule CD1d has been described to acquire its glycolipid ligands +

Atg6/Beclin-1 or Atg7 +

Atg5-/-

SP

Th2

CD4+

Atg5-/-

Atg5-/-

SP

CTL

CD8+

Atg5-/-

Atg5-/-

Pro-B

Pre-B

B-1

Figure 5 Requirement for macroautophagy for B and T cell development and survival. In the absence of macroautophagy, peripheral T cell numbers are severely diminished. Th2-polarized CD4+ T cells, however, survive nutrient depletion better after inhibition of macroautophagy via the siRNA-mediated silencing of Atg6/Beclin-1 and Atg7. On the contrary, the expansion of CD8+ T cells is compromised without Atg5, resulting in macroautophagy loss. With respect to B cell development, macroautophagy is required for the transition from pre- to pro-B cells. In addition, B-1 cells need macroautophagy for their survival. 440

Munz ¨

in MIICs (141, 142). Because autophagosomes frequently fuse with the MIICs and because lysosomal hydrolysis allows for lipid processing, macroautophagy might deliver glycolipid ligands for CD1d molecules, which then could be presented to NKT cells. Therefore, autophagy might contribute to antigen presentation to innate and adaptive lymphocytes by different mechanisms, ranging from delivering intracellular material for MHC class II and CD1d presentation to rendering dying cells more susceptible for phagocytosis and cross-presentation.

MACROAUTOPHAGY DURING T AND B CELL DEVELOPMENT AND IMMUNE RESPONSES Another mechanism by which macroautophagy inﬂuences adaptive immunity is by inﬂuencing the development and survival of adaptive lymphocytes (Figure 5). Whereas plasmacytoid DCs seem to develop normally in the absence of macroautophagy caused by Atg5 deﬁciency (75), macroautophagy-deﬁcient B and T cell differentiation and immune responses are deregulated. Both CD4+ and CD8+ T cell development is compromised in mice, with conditional deletion of Atg5 in T cells (143). In addition to reduced peripheral Atg5−/− T cell numbers, these macroautophagy-deﬁcient T cells also proliferate less in response to stimulation. Therefore, the prosurvival functions of macroautophagy seemed required both during T cell development for recent thymic e´ migr´es and during expansion in the periphery. However, macroautophagy might not be beneﬁcial for all and especially for differently polarized immune responses. In humans, Th2polarized CD4+ T cells, which are thought to support humoral immune responses most efﬁciently, seemed to accumulate autophagosomes to higher and probably even unhealthy levels (144). In these cells, the siRNA-mediated silencing of Atg7 and Atg6/Beclin-1 protects from cell death after nutrient withdrawal, whereas Th1-polarized T cells, which conversely support primarily cell-mediated immune responses, might be protected from cell

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death by macroautophagy. These data suggest that Th2-polarized cells already ﬁght for their survival with higher macroautophagy levels, and nutrient withdrawal drives them more readily into cell death via macroautophagy. Similar to T cell development, pro-B cells require macroautophagy during their transition to preB cells, which results in decreased peripheral Atg5-deﬁcient B1 cell numbers (145). B-1a cell survival is further compromised in the absence of macroautophagy. These studies point toward a role for macroautophagy in the survival of T and B cells during selection processes in their development. Furthermore, these lymphocytes with strong proliferative capacity might require macroautophagy again during immune responses to expand to protective levels.

POSSIBLE ROLES OF MACROAUTOPHAGY IN IMMUNITY AND TOLERANCE INDUCTION OF THE ADAPTIVE IMMUNE SYSTEM Macroautophagy contributes to immunity at three levels. It directly removes intracellular pathogens, visualizes these to the adaptive immune system by facilitating antigen presentation onto MHC class II molecules, and ﬁnally supports adaptive lymphocyte survival for optimal expansion during protective immune responses. Although considerable evidence for these protective functions of macroautophagy has been collected, a role during the maintenance of tolerance against self-tissues is just slowly emerging. Two main mechanisms guard humans from autoreactivity by T cells. These are central and peripheral tolerance (146, 147). During central tolerance induction, positive selection in the thymic cortex and negative selection in the thymic medulla ensure that T cells have low afﬁnity to selfMHC molecules (positive selection), but those that recognize self-peptides strongly get eliminated (negative selection). The cell types involved in this education process are cortical and medullary thymic epithelial cells and

thymic DCs. Whereas DCs can efﬁciently process self-antigens for MHC class II presentation after endocytosis, thymic epithelial cells have less endocytic potential. They might compensate for this by their increased macroautophagic activity. Indeed, the thymic cortical epithelium of GFP-LC3 transgenic macroautophagy reporter mice demonstrated one of the highest levels of autophagosome content among all analyzed tissues (12). Interestingly, autophagosome accumulation was particularly pronounced at an early age, paralleling the agedependent decline of thymic T cell output. Indeed, positive and negative T cell selection is compromised in macroautophagy-deﬁcient thymii (148). A recent study reported that two out of ﬁve transgenic CD4+ T cell speciﬁcities were inefﬁciently selected through Atg5negative thymic tissue. Furthermore, mice with Atg5-deﬁcient thymii developed autoimmunity. Therefore, self-antigen presentation on MHC class II molecules after macroautophagy seems required for central tolerance induction during CD4+ T cell development. Autoreactive T cell speciﬁcities that escape negative selection in the thymus, however, need to be silenced by peripheral tolerance induction. Two main cell types have been implicated in mediating this process: immature DCs and lymph node stromal cells (147, 149). We have demonstrated considerable macroautophagy levels in immature DCs (13). Therefore, self-antigen presentation on MHC class II molecules after autophagy might contribute to central and peripheral tolerance induction in the CD4+ T cell compartment. Further evidence that macroautophagy is important for tolerance maintenance comes from genetic studies in one inﬂammatory bowel disease, Crohn’s disease. Single-nucleotide polymorphisms in Atg16L1 (67, 69, 150) and IRGM (68) were found to be associated with this disease. It is unclear if these mutations result in macroautophagy deﬁciency. Indeed, it has been demonstrated that at least the T300A Atg16L1 variant is not a loss-of-function mutant. Therefore, it is tempting to speculate that these mutations either impair speciﬁc

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Peripheral tolerance: elimination of autoreactive lymphocyte reactivity in peripheral tissues Central tolerance: thymic education of T cells

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autophagic innate resistance mechanisms to gut commensals, allowing them to cause inﬂammation after invasion, or compromise tolerance by self-antigen presentation on MHC class II molecules at this mucosal surface, which is then easily broken by proinﬂammatory microbial stimuli at this site. A third mechanism by which macroautophagy might support tolerance is by facilitating apoptotic body clearance (139, 140). Failure of apoptotic body clearance has been implicated in the pathogenesis of systemic lupus erythematosus (151). The accumulation of apoptotic bodies in systemic lupus erythematosus might provide an elevated self-antigen reservoir for autoantibody development. Possibly counteracting this mechanism, macroautophagy seems to be required to expose phosphatidylserine on the surface of dying cells for efﬁcient uptake by phagocytes (139, 140). Indeed, Atg5 or Atg6/Beclin-1 cells were less efﬁciently phagocytosed after apoptosis. Thus, macroautophagy might facilitate tolerance maintenance in the adaptive immune system at several levels by processing antigens for MHC class II presentation during central and peripheral tolerance induction, by balancing immune activation and ignorance against gut commensals, and by facilitating self-antigen removal to avoid buildup for autoreactive B cell stimulation.

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CONCLUDING REMARKS The characterization of the molecular machinery for autophagy allows for the evaluation of its contribution to innate and adaptive immunity. This pathway seems to be used for the clearance of intracellular pathogens and the processing of pathogenic constituents for activation of the adaptive immune system. Similar to proteasomal proteolysis for antigen presentation on MHC class I molecules to CD8+ T cells, autophagy might play a crucial role both in shaping the CD4+ T cell repertoire via self-antigen processing onto MHC class II molecules for central and peripheral tolerance induction and in guiding CD4+ T cell responses through microbial antigen presentation on MHC class II molecules. Furthermore, autophagy contributes with its prosurvival function to the metabolic ﬁtness of immune system components, allowing for efﬁcient expansion of adaptive lymphocytes during immune responses. These supportive roles of autophagy for innate and adaptive immunity have now been documented in individual model systems, but their exact role during immune responses against pathogens and tumors in vivo needs to be investigated next. Therapeutic interventions into this pathway should be considered only with a detailed understanding of how autophagy interferes with immunity during human disease.

DISCLOSURE STATEMENT The author is not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS My research is supported by the Dana Foundation’s Neuroimmunology program, the Arnold and Mabel Beckman Foundation, the Alexandrine and Alexander Sinsheimer Foundation, the Burroughs Wellcome Fund, the Starr Foundation, the National Cancer Institute (R01CA108609 and R01CA101741), the National Institute of Allergy and Infectious Diseases (RFP-NIH-NIAIDDAIDS-BAA-06-19), the Foundation for the National Institutes of Health (Grand Challenges in Global Health), and an Institutional Clinical and Translational Science Award (to the Rockefeller University Hospital). 442

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13. Quantification of autophagosome fusion with MIICs.

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58. Demonstrates that macroautophagy can clear pathogenconditioned phagosomes.

69. Together with Refs. 67 and 68, demonstrates that a mutation in Atg16L1 is associated with Crohn’s disease.

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Alternative Activation of Macrophages: An Immunologic Functional Perspective Fernando O. Martinez,1 Laura Helming,2 and Siamon Gordon1 1

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom; email: [email protected], [email protected]

2

Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany

Annu. Rev. Immunol. 2009. 27:451–83

Key Words

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

IL-4, IL-13, M2, macrophage polarization, parasite infection, allergy

This article’s doi: 10.1146/annurev.immunol.021908.132532

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0451$20.00

Macrophages are innate immune cells with well-established roles in the primary response to pathogens, but also in tissue homeostasis, coordination of the adaptive immune response, inﬂammation, resolution, and repair. These cells recognize danger signals through receptors capable of inducing specialized activation programs. The classically known macrophage activation is induced by IFN-γ, which triggers a harsh proinﬂammatory response that is required to kill intracellular pathogens. Macrophages also undergo alternative activation by IL-4 and IL-13, which trigger a different phenotype that is important for the immune response to parasites. Here we review the cellular sources of these cytokines, receptor signaling pathways, and induced markers and gene signatures. We draw attention to discrepancies found between mouse and human models of alternative activation. The evidence for in vivo alternative activation of macrophages is also analyzed, with nematode infection as prototypic disease. Finally, we revisit the concept of macrophage activation in the context of the immune response.

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INTRODUCTION Macrophages (Ms) were initially recognized by Elie Metchnikoff as phagocytic cells responsible for pathogen elimination and housekeeping functions in a wide range of organisms, from invertebrates to vertebrates. In 1905, he summarized the evidence that phagocytic mononuclear cells from animals resistant to certain bacterial infections were more adept at killing those and unrelated bacteria, setting the basis for the concept of “macrophage activation” (1). However, 60 more years of combined work were required to deﬁne how Ms become more efﬁcient bacterial killers. In their work regarding M activity in acquired resistance to intracellular bacteria, Mackaness, North, and their colleagues realized that protection against infection was mediated not only by humoral, but also by independent cellular factors (2, 3). In this response they deﬁned a form of acquired resistance to intracellular bacteria that could not be passively transferred with serum, yet induced a spectrum of consistent morphologic and functional changes in the antibacterial activity of Ms. Importantly, they noted that the antibacterial mechanism was not directed exclusively against the organism that provoked it. Bloom, Bennett, and David were among those who helped to identify lymphocytes as the major antigen-speciﬁc cells responsible for M microbicidal activation and consequent transfer of resistance against families of different intracellular pathogens (4, 5). Soon thereafter, the key factor for collaboration between these cells was discovered: interferon (IFN)-γ (6). IFN-γ is produced by activated CD4+ T helper 1 cells (Th1), CD8+ T cytotoxic 1 (Tc1) cells, and natural killer (NK) cells. This cytokine converts resting Ms into potent cells with increased antigen presenting capacity, increased synthesis of proinﬂammatory cytokines and toxic mediators, and augmented complement-mediated phagocytosis. Thus, Ms acquire the capacity for killing of bacteria, especially intracellular pathogens, and perhaps tumors. As the ﬁrst type of antimicrobial M activation to be rec-

MΦ: macrophage

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Th: T helper cell (Th1, Th2, Th17)

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ognized, it became known as “classical activation of Ms.” In 1982, the immunological counterpart of IFN-γ, interleukin (IL)-4, was identiﬁed by Howard, Vitetta, Paul, and colleagues (7). Coffman, Mosmann, and collaborators provided evidence that IFN-γ and IL-4 are produced by mutually inhibitory CD4+ T helper cells: Th1 and Th2 (8). In naive T helper cells, IL-4 and IFN-γ genes are silent; upon TCR engagement and costimulation Th cells begin to choose between Th1 and Th2 cell fates. Knowledge of mutual exclusion led to the discovery that Th2 cells also produce IL10 to suppress Th1 cells (8). Th2 cells also secrete IL-13, a cytokine that partially shares ligand binding receptor complexes with IL-4 (9). In contrast to IFN-γ, IL-4 and IL-13 mediate immune responses typically characterized by eosinophilia, basophilia, mastocytosis, enhanced B cell class switching, and antibody production, with consequent plasma accumulation of IgE and IgG1. Th2 responses are essential for the control of extracellular parasites, including helminths, protozoa, and fungi, but also contribute to allergy, increased susceptibility to other pathogens, and complications of infection such as ﬁbrosis (Table 1). Initial observations regarding the role of IL-4 in M activation showed that this cytokine was able to inhibit the respiratory burst and the production of IL-1β and IL-8 (10). It was also shown that IL-4 induced MHC class II expression, and M-M fusion and that IL-4 came to play an essential role in M-mediated control of the protozoon Trypanosoma cruzi (11–13). Importantly, it was found that IL-13 induces both redundant and nonredundant effects to those of IL-4 in Ms (9). With the ﬁnding in 1992 of upregulation of mannose receptor (MRC1) as a distinctive marker of IL-4-activated Ms, together with the induction of MHC class II antigens, the concept of alternative activation was proposed, stating, “IL-4 in an inﬂammatory focus would cause recruited macrophages to acquire an entirely different phagocytic receptor and secretory capability compared with macrophages classically

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Table 1

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Main features of IL-4 and IL-13 gene regulation and cellular effects

Cytokine family

IL-4 and IL-13 are prototypical four-helix bundle short-chain cytokines, a characteristic of ligands of the class 1 cytokine superfamily

Gene location

The gene for IL-4 resides on mouse chromosome 11 and on the long arm of human chromosome 5. The IL-13 gene lies immediately downstream of the IL-4 locus

Gene regulation

Studies of DNAse1 hypersensitivity, DNA methylation, and permissive marks in naive T cells show that speciﬁcation of the IL-4/IL-13 transcriptional state is driven by an ordered sequence of regulatory element activation/deactivation events. At the naive stage, preactivation of hypersensitivity site V (HSV) sufﬁces to maintain low levels of IL-4 and IL-13 transcriptional permissivity

Receptor

For IL-4, the receptors are type I and type II. For IL-13, the receptors are type II and IL-13Rα2

Effect on B cells

IL-4/IL-13 induce the isotype switch and secretion of IgE by B lymphocytes

Effect on T cells

IL-4 drives the differentiation of naive Th0 lymphocytes into Th2 lymphocytes and can prevent apoptosis in T cells. T lymphocytes lack IL-13 receptors

Effect on MΦs

IL-4/IL-13 enhance the capacity for ﬂuid-phase pinocytosis and endocytosis IL-4/IL-13 inhibit autophagy in Ms IL-4/IL-13 induce M fusion/giant cell formation IL-4/IL-13 inhibit NO production/increase arginase activity IL-4/IL-13 treatment leads to enhances MHCII expression/antigen presentation IL-4/IL-13 can inhibit macrophage-mediated killing of pathogens IL-4 induces the expression of mediators of tissue remodeling and inhibits phagocytosis of latex beads. Effects of IL-13 on tissue remodeling or phagocytosis have not been determined

Effect on other cells

IL-4/IL-13 promote ﬁbroblast proliferation, and collagen synthesis IL-4/IL-13 augment the expression of adhesion molecules and proﬁbrotic cytokines from ﬁbroblasts IL-4/IL-13 augment the ability of human mesenchymal cells to contract collagen gels IL-4/IL-13 inhibit bone resorption by osteoclasts

activated by IFN-γ treatment or BCG infection” (14). In the following years, we learned that the phagocytic and secretory proﬁle of Ms could be further modiﬁed by many other self and pathogen-derived signals (15–17). The main stimuli associated with distinctive M phenotypes include glucocorticoids (GC) recognized by the GCR (glucocorticoid receptor), IL-10 recognized by IL-10R1, and immune complexes recognized by the Fc receptor family, among others. The fact that these phenotypes are all alternatives to classical activation and show a partial overlap with the effects of IL-4 and IL-13 has fuelled confusion in the ﬁeld over the past decade. Since the discovery of activation and its heterogeneity, several classiﬁcation schemes have been proposed, the most recent of which deﬁnes classically activated M (caM) as M1, and the group of non-caM as M2a-c (16). Before discussing the uniqueness of IL-4/IL-13 or IFN-γ activa-

tion in relation to other forms of M stimulation, and the classiﬁcation system in use, we review and clarify current knowledge concerning IL-4-/IL-13-activated Ms, based on recent ﬁndings regarding cytokine production, signaling, and effects on Ms. In addition, we assess primary evidence for alternative activation in vivo, with emphasis on parasitic disorders and allergy, and discuss where we are in the ﬁeld, critically analyzing the less developed areas.

IL-4 AND IL-13 RECEPTORS AND SIGNALING IL-4 and IL-13 Receptors and Adaptors IL-4 is recognized by the membrane receptor IL-4Rα, a unique member of the common gamma chain (γc ) family of receptors (18), characterized by conserved structural motifs in the extracellular region (19). These motifs include www.annualreviews.org • Alternative Activation of Macrophages

GC: glucocorticoids caMΦ: classically activated macrophage γc : IL-2 receptor common γ-chain

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STAT6: signal transducers and activators of transcription protein 6

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conserved paired cysteine residues and, in the membrane proximal region, a WSXWS motif required for the conformation of the receptor chain, which mediates speciﬁc cytokine binding (20). The IL-4Rα chain binds IL-4 with high afﬁnity (21), leading to dimerization with another protein to form either type I or type II receptors (Figure 1a). In most cells of the hemopoietic lineage, the type I receptor arises by recruitment of the γc chain (22, 23). In nonhemopoietic and myeloid hemopoietic cells, IL-4Rα can recruit IL-13Rα1 (24), forming the type II receptor. Dimerization leading to the formation of either type I or type II receptors induces a cytoplasmic signaling cascade that involves the Janus kinase family (25–27), the insulin receptor substrate family (23), and the phosphoinositide 3-kinase (PI3K) pathway. Janus kinase activation leads to phosphorylation of STAT6 (signal transducer and activator of transcription 6), which dimerizes, migrates to the nucleus, and binds to promoters of genes (28) (Figure 1b). The type II receptor is the main functional receptor for IL-13 (21). In addition, IL-13 is recognized with high afﬁnity by the IL-13Rα2 receptor (29), endowed with a very short intracellular domain. Kawakami and colleagues (29) found that IL-13Rα2 undergoes internalization after ligand binding without causing signaling and proposed a role as decoy receptor. Furthermore, while IL-13 efﬁciently caused activation of STAT6 protein in cells transfected with the IL-13Rα1 and IL-4Rα chains, IL-13Rα2 inhibited this activation (29). Novel ﬁndings challenge the status of the IL-13Rα2 as a decoy receptor in Ms (30). Fichtner-Feigl and colleagues (30) showed that IL-13 and tumor necrosis factor (TNF) cooperate to induce the expression of IL-13Rα2; IL-13 then signals through IL-13Rα2 to activate an AP-1 variant containing c-jun and Fra-2, which activates the promoter of tumor growth factor (TGF)β, leading to its production. Prevention of IL-13Rα2 expression, gene silencing, or blockade of IL-13Rα2 signaling led to marked downregulation of TGF-β production in oxazolone-induced colitis and bleomycinMartinez

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induced lung ﬁbrosis (30). These data suggest that IL-13Rα2 signaling may become functional during prolonged inﬂammation (30). Although the signaling of IL-4 and IL-13 has been considered equivalent, there are differences between them in terms of signaling intensity and kinetics. The epithelial carcinoma cell line A549 has been a useful model to study signaling by the common type II receptor, since it expresses IL-4Rα and IL-13Rα1, but not the γc or the decoy receptor IL-13Rα2 (31). In this model, IL-4 induced STAT6 phosphorylation at ﬁve- to tenfold lower concentrations than IL-13. IL-4 signaling in the human B cell line Ramos, which expresses γc chain and IL-4Rα, stimulated tyrosine phosphorylation of STAT6 with a dose-response similar to that observed in A549. At all concentrations, IL-4 rapidly induced tyrosine phosphorylation of STAT6 in A549, reaching plateau levels between 10– 15 min of stimulation, whereas the response to IL-13 was substantially slower. The relative delay in STAT6 phosphorylation induced by IL-13 via the type II receptor complex was most apparent at lower concentrations of the cytokines (31).

Interaction Between IL-4/IL-13 Pathway and Other Signaling Pathways The IL-4/IL-13 cascades depend on and contribute to other signaling pathways, reminiscent of the cross talk between Toll-like receptor (TLR) and IFN pathways. However, since these ﬁndings are relatively new, the relation between the pathways is not always clear. Emergent examples are represented by interaction with nuclear receptors such as the peroxisome proliferator-activated receptors, PPARδ and PPARγ and the GCR, but also membrane molecules and cytokines such as galectin-3, IL-10, and IL-21, among others. PPARδ and PPARγ seem to coordinate the M transcriptional response to IL-4 and IL-13 at the gene transcription level (32–34). Studies with PPARδ−/− , PPARγ−/− , and PPARδ/γ−/− Ms reveal that both receptors are required

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a IL-4

IL-4

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Type I

IL-13

Type II

Type II

IL-4/IL-13

b

JAK3

JAK1

IRS2

IRS2 STAT6 IRS2 GRB2

STAT6 STAT6 SHC

STAT6 STAT6

PI3K

STAT6 STAT6

Gene expression Figure 1 Recognition and signaling machinery of IL-4 and IL-13. (a) Recently, LaPorte and colleagues (31) deﬁned the three-dimensional structures of the type I (IL-4Rα/IL-4/γc ) and type II (IL-4Rα/IL-4/IL-13Rα1 or IL13Rα1/IL-13/IL-4Rα) ternary signaling complexes (the order shown in parentheses coincides with the order of complex assembly). In these models, the type I complex shows the structural basis for γc ability to recognize six different γc cytokines, whereas type II complexes show an unusual top-mounted Ig-like domain on IL13Rα1 for a novel mode of cytokine engagement, perhaps responsible for the reversal in the IL-4 versus IL-13 ternary complex assembly (21). IL-4Rα is depicted in blue for the three models; γc and IL-13Rα1 are brown for type I and type II complexes, respectively; and IL-4 and IL-13 are fuchsia. (b) Dimerization of the type I and II receptors activates the Janus kinases, Jak1, Jak3, and Jak1, Jak2, Tyk2, respectively (25–27). IRS2, the main member of the insulin receptor substrate family in hemopoietic cells, is also recruited to the receptor complex (23). Phosphorylated IRS activates PI3K and the adaptor Grb2 (18). STAT6 (28) is recruited to the receptor complex and tyrosine phosphorylated. Phosphorylated STAT6 dimerizes, migrates to the nucleus, and binds to promoters of genes (28). Additional signals may be mediated by the recruitment of the adaptor protein Shc. www.annualreviews.org • Alternative Activation of Macrophages

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aaMΦ: alternatively activated macrophage

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for optimal expression of alternative activation markers. Importantly, IL-4 induces the production of PPAR ligands in Ms by induction of the 12/15-lipoxygenase (16). The KO (knockout) mice show that PPARγ regulates primarily metabolic programs in alternatively activated Ms (aaMs), whereas PPARδ is required for the full expression of their immune phenotype, including expression of pattern-recognition receptors (PRRs), and costimulatory molecules and suppression of the M-mediated inﬂammatory response (32–34). In contrast to the PPARs, GCs interact with IL-4 in a synergistic fashion, with the combination of stimuli inducing special features. IL-4 per se induces the loss of surface expression of TGF-βRII, whereas simultaneous stimulation with relatively low concentrations of the GC dexamethasone (1 × 10−8 M) sufﬁces to maintain detectable TGF-βRII on the surface (35). Pharmacological concentrations of dexamethasone lead to enhanced surface expression and prolonged TGF-β-mediated signaling. A similar mechanism is responsible for the expression and function of the membrane protein stabilin1. The combination of GC and IL-4/IL-13 induces an increase and stabilization of the levels of its messenger and protein but, more importantly, activates two distinctive intracellular trafﬁcking pathways for receptor-mediated endocytosis and recycling, resulting in stabilin1 shuttling between the endosomal compartment and the trans-Golgi network. The major role of stabilin-1 seems to be that of a homeostatic scavenger receptor for endogenous signals such as acLDL (acetylated low-density lipoprotein), SI-CLP (chitinase-like protein), and SPARC (secreted protein, acidic, cysteinerich), the delivery of newly synthesized proteins from biosynthetic to secretory pathways, and perhaps mediation of leukocyte adhesion and transmigration (36). Other molecules also inﬂuence the expression of aaM genes in vivo. Galectin-3 is a membrane molecule that participates in a feedback loop that causes sustained PI3K activation via activation of CD98. siRNA-targeted depletion of galectin-3, murine KO models, and Martinez

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speciﬁc galectin-3 inhibitors block the expression of the main aaM markers, thus supporting a role as a key mechanism in IL-4/IL-13 pathways (37). These data conﬁrm the notion that PI3K signaling plays an important role in alternative M activation in vivo. SHIP1 (SH2-containing inositol-5-phosphatase) is a potent negative regulator of the PI3K pathway in hemopoietic cells. Ms in mice with a targeted deletion of SHIP1 had increased expression of alternative activation markers, a property that appears to be dependent on TGF-β present in vivo (38). Ablation of the well-known cytokine IL-10 can also inhibit the upregulation of aaM markers in response to African trypanosomiasis (39). The increase in aaM markers coincides with the appearance of IL-10, which could suggest dependence on IL-10. In this IL10 KO experimental model, of a dozen aaM markers tested, all returned to the levels found in noninfected wild-type (WT) mice. Note that IL-21 receptor KO mice are also deﬁcient in pulmonary aaMs following infection by Nippostrongylus brasiliensis and the ﬁlarial nematode Schistosoma mansoni (40). Does this make IL-10, IL-21, and galectin-3 subcomponents of the IL-4 pathway, as with IFN-β for the IFN-γ and TLR4 pathways? Accurate interpretation of these results and critical analysis of the pathways will be essential to understand the contribution of IL-4/IL-13, and other signals, to alternative activation of Ms in the development of a Th2 immune response, where multiple signals are produced both sequentially and in unison.

THE CELLS BEHIND MΦ ALTERNATIVE ACTIVATION The main characteristics of IL-4 and IL-13, as well as their sources in inﬂammation, have long been known (Table 1). Several cell types produce IL-4 and IL-13, including conventional CD4+ Th2 and CD8+ T cells (41, 42), NKT cells (43), basophils (44), mast cells (44, 45), and eosinophils (46), which suggests that alternative activation can be of both innate and

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acquired origin. Their production is allele restricted in Th2 cells, whereas in innate immune cells the expression is biallelic (47); major epigenetic alterations involving chromatin reorganization and transcription factor activation control their expression. Among other mechanisms, synergistic activation of the 3 IL-4 and CNS1 (conserved noncoding sequence-1) enhancers during Th2 priming leads to high levels of IL-4 and IL-13 transcriptional permissivity, rather than to transcription itself, which correlates with an enhanced capacity of Th2 cells to express either, or both, cytokines (48). Thus, the studies presented here, although focused on IL-4, possibly also reﬂect the production of its immediate neighbor, IL-13. This supposition is strengthened by the fact that many of the effects observed in these models, such as ﬁbrosis, mucus secretion, and parasite expulsion, depend on IL-13 rather than IL-4 (49). Although the origin of the cytokines is broadly known, the precise contribution of each cell type to the bulk of cytokine produced in acute and chronic inﬂammatory settings remains elusive. Helminthiasis and asthma are the most common Th2 disease models used to study IL-4 and IL-13 production and consequent M alternative activation. The involvement of the Th2 arm of the immune system in tissue repair is less clear, although Th2 cytokines, especially IL-13, have been associated with ﬁbrosis, essential for parasite containment, but also for wound healing. Consequently, relatively few studies have addressed this question. Useful transgenic mice have been developed for components of the IL-4/IL-13 pathways, although only recently has IL-4 expression been associated with green ﬂuorescent protein (GFP) expression: GFP+ IL-4+ (50) and the 4get mouse (51) (Table 2). IL-4 is produced at very low concentrations under homeostatic conditions in 4get mice. Baseline GFP expression is approximately 1% of spleen, lung, mesenteric, and peripheral lymph node CD4+ cells (51). This unexpected physiologic expression of the IL-4 gene may account for site-restricted alternative activation of Ms. Among dispersed lung cells,

90% of the GFP+ cells were CD4− . IL-4+ cells increased in response to tissue injury as demonstrated by Loke et al. (52), using sterile peritoneal surgery on 4get mice as the model. Post surgery, there was a sharp accumulation of IL-4-expressing cells in the peritoneal cavity of 4get mice, mainly represented by >90% mature SiglecF+ eosinophils. This early IL-4 response of innate origin was of short duration, while in the presence of the ﬁlarial nematode Brugia malayi it was prolonged, and the cell composition shifted from 90% eosinophils to 50% eosinophils, 40% mast cells, and 10% Th2 cells. As expected, this new inﬁltrate resulted in progression to a late, increased Th2 response that, however, shared M effector functions with the early response. Experiments done in RAG−/− mice, which lack B or T cells, showed no signiﬁcant differences from WT animals in the early effector response, supporting its association with cells of innate origin (52). N. brasiliensis, another commonly studied nematode, causes infections typically associated with intestinal pathology, although the host can develop, in addition, a profound pulmonary mucus response (51, 53, 54). Infection of 4get mice with this pathogen showed that eosinophils were the most prevalent cell type, increasing up to 1000 times, with Th2 cells and basophils comprising three and ten times lower cell numbers, respectively (55). After ten days of infection, 40% of the total CD4+ T cells in the lung spontaneously expressed GFP. Importantly, CD4+ T cells in spleen and draining mesenteric lymph nodes were 15%, with a signiﬁcant 5% CD4+ cell accumulation in nondraining peripheral lymph nodes (51). In response to the nematode Heligmosomoides polygyrus, the cell inﬁltrate was similar to that induced by N. brasiliensis. In this study, the frequency of CD4+ /GFP+ Th2 cells increased in peripheral blood lymphocytes and in all lymphoid organs analyzed, including the draining mesenteric lymph nodes, spleen, and Peyer’s patches, but also in some tertiary sites such as the lung (56). These results show that IL-4 www.annualreviews.org • Alternative Activation of Macrophages

GFP: green ﬂuorescent protein

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Table 2

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Principal animal models developed to study the signaling and effects of IL-4 and IL-131

Molecule

Transgenesis details

Alternative activation phenotype

References

IL-4 KO

IL4tm1Cgn —Disruption by insertion of vector into the ﬁrst exon of the gene IL4tm1Kopf —Disruption by insertion of vector into the third exon of the gene IL4tm1Nnt —Disruption caused by insertion of vector into exon 3 of the gene

Normal expulsion of N. brasiliensis Impaired induction of pulmonary granuloma formation in response to S. mansoni eggs Increased resistance to Leishmania major infection relative to BALB/c control mice Exacerbated EAE Conﬂicting data regarding eosinophilia in asthma

152, 153

IL-13 KO

IL13tm2Anjm —Disruption caused by insertion of vector into exon 3

Failure to expel N. brasiliensis Impaired induction of pulmonary granuloma formation in response to S. mansoni eggs Decreased ﬁbrosis in response to injury

154

IL-4/IL-13 KO

IL4/IL13tm3Anjm —Deletion of a 15-kb region from exon 3 of the Il13 locus to intron 3 of the IL4 locus

Failure to expel N. brasiliensis Induction of pulmonary granuloma formation in response to S. mansoni eggs is abolished Decreased ﬁbrosis in response to injury

154

IL-4Rα KO

IL4ratm1Fbb —Exons 7 through 9 replaced with a single loxP site IL4ratm1Sz —Insertion of vector replaced exons 7–9 of the gene

Failure to expel N. brasiliensis Increased resistance to L. major infection relative to BALB/c control mice Decreased granulomatous pathology, decreased ﬁbrosis, and increased mortality after infection with S. mansoni Attenuated asthma phenotype

123

STAT6 KO

Stat6tm1Aki —The 3 exons encoding the SH2 domain were replaced Stat6tm1Gru —The region encoding the SH2 domain was replaced Stat6tm1Jni —Disruption was caused by insertion of vector into the ﬁrst coding exon

Failure to expel N. brasiliensis Abolition of eosinophilia in asthma

155

GFP+ IL-4+

Hu-Li and colleagues engineered a gene-targeted mouse in which the ﬁrst exon and 178 nucleotides of the ﬁrst intron of the IL-4 gene were replaced by the green ﬂuorescence protein (GFP)

In heterozygous mice, no impairment of the type II response required for OVA allergic airway inﬂammation despite gene disruption

50

4get

Mohrs et al. designed a bicistronic IL-4 reporter in which the GFP was introduced without deleting IL-4, designated IL-4/GFP-enhanced transcript mice (4get)

IL-4 response appears fully conserved

51

LysMCreIL4Rα−/flox

IL4ratm2Fbb —Exons 7–9 ﬂoxed by insertion of loxP sites Intercross between hemizygous IL-4Rα−/ﬂox mice (bearing one ﬂoxed and one disrupted IL-4Rα allele) and transgenic LysMCre mice on an IL-4Rα−/− background

Increased susceptibility to infection with S. mansoni: increased mortality (associated with increased Th1 cytokines, hepatic and intestinal histopathology, increased NO and sepsis) but normal ﬁbrosis Increased resistance/delayed disease progression in cutaneous leishmaniasis

132

1

Further information can be found in the MGI website at http://www.informatics.jax.org.

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tions. ELISAs quantifying IL-13 and IL-4 concentrations showed that IL-13 was tenfold and fourfold higher than IL-4 in media from 3T3L1 adipocytes and primary adipocytes, respectively. The IL-13 produced by adipocytes is relevant to the homeostasis of adipose tissue and regulation of diseases, such as control of insulin resistance associated with the metabolic syndrome.

can be produced outside the primary inﬂammatory locus, thus possibly affecting the activation of widely distributed Ms in secondary and even tertiary organs. While in 4get mice the predominant myeloid cell making IL-4 is the eosinophil, the same pathogen in GFP+ IL-4+ showed that basophils were far and away the most numerous IL-4-producing cells (57). The nature of these differences remains to be deﬁned. GFP+ IL-4+ mice were also used by Chen and colleagues (47) to study the production of IL-4 in an OVA-induced lung allergy model. Despite the gene disruption in GFP+ IL-4+ heterozygous mice, the type II response required for OVA allergic airway inﬂammation was not impaired. The authors found accumulation of GFP+ cells, and thus of IL-4-positive cells, in the bronchoalveolar lavage, where the CD4+ GFP+ cells were twice the number of CD4− GFP+ cells. Among the CD4− GFP+ population, 93% of the cells were eosinophils, 4% monocyte-like cells, and 2.6% blast-like cells. In the Th2 response induced by alum adjuvants, the IL-4 reporter signal was restricted to CD4+ T cells in draining lymph nodes throughout the duration of the experiments, without apparent involvement of innate immune sources (58) (see Table 3). Kang and colleagues (59) demonstrated that in addition to hemopoietic cells, adipocytes are a source of Th2 cytokines, especially IL-13. In 3T3-L1 adipocyte cell lines, expression of IL-13 is dramatically induced upon differentiation from preadipocytes to adipocytes, with similar results in differentiated human adipocytes. IL-4 was also induced in differentiated adipocyte cell lines but was undetectable in human adipocytes under the same condiTable 3

EFFECTOR FUNCTIONS OF ALTERNATIVELY ACTIVATED MΦS IN THE TH2 IMMUNE RESPONSE The connection of classical activation with the well-understood Th1 response, ﬁrst demonstrated at cellular levels and then in molecular detail, soon made it broadly accepted. Alternative activation has remained ill deﬁned for many years, perhaps inﬂuenced by the fact that in contrast to classical activation, it started as an in vitro paradigm, and for years little was done to understand the effector function of Ms in vivo. The goal of the Th2 response is the elimination and control of infection by extracellular pathogens such as helminths. The size of these pathogens impedes their phagocytosis, and thus Ms and dendritic cells (DCs) developed other functions to sample, present their antigens, and perhaps eliminate them. It has been hypothesized that Th2 immune responses control helminth infection by challenging and directly causing damage to the parasites, which in turn deviate their priorities from reproduction to defense; intense mucus production and increased muscle motility promote physical expulsion of the invading organisms. How Ms contribute to this response should

Summary of IL-4 and IL-13 cellular sources in diverse disease models

Cell type

Basal

Injury

Helminths

Asthma

Eosinophils

+

+++

++++++

+++

Basophils

?

?

+++

?

Th2 cells

+

–

+++

++++++

Mast cells

?

?

?

?

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be a direct reﬂection of the changes induced by IL-4 and IL-13 in their general functions and gene expression programs, as discussed in the next section.

Direct Effects of IL-4 on MΦ Function: Phagocytosis, Endocytosis, and Autophagy

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Similarly to IFN-γ, IL-4 and IL-13 modify the function of Ms at various levels, with some mechanisms strictly dependent on direct signaling and others also requiring de novo gene expression. In contrast to IFN-γ, which increases phagocytosis via selected receptors, IL-4, through direct PI3K activation, increases the capacity for ﬂuid-phase pinocytosis, MRC1-dependent endocytosis, as well as MRC1-independent micro- and macropinocytosis (60, 61). At the cellular level, IL-4 increases tubular vesicle formation at the pericentriolar region, associated with trafﬁcking of recycling and early endosomes, concurrent with decreased particle sorting to lysosomes (60). The combination of IL-4 with PGE2 increases the size of early endosomes, endoplasmic reticulum, and Golgi compartments, in parallel with MRC1-mediated and ﬂuid-phase endocytosis (62). IL-4 in combination with GM-CSF also affects lamellipodia formation in Ms via Rac-1 activation, without consequences for the uptake of latex beads (63). In contrast, another study shows that phagocytosis of latex beads is decreased in aaM (64). The endosomal stimulation by IL-4 may support increased antigen uptake and presentation of soluble extracellular antigens, providing a mechanism for cytokinedependent enhancement of antigen uptake in bystander Ms (60). Increased sampling of the extracellular environment by Ms and DCs is crucial for MHC class II presentation of pathogens, such as helminths, that cannot enter the phagocytic pathway. Autophagy, a process that resembles phagocytosis, is a catabolic mechanism involving the degradation of a cell’s own components through its lysosomal machinery. Autophagy is important for innate immunity against intra460

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cellular pathogens such as Mycobacterium tuberculosis (65). IL-4 inhibits autophagosome formation induced by starvation and reduces the number of IFN-γ-induced autophagic vacuoles (65). Importantly, during mycobacterial infection, alternative activation of Ms does not affect phagosome maturation, but inhibits autophagy-dependent maturation and killing of mycobacteria (66). IL-4 may spare investing energy and resources in presentation of intracellular antigens in the antihelminthic response.

IL-4 and MΦ Fusion IL-4 and IL-13 induce M fusion with formation of multinucleated giant cells in vitro (67). This phenomenon can be observed during granulomatous responses induced by infection with several pathogens, e.g., S. mansoni and M. tuberculosis, and is also present during the foreign body reaction. M fusion and therefore multinucleation is also the basis of the formation of osteoclasts induced by RANKL and M-CSF (49, 50). While osteoclasts have the capacity to degrade bone, we can only speculate that IL-4- and IL-13-induced polykaryons may acquire the ability to degrade large components that cannot otherwise be internalized by individual Ms. This may be important for the degradation of large extracellular parasites and foreign materials such as implants. However, M fusion could have other functional consequences such as complementation of the properties of individual Ms and enhanced/decreased antigen presentation, pathogen killing, or secretory activity. The potential beneﬁts or costs of fusion and the contribution of IL-4/IL-13 to M fusion in vivo remain largely unknown. Given that alternative activation induced by IL-4 and IL-13 does not induce giant cell formation in all situations, we can postulate that extra signaling pathways are required, which could originate from the recognition by Ms of foreign materials such as schistosomal eggs, and mycobacteria, among others (68, 69). In line with this hypothesis, adhesion to a permissive substratum but also the inﬂammatory status of the Ms are

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important determinants of efﬁcient IL-4induced giant cell formation (69). The molecular mechanism of M fusion induced by IL-4 is also unknown; however, it is clear that alternative activation leads to the induction of fusogenic molecules on Ms (69). In fact, recent reports identify in mouse the seven-transmembrane receptor DC-STAMP (dendritic cell–speciﬁc transmembrane protein) and Cadherin-E as IL-4-induced fusogenic molecules (64, 70).

IL-4 and IL-13 Gene Signatures in MΦs: A Species Matter Although the signaling cascade induced by IL-4/IL-13 modulates key effector functions of Ms such as endocytosis, those effects cannot be compared with the repercussions of de novo IL-4-associated gene programs (16, 71– 74). These gene expression programs determine to a great extent the altered functions acquired by aaMs. In the past decade, information about IL-4 effects on M gene expression has grown exponentially owing to the sequencing of the complete human and murine genomes and the establishment of tools to assess gene expression at full genomic level. Efforts by several investigators have yielded comprehensive gene proﬁles of IL-4- and IL-13-stimulated Ms, providing novel candidate markers for alternative activation while clarifying differences among species and between these cytokines and other inﬂammatory factors (16, 71–74). Importantly, although the general functions and behavior of murine and human aaMs are expected to be highly conserved, our own unpublished data suggest that the genes modulated by IL-4 in each species are different to some extent. Quantitatively, the changes in the M transcriptome induced by IL-4 and IL-13 alone are more restricted than the gene expression programs induced by LPS or LPS in combination with IFN-γ, but similar in magnitude to the effect of IL-10, IFN-γ alone, oxidized LDL, and GCs (16). Qualitatively, although distinctive, the nature of IL-4 and IL-13 proﬁles at times

overlaps with those of other cytokines, e.g., IL-10 and GC (16). Before discussing the main gene signatures associated with IL-4 and IL-13, note that most of our current knowledge of aaM gene transcription originates from studies carried out in murine models, whereas human studies are scanty (Table 2). Thus, it will be crucial to keep updating this subject as further evidence is produced. Among the group of genes extensively studied in mouse Ms, arginase 1 (ARG1) is a prototypic alternative activation marker. The expression of ARG1 induces a shift of arginine metabolism from the IFN-γ-induced production of NO via iNOS toward production of ornithine and polyamines, which are important for wound healing (75). Similar to IFN-γ-driven tryptophan depletion, arginine depletion in IL-4-treated Ms can also lead to inhibition of T cell proliferation (76). Although homologs exist in humans, both ARG1 and iNOS induction are conﬁned mainly to murine Ms (77, 78). Other murine restricted markers lack homologs in humans including YM1 and YM2 (CHI3l3 and CHI3l4), members of the chitinase family (79, 80), and FIZZ1 (found in inﬂammatory zone 1, RETNLA) (80, 81). YM1 and YM2 are members of the glycosyl hydrolase 18 family, which includes other hydrolases such as the acidic mammalian chitinase (AMCase), an enzyme able to hydrolyze chitin, a polymer of N-acetylglucosamine. Chitin is not expressed in mammalian systems but is abundant in the structural coat of fungi, the exoskeleton of many arthropods, and parasitic nematodes. Both YM1 and YM2 lack chitinase activity, and although it has been hypothesized that they function as lectin binding proteins or cytokines, no clear evidence exists in support of this function. Human Ms also have restricted expression of alternative activation markers. An interesting gene signature present in human but not in mouse aaMs is that of nucleotide G protein–coupled receptors (GPCRs). IL-4 activation is characterized by the expression of a restricted group of GPCRs, ﬁve of which constitute nucleotide and sugar nucleotide www.annualreviews.org • Alternative Activation of Macrophages

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receptors: GPR86, GPR105 (also P2Y14), P2Y8, P2Y11, and P2Y12 (16). Three of these, P2Y12, GPR105, and GPR86, are linked on chromosome 3. Preliminary data suggest that nucleotides and sugar nucleotides are released during cell injury and perhaps by pathogens, and thus IL-4 can make Ms responsive to a whole new set of environmental signals (82, 83). One postulate of the concept of M alternative activation is the acquisition of an entirely different phagocytic receptor repertoire. Of all the murine and human alternative activation– associated receptors, MRC1 has been most comprehensively studied. It is detected in most tissue Ms but also in hepatic and lymphatic endothelia, as well as in glomerular mesangial cells. MRC1 was identiﬁed because of its ability to recognize endogenous glycoproteins, such as lysosomal hydrolases, but MRC1 also mediates the uptake of mannosylated antigens, cell-cell contact through its interaction with l-selectin, binding and uptake of agalactosyl IgG, and importantly, pathogen recognition. MRC1 interacts with a wide range of microorganisms including the fungus Candida albicans and several species of mycobacteria and Leishmania (84). Current data suggest a role for MRC1 in cell signaling, perhaps associated mainly with inhibition of IL-12 production in response to endotoxin and production of the antiinﬂammatory cytokines IL-10 and IL-1Ra (IL-1β receptor antagonist) and of the nonsignaling IL-1RII (IL-1β decoy receptor) (85). In addition to upregulating MRC1 expression, IL-4 and IL-13 regulate a broad set of phagocytic receptors. IL-4 and IL-13 upregulate M scavenger receptor 1 (MSR1) with clear roles in pathogen recognition and lipoprotein clearance, the C-type lectin-like receptor Dectin-1, with speciﬁcity for β-1,3- and β1,6-linked glucans, found in fungi and some bacteria, and a known role in TLR2 signaling (85). DC-SIGN, also an alternative activation marker, was ﬁrst identiﬁed through its binding to ICAM-2 and 3, facilitating antigen presentation and costimulation (85). This receptor plays a role in innate recognition of a plethora of pathogens including M. tuberculosis,

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C. albicans, Helicobacter pylori, Leishmania mexicana, and S. mansoni. Less well described markers of human M alternative activation are DCIR and DCL-1 (71, 86–89). IL-4 and IL-13 induce the expression of MGL1/2 (CLEC10A in humans) and CD23 in both species. Functionally, murine MGL has been documented to be involved in the recognition and endocytosis of galactosylated glycoproteins (90, 91), where MGL1 binds preferentially to Lewis X moieties and MGL2 shows speciﬁcity for α-GalNAc or β-GalNAc residues (92). MGL1 has been implicated in M homing, granuloma maintenance (93–96), and suppression of proinﬂammatory cytokines (97). The other postulate of the alternative activation concept is the acquisition by Ms of a secretory repertoire entirely different from that of classical activation. It is well accepted that IL-4 and IL-13 mediate repression of proinﬂammatory cytokines and induction of an antiinﬂammatory milieu (16). In addition to antagonizing the secretion of IL-12, IL-1β, TNF, and IL-8 induced by classical activation and other proinﬂammatory factors (11, 98), IL-4 and IL-13 have other means to downregulate a pro-Th1 response. In humans, aaMs express less IL-1β but also more IL-1RII and IL-1Ra (99). Downregulation of caspase-1, the enzyme responsible for the activation of IL-1β, contributes to downregulation of its secretion (72); importantly, this system also processes IL-18. However, this property may change in certain conditions, in which IL-4 may increase the production of inﬂammatory cytokines (100). Human and murine aaMs secrete a number of ﬁbrogenic factors such as ﬁbronectin 1 (FN1) and matrix associated protein βIG-H3 (74, 101), and the coagulation factor XIII (F13A1) (102), which bears transglutaminase activity. They also produce insulin-like growth factor 1 (IGF1) (71, 103) and platelet-derived growth factor C, which provide signals for tissue proliferation and repair (101). One function of IFN-γ is ampliﬁcation of the inﬂammatory inﬁltrate, through production of selected chemokines. Importantly, one

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of the stronger gene signatures also found in IL-4- and IL-13-activated Ms is that of a distinct set of chemokines (71). Compared with other stimuli, IFN-γ and IL-4/IL13 are the strongest for selective chemokine induction (71). IL-4 upregulates a group of six chemokines in human Ms, including CCL13, CCL14, CCL17, CCL18, CCL22, and CCL24. Three of the upregulated human chemokines lack murine orthologs: CCL14 (only human), CCL18 (pseudogene in the mouse), and CCL23 (low homology to murine CCL6), whereas CCL17 and CCL24, which do have murine orthologs, are exclusively upregulated in humans. In mouse Ms, IL-4 induces CCL2, which represents the homolog of human CCL13. Information about regulation of other murine chemokines is lacking. The chemokines produced by both species target a conserved set of chemokine receptors: CCR1, CCR2, CCR3, and CCR4. CCR1 and CCR2 are mainly ex-

a

pressed by monocytes, basophils, monocytes, and memory T cells, whereas CCR3 characterizes Th2 cells, eosinophils, and basophils, and CCR4 characterizes only Th2 cells. Furthermore, several aaM CC-chemokines act as natural antagonists of the IFN-responsive chemokine CXCL10, competing with moderate afﬁnity for the binding to CXCR3 (104). CC-chemokines such as CCL2 and CCL13 not only attract selected leukocytes, but also dampen the accumulation of mRNA for IL12 (105) and act as activators of eosinophil effector functions (106). The role of this set of chemokines is well documented in Th2 human and murine diseases such as the immune response to Leishmania major (107), eosinophilrich interstitial lung granulomas induced by antigens of S. mansoni eggs (108), and in asthmatic lungs (109). A species-speciﬁc diagram of the main alternative activation gene markers is shown in Figure 2.

b

Eosinophil

Eosinophil

Basophil

Basophil Th2 CCL13 CCL14 CCL17 CCL22

Th2 CCL2 CCL7

Human macrophage

HGL1 MRC1

IGF1 FXIII FN1

MGL1 MRC1

STAT6

MHCII

YM1 YM2 FIZZ1

Murine macrophage STAT6 ARG1

STAB1

Proline

GPR105 ? Fusogenic molecules

DC-STAMP

E-cadherin

Figure 2 Human and murine macrophage alternative activation markers. M alternative activation is associated with changes in M function and distinctive gene signatures. Although the alternative activation markers for human (a) and murine (b) Ms have long been considered analogous, there are differences between species. IL-4 and IL-13 induce genes conserved in both species such as MHC class II (MHCII) and MRC1, as well as divergent ones such as ARG1, YM1 in mouse, and GPR105 in humans. Despite gene evolution, there is conservation of function, i.e., different chemokines attract similar cellular inﬁltrates in both species. Hitherto, fusogenic molecules have been identiﬁed only in mouse. The divergences between species and their functional repercussion require further investigation.

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BONA FIDE ALTERNATIVELY ACTIVATED MΦS IN VIVO Th2 responses are mainly directed toward extracellular parasites such as helminths and some protozoa, while creating optimal conditions for proliferation of pathogens whose elimination requires a Th1 response. Intracellular pathogens have, as a result, created intricate mechanisms to promote Th2 responses and avoid Th1 immune control. Genetic and environmental factors also affect Th2 responses, inducing a set of allergic diseases. The Ms present in these pathologies represent bona ﬁde examples of aaMs. Other non-Th2 pathologies are characterized by Ms with very speciﬁc phenotypes, sometimes with suppressive phenotype and markers in common with IL-4- and IL-13-treated Ms, such as endotoxin-tolerant Ms, and tumor-associated Ms (TAMs), among others. As a consequence, they have also been called alternatively activated Ms, but they may not share all their features, deﬁned above.

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APC: antigenpresenting cell

Necessary Alternative Activation Helminths are parasitic worms that represent the most common infectious agent in developing countries, affecting close to three billion people. Helminths comprise two phyla: nematodes and platyhelminths, further divided into cestodes and trematodes. Protection against both phyla depends on IL-4 and IL-13. SevTable 4

In vivo evidence for M alternative activation

Pathogen

Model/host

aaMΦ marker

References

Brugia malayi

Mouse

Ym1, Fizz1, Arg1

Schistosoma mansoni

Mouse

Ym1, Fizz1, Arg1

Fasciola hepatica

Mouse/sheep

Ov-YM1.Mu-ARG1, chitinase activity

156, 157

Taenia crassiceps

Mouse

Ym1, Fizz1, Arg1, Mrc1

119, 158

Trichinella spiralis

Guinea pig

Arg1

159

Nippostrongylus brasiliensis

Mouse

Ym1, Fizz1, Arg1, Mrc1

160

Heligmosomoides polygyrus

Mouse

Ym1

161

Hymenolepis diminuta

Mouse

Arg1, Fizz1

162

Toxocara canis

Mouse

IL-10, TGF-β, IL-12 Null

163

Trichuris muris

Mouse

Fizz1, Mrc1

164

Abbreviations: ov-ovine, mu-murine. 464

eral murine models have been used to study the immune response to gastrointestinal nematodes and the contribution and phenotype of the participating Ms (Table 4). Experimental infection by N. brasiliensis is performed subcutaneously, and as the parasites mature, they migrate and enter the lungs where they induce strong, polarized Th2 responses in situ and in lung-associated lymph nodes (51, 53, 54). Lung pathology is associated with a strong induction of alternative activation in alveolar Ms, as shown by elevated expression of YM1 and FIZZ1 (40, 54). In this model, aaMs do not participate in worm expulsion following primary infection (110, 111), while examination of lung pathology in response to migrating larvae from N. brasiliensis and also in the memory response to another nematode, H. polygyrus, demonstrates a clear contribution of aaMs (111). Chronic induction of aaMs drives lung ﬁbrosis, consistent with the wound-healing functions of aaMs induced by the ﬁlarial nematodes B. malayi, and also drives inhibition of inappropriate CD4+ proliferative responses in the later stages of Litomosoides sigmodontis (112, 113). Though mice are usually able to resolve primary gastrointestinal nematode infections, reinfection with H. polygyrus has shown aaMs to be critical for induction of effective memory responses to secondary infection (111). This ﬁnding is in agreement with the requirement of antigenpresenting cells (APCs), and speciﬁcally of

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M-derived cytokines, as secondary signals for T cell activation. In contrast to the data for N. brasiliensis, in this model memory CD4+ lymphocytes induce a rapid recruitment of aaMs to the intestine, where M and arginase depletion inhibits the host’s ability to expel worms. The other major group of helminths are trematodes and cestodes. Experimental infection of mice with the trematode S. mansoni results in a Th2 cytokine response important for effective formation of granulomas surrounding parasite eggs, and also expulsion of eggs in the feces (114, 115). S. mansoni–infected IL-4Rα−/− and LysMCre IL-4Rα−/ﬂox mice are unable to survive acute schistosomiasis, since Th2 progression confers aaM-dependent protection; their absence results in a Th1 response, as seen in IL-4Rα−/− mice (110). In this setting, Ms express the alternative activation markers ARG1, YM1, and FIZZ1. It has been proposed that aaMs mediate repair of tissue damage and dampen excessive inﬂammation resulting from traversal of parasite eggs across the intestine, and are therefore important for host survival from acute schistosomiasis (110). Similar mechanisms may operate in humans, where infection by S. mansoni correlates with production of TNF-α, IL-4, and IL-5. A positive correlation is found between cytokine secretion and severity of the disease, measured as periportal ﬁbrosis versus intestinal pathology (116). Infection of mice with the cestode Taenia crassiceps induces a mixed Th1/Th2 immune response (117), and unlike the case of infection with the other helminths discussed, caMs play a major role in clearing T. crassiceps infection (118). However, when the infection progresses to a chronic stage, Th2 cytokine dominance results in aaM recruitment that controls excessive inﬂammatory responses and increased CD4+ apoptosis (119–122).

Unwanted Alternative Activation Asthma is a chronic inﬂammatory disease of the airways characterized by IgE production, goblet cell metaplasia, airway smooth muscle

proliferation and hyperplasia, and airway hyperresponsiveness, together with enhanced expression of selected cytokines and chemokines. There is overwhelming evidence that chronic allergic inﬂammatory processes depend on expression of the Th2 cytokines IL-4, IL-5, IL-9, and IL-13 (123, 124), highly expressed in airway tissues from asthmatic patients. In murine models of asthma, alveolar Ms express the alternative activation markers Ym1, FIZZ1, and ARG1 (47, 125). There are other congenital noninfectious autoimmune and idiopathic inﬂammatory diseases, with increased levels of IL-4 and IL13, generally accompanied by eosinophilia, impaired cellular responses, and increases in one or more B cell activities, such as hypergammaglobulinemia, autoantibody production, or particularly increased IgE production. Examples are Ommen’s syndrome, the hypereosinophilic syndrome (HES), Kimura’s disease, and Job’s and Wells’ syndromes. Unfortunately, as for most human-related pathologies, no studies address their association with alternative activation of Ms. Viruses beneﬁt from a Th2 environment, and a remarkable example is that of dengue virus, a mosquito-borne ﬂavivirus that can cause hemorrhagic fever in humans. This virus is transmitted to humans by the mosquito Aedes aegypti; its salivary factors induce a shift in cytokine expression with downregulation of the type I and type II IFN response and enhanced expression of Th2 cytokines (126). These Th2 cytokines not only dampen the Th1 response required for viral elimination but also induce MRC1, which binds all four serotypes of dengue virus and can be exploited by the virus to infect Ms (127). Murine gamma herpes virus 68 (MHV-68), from the same family of the human Kaposi’s sarcoma–associated herpesvirus and Epstein-Barr virus, induces latent infection in lymphoid tissue in IFN-γR KO. In these mice, infection results in transient ﬁbrosis in multiple organs, with the spleen severely affected (128). Fibrosis is preceded by inﬁltration of germinal centers by different subsets of splenic Ms. These Ms express high levels of ARG1 and murine M www.annualreviews.org • Alternative Activation of Macrophages

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markers F4/80, ER-TR9, and MOMA-1. Other genes upregulated are FIZZ1, tissue inhibitor of metalloproteinase-1, matrix metalloproteinase12, FN1, and F13A1. This system provides an important model for studying the pathogenesis of ﬁbrosis initiated by a latent herpesvirus infection and may explain complications that arise in the event of unwanted Th2 responses. In fungal infections, such as the welldescribed Candidiasis, Th1 responses correlate with host reactivity and asymptomatic or mild forms of the infection, in contrast to the correlation between Th2 cell responses, severe disease, and the establishment of fungal allergies (129). A novel example is Cryptococcus neoformans– induced bronchopulmonary disease, in which IL-4- and IL-13-mediated immune mechanisms exacerbate disease. Intranasal infection with C. neoformans of IL-4Rα1, IL-4, and IL-13 KO animals and of IL-13 transgenic and WT mice showed that lack of the receptor or of the cytokine correlates with signiﬁcantly milder pathology, whereas overexpression was correlated with higher fungal brain burden and consequent cerebral lesions. Furthermore, IL-13+ transgenic mice harbored large pseudocystic lesions in the CNS parenchyma, bordered by voluminous foamy aaMs containing intracellular cryptococci, without signiﬁcant microglial activation. In WT mice, aaMs tightly bordered pseudocystic lesions, and these mice, in addition, showed microglial cell activation. As expected in resistant IL-4−/− , IL-13−/− , and IL-4Rα−/− mice, no alternative activation markers were discernible. Microglial cells of all mouse genotypes neither internalized cryptococci nor expressed markers of alternative activation, although they displayed similar IL-4Rα expression as Ms. These data provide the ﬁrst evidence of the development of aaM in a CNS infectious disease model and point to distinct roles of Ms versus microglia in the CNS immune response against C. neoformans (130). Protozoans constitute a group of pathogens that appear to require Th1 responses to be killed. In murine models, for example, successful resolution of cutaneous infection with the

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protozoan L. major depends on the host launching an effective Th1 response, enabling caMs to phagocytose and kill the parasites (131–133). In the Th2-biased BALB/c, mice develop fatal progressive disease within 3 months of infection. During this time period, deletion of either IL-4 or its receptor made it possible to contain infection, with reduced footpad swelling and parasite load, moderate histopathology, and reduced Th1 response (134). However, these mutant mice were not able to heal as completely as the infected Th1-biased C57BL/6 mice, indicating that additional factors are necessary for subsequent healing and elimination of the pathogen. In longer infection time points such as 6 months, IL-4Rα−/− mice developed progressive disease with massive footpad swelling, ulcerative and necrotic lesions with subsequent destruction of connective tissue and bones, as well as dissemination into organs and consequent mortality. In striking contrast, IL-4−/− mice maintained control of infection on a moderate level but were unable to clear the pathogen, suggesting that IL-13 and not IL-4 plays a protective role during chronic leishmaniasis (135). In general, alternative activation induced by Th2 responses against helminth infections, asthma, and other pathologies can modulate the immune response in a “bystander” fashion, perhaps explaining the high rate of concomitant infections in developing countries, where secondary infections of protozoan may be facilitated. Epidemiologic studies show common coinfections of helminth and protozoa in humans, including Schistosoma/ leishmaniasis, Litomosoides/leishmaniasis, Taenia/leishmaniasis, Taenia/trypanosomiasis, ﬁlaria/malaria, or helminth/tuberculosis (136).

Non-Th2-Related Alternatively Activated MΦs: Do They Exist? Other forms of M phenotypes have been categorized as aaM. These include Ms present during tissue repair and those of several chronic pathologies such as cancer and atherosclerosis. aaMs are expected to have strong proﬁbrotic

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properties, as demonstrated in studies with IL-4 and IL-13 overexpression. However, no clear data show a direct correlation between these diseases and tissue repair. An interesting ﬁnding derives from a sterile tissue injury model study by Loke et al. (52), brieﬂy discussed above. In this model, sterile sham surgery induces a wave of innate cells, mainly eosinophils, able to secrete IL-4. IL-4 secretion correlates with M expression of the alternative activation markers ARG1 and YM1, among others. As such, it was proposed that activation driven by innate immune cells, may be an essential component in tissue repair. Further data are required to strengthen these observations. TAMs have also been described as aaM (137, 138). No consistent evidence connects IL-4 or IL-13 with tumor progression or the tumor microenvironment, indicating that other cytokines and mechanisms may inﬂuence the phenotype of TAMs. In murine and human TAMs, increased levels of p50, the inhibitory subunit of NF-κB, reduce the production of MyD88-dependent IL-12p70 and TNF (139, 140). Importantly, MyD88-independent IRF-3, which induces IL-10 and chemokines, is preserved, leading to dampening of the inﬂammation. The tumor microenvironment is essential for maintenance of this phenotype, which is lost in culture after 24 h. In accordance with the defects in the NF-κB pathway, TAMs express high levels of IL-10, Dectin-1, MGL1, TGF-β, and SRA; high levels of CXCL10, CXCL9, and other IFN-responsive genes; and low levels of IL-12, TNF-α, and NOS2 (139, 140). Thus, TAMs represent a unique M population, without a clear correlation with IL-4 or IL-13 stimulation. TAMs are characterized by expression of genes in common with aaMs, probably the same genes that IL-4 modulates to control inﬂammation, but also coexpress IFN-γ-inducible chemokines and other genes (139). Atherosclerotic plaques are characterized by a prominent M inﬁltrate. These Ms display a foamy appearance, owing to accumulation of intracellular lipid droplets. The role of IL-4 in atherosclerosis has been investigated

in several mouse models; however, the correlation between these cytokines and the disease is not clear. While exogenous IL-4 has protective or deleterious effects, depending on the mouse model used, most KO models for IL-4 components tend to develop milder disease (141, 142). The IL-4−/− /ApoE−/− -mice have a signiﬁcant temporary reduction in the aortic root plaque area compared with ApoE−/− -mice. Immune cell–speciﬁc deﬁciency of IL-4 in hypercholesterolemic, cholate-fed female LDLR−/− mice provokes a reduction in atherosclerosis in the arch/thoracic regions of the aorta, but not in the aortic root. Accelerated fatty streak formation induced by heat shock protein 65 or M. tuberculosis is reduced in IL-4−/− mice compared with lesions in WT mice. Taken together, the majority of ﬁndings would suggest a proatherogenic role for endogenous IL-4; this effect appears to be greatly inﬂuenced by the vascular site and the disease stage. Importantly, although IL-4 may play a role in the disease itself, in vitro studies of human and mouse foam cell M models do not show a signiﬁcant overlap between the gene expression proﬁle of these cells and aaMs.

MΦ ACTIVATION REVISITED Ms appear early in development, occupying strategic positions in different organs, and they persist throughout adult life. As part of their innate immune role, they act as sentinels in tissues, encountering pathogens and orchestrating the attraction and development of more complex acquired responses. Ms are not the only effector cells of the immune system; DCs, neutrophils, basophils, eosinophils, and T and B cells all participate in the defense against pathogens. As such, the view of the immune response as mere interaction between the APC and the T cell is somewhat limited. DCs and Ms recognize pathogen or injury signals and then provide signals to initiate the inﬂammatory response. This initial interaction between pathogen-conserved moieties and the M induces a primed state in Ms. The receptors for this initial activation include mainly PRRs for PAMPs, such as TLRs, NALPs, and other www.annualreviews.org • Alternative Activation of Macrophages

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Macrophage signaling receptors in the context of Th1 or Th2 responses

Selected receptors

Role

Ligands

IL-12/IL-10 balance contribution

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Interleukin-1 and Toll-Like Receptor Superfamily • TLRs are the best characterized signal-generating receptors among the PRRs; they initiate key inﬂammatory responses and also shape adaptive pro-Th1 immunity • TLRs initiate shared and distinct signaling pathways by recruiting different combinations of the TIR domain–containing adaptor molecules: MyD88, TIRAP (MAL), TRIF (TICAM1), and TRAM • Strong ﬁrst stimulation of the TLR can induce TLR-signaling inhibitors, such as MyD88s (a splice variant of MyD88) and IRAKM, and as a result inhibit signaling by the second TLR stimulation until the inhibitors disappear, whereas weak sequential TLR stimulations can boost the signaling and thus the immune response TLR4

Critical for host defense against Gram-negative bacteria in both mice and humans

LPS from Gram-negative bacteria, mannan from Candida albicans, GIPLs from Trypanosoma, viral envelope proteins from RSV and MMTV

TLR4 stimulation induces in M the expression of inﬂammatory cytokines such as IL-6, type I IFNs, TNF-α, and importantly IL-12. However, context-dependent TLR4-PI3K stimulation may induce IL-10 and inhibit IL-12 in DCs

TLR1, 2, 6

TLR2 is essential for host defense against Gram-positive bacteria. This receptor collaborates with other TLRs or non-TLR PRRs, and this collaboration can also modulate signaling outcomes

TLR1: triacyl lipopeptides from bacteria and mycobacteria. TLR2: LTA from Gram-positive bacteria, yeast zymosan, lipopeptides (Pam3CSK4, MALP2), lipoarabinomannan from mycobacteria TLR6: diacyl lipopeptides from Mycoplasma, LTA from Gram-positive bacteria, yeast zymosan

In DCs, prolonged ERK activation and the subsequent, enhanced induction of c-Fos (an AP-1 component) upon TLR2 stimulation seems to play a signiﬁcant role in the production of a high level of IL-10 and little IL-12

NOD-Like Receptor Family • NLRs comprise a family of cytosolic proteins that play a pivotal role in the recognition of intracellular PAMPs, mediating protective immune responses elicited by intracellular pathogens or endogenous danger signals, and act in synergy with various TLRs to enhance immune responses in APCs NOD1, 2

NOD1: acts as a sensor for Gram-negative bacteria NOD2: acts as a general sensor for most bacteria

NOD1: recognizes the peptide γ-D-glutamyl-meso-diaminopimelic acid, and mainly acts as a sensor for Gram-negative bacteria NOD2: recognizes muramyl dipeptide MDP, NAG-NAM-lalanyl-isoglutamine

NOD1 and NOD2 act in synergy with TLR3, 4, and 9 in human DCs to induce IL-12p70 production and promote Th1 cell differentiation

(Continued )

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Table 5 Selected receptors

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(Continued ) Role

Ligands

IL-12/IL-10 balance contribution

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C-Type Lectin Receptor Family • The C-type lectins recognize speciﬁc pathogen-associated carbohydrate structures • C-type lectins were originally named to reﬂect the special importance of Ca2+ in carbohydrate binding MRC1

MRC1 displays Ca2+ -dependent lectin activity toward terminal mannose and acts as general sensor for different intracellular parasites and phagocytic receptors

Endogenous and exogenous ligands bearing mannose, fucose, N-acetyl glucosamine, and sulfated sugars

Stimulation with the anti-mannose receptor antibody blocks induction of the IL-12

Dectin-1

Appears to deal primarily with fungal infection although it may also recognize other ligands

Has speciﬁcity for β-1,3- and -1,6-linked glucans found in fungi, plant cell walls, and some bacteria but does not have afﬁnity for monosaccharides or glucans with different linkages

Dectin-1 and TLR2 collaborate synergistically to downregulate proinﬂammatory cytokines such as TNF-α and/or IL-12 and induce IL-10 production

Nuclear Receptors • Many nuclear receptors such as glucocorticoid receptor (GR), PPARs, and LXRs have strong antiinﬂammatory functions that are mediated by negatively regulating the expression of certain inﬂammatory responsive genes • Ligand-dependent transrepression is thought to be the major mechanism for such gene regulation VDR

Vit D3 is essential for calcium and phosphorus homeostasis

1,25OH 2D3

VDR stimulation inhibits IL-12 production by interfering with NF-κB signaling

RXR

Vit A is a known potentiator of the immune system

9-cis-RA, FAs, methoprene acid, DHA

Retinoic acid enhances the production of IL-10 while reducing IL-12 secretion

Complement Receptors • The complement inﬂammatory cascade is part of the phylogenetically ancient innate immune response and is crucial for our natural ability to ward off infection • It is important for defense against microbial infections because it triggers the generation of a membranolytic complex (C5b9 complex) at the surface of the pathogen and C fragments (named opsonins, i.e., C1q, C3b, and iC3b) that interact with C cell surface receptors (CR1, CR3, and CR4) to promote phagocytosis • Soluble C anaphylatoxins (C4a, C3a, and C5a) greatly control the local proinﬂammatory response through chemotaxis and activation of leukocytes gC1qR

gC1qR plays a pivotal role in the regulation of inﬂammatory and antiviral T cell responses

C1q

C1q can downregulate the TLR4-mediated production of IL-12 but not of other inﬂammatory cytokines such as IL-6 or TNF-α in human monocytes

C5aR

C5a is a potent chemoattractant for neutrophils and monocytes, and signaling is mediated by a 7TM GPCR

C5a

Selectively suppresses TLR4-mediated expression of IL-12 family members IL-12, IL-23, and IL-27, but not of other cytokines such as TNF-α or IL-10

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IL-4 increases the expression of MRC1, SR-A, Dectin-1, DC-SIGN, DCIR, DCL-1, and CLECSF13. IL-4/IL-13 increases endocytosis of mannosylated ligands and other receptor-mediated endocytic processes, and downregulates latex bead phagocytosis.

Increases MHC class II and presentation to Th2 cells

Phagocytosis/ endocytosis

Antigen presentation

IL-4RI, II

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GCs inhibit antigen processing and presentation by several mechanisms, including proteasome suppression

GCs increase the expression of MRC1 and CD163. GCs increase phagocytosis of latex beads, complementmediated endocytosis, as well as phagocytosis of apoptotic cells.

GCR

Increases antigen presentation through upregulation of costimulatory molecules. TLRs have a critical role in selecting antigens for presentation via MHC class II molecules after the phagocytosis of particles

TLR signaling enhances phagosome maturation, regulates the expression of phagocytic receptors, such as SRA and MARCO, and induces activating Fcγ receptor expression.

TLR4

Receptor complex stimulated

Type II Ms express high levels of costimulatory molecules and are efﬁcient APC to Th2 cells

No information regarding phagocytosis was found. However, these macrophages are well-known producers of IL-10, which may recreate a prophagocytic proﬁle.

FcR+ TLR4

Increases class I and II antigenpresenting complexes and costimulatory molecules. Also stimulates de novo synthesis of the immunoproteasome. MHC class II loading is increased by synthesis of cathepsins B, H, and L

Increases complement secretion and complement receptor surface expression on mononuclear phagocytes, enhancing complementmediated phagocytosis.

IFN-γ+ TLR4

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Inhibits presentation through downregulation of MHC class II and costimulatory molecules

IL-10 increases the expression of FcγRI, II, and III, Marco, and CD163. Exposure of monocytes to a combination of IL-10 and IL-4 results in a synergistic effect on CR-mediated ingestion without membrane expression changes. Anti-IL-10 mAb signiﬁcantly reduces monocytes’ capacity to ingest IgG- or C3b/C3bi-coated particles, suggesting a role for IL-10.

IL-10R

Comparison between the best-described M activation phenotypes ARI

Function

Table 6

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Induces chemokines such as CCL2, CCL13, and CCL17, which coordinate the recruitment of eosinophils, basophils, and some polarized Th2 cells, through activity on CCR3 and is involved in proangiogenic networks Induces IL-1Ra, IL-10, TGF-β and antagonizes LPSand IFN-responsive chemokines Decreases microbicidal functions in Ms

Induces cell fusion

Production of chemokines

Cytokine production

Microbicidal properties

Cell fusion

Does not induce fusion

Decreases microbicidal functions in Ms

Does not induce fusion

Decreases microbicidal functions in Ms

Does not induce fusion

Induces NADPH oxidase complex (ROS generation)

Induces IFN-β, IL-6, TNF-α, IL-1β

Directly increases the production of CCL2, CCL3, and CCL5. Induces CXCL9, CXCL10, and CXCL11 through IFN-β production

Not determined

Not determined

Induces switch from IL-12 to IL-10 production

Increases production of CCL1, CXCL3, and CCL20

Does not induce fusion

Induces ROS and RNI generation. Other induced mechanisms are tryptophan depletion by IDO

IL-12, IL-6, TNF-α, IL-1β, IL-15

Increases production of CXCL9, CXCL10, CXCL11, and CCL2-5 and is known to recruit monocytes, Tc, and Th1 cells

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Induces IL-10 and TGF and antagonizes most LPS-, IL-4- and IFN-responsive chemokines

Inhibits production of the Th1 chemokines CXCL9, CXCL10, CXCL11, CCL5, and CCL24

ARI

Induces IL-10 and antagonizes most LPS- and IFN-responsive chemokines

Increases production of CXCL13, CXCL4, CCL23, and CCL18

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surface and cytosolic receptors. The best known PRRs are often responsible for an innate proTh1 form activation of Ms based on the selective expression of IL-12, decreased IL-10 levels, and expression of chemokines and other factors that mediate recruitment and activation of selective accessory cells, as NK cells, eosinophils, and basophils, among others (Table 5). Little is known about PRRs that initiate Th2 responses, although a few novel results demonstrate the presence of pro-Th2 PAMPs and TLRs in parasites and Ms, respectively. With the recruitment of IFN-γ- or IL-4 secreting cells of the innate system, conditions are created for the further development of either a Th1 or a Th2 milieu. The appearance in the inﬂammatory focus of either of these cytokines induces either classical or alternative ac-

a

b

Th1 response

IFN-γ

Th2 response

IL-12 PAMPs

Th1

IL-4, IL-13

M1 response

tivation, ﬁrst in an innate manner (given the origin of the cytokine) and subsequently in an acquired process (Figure 3). For T cells, a twosignal activation model is currently accepted, in which the ﬁrst signal originates from ligation of the T cell receptor (TCR) by APC MHCpeptide and the second signal, in the form of a cytokine, is provided by the APC, Ms, or DCs (Figure 3). Thus, T cell and M activation seems to occur in an interdependent two-step fashion. This staged activation may also apply to DCs. Signiﬁcantly, in addition to IFN-γ or IL-4/IL-13 signal recognition complexes, there is a group of M signaling receptors whose ligation also leads to M activation. These receptors respond to a wide variety of stimuli of both innate and acquired origin, including the cytokines IL-10, IL-1, IL-6, TNF, IL-17, and

IL-10 PAMPs

Th2

M2 response

Macrophage

Macrophage

Basophil

PMN IFN-γ

IL-4, IL-13 NK

NK Eosinophil

Figure 3 Two-signal macrophage activation model in the context of the immune response. M full activation requires two major signals, recognition of PAMPs by PRRs and stimulation with IFN-γ or IL-4, in a cycle that determines the development of speciﬁcity and effector functions in the immune response. For Th1 responses (a), PRR-induced chemokines attract IFN-γ-producing innate immune NK cells and also naive T lymphocytes. Innate IFN-γ and PAMPs induce a ﬁrst wave of classical activation in Ms, stimulating IL-12 secretion, an important signal for Th1 activation. With Th1 activation, greater levels of IFN-γ induce long-lasting classical activation of Ms while a full cytotoxic T cell response is mounted. For Th2 responses (b), uncharacterized PRRs induce the recruitment of IL-4 and IL-13, producing innate immune cells such as eosinophils and basophils and naive T lymphocytes. IL-4 and IL-13 produced by innate immune cells induce a ﬁrst wave of innate alternative activation in Ms but also provide the secondary signal for Th2 development. Innate IL-4 and PAMPs may synergize to stimulate IL-10 secretion from Ms, which provides a signal for Th1 repression. IL-10 produced by Ms may also induce the development of repressor T cells, which oppose Th1 activation. 472

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IL-25. Stimuli such as IL-10, GC, and Fc receptor ligands induce responses biased toward a Th2 proﬁle, and they have been grouped as part of alternative activation. The phenotype induced in Ms by this last group of stimuli overlaps partially with that of IL-4 or IL-13, but only in limited genes and features (Table 6). Confusion arises from the fact that the term “activation” may be correctly used at both an immune response level, as we use it throughout this manuscript, and from a wider Mcentered point of view. In the ﬁrst application, activation is seen as the acquisition of resistance against speciﬁc pathogens, in response to clearly discernible arms of acquired immunity, Th1 and Th2 responses (Figure 3), whereas in the second case, it is considered as any stimulus that moves Ms from their resting condition (Table 5). At the level of the immune response are Th1, Th2, Th17, and a set of immune-suppressive T cells, whose main characteristic is their mutual exclusion (143). These immune response speciﬁcity–determining cytokines have evolved to deal with either activation against speciﬁc pathogens or deactivation of the immune response and resolution. As such, it is natural to suppose that they induce four main effector phenotypes in Ms. While classical and alternative activation of Ms and their co-T cell Th1 and Th2 partners are in part understood, the role of IL-17 and other polarized T cell products in Ms remains largely unknown. The other stimuli are also important because they play a deﬁnitive role in modiﬁcation of the M phenotype in the course of an immune response and may be abundant in speciﬁc pathologies; however, they do not represent an immunologic equivalent to the Th cytokines. In 1999, Goerdt and colleagues (144) proposed a classiﬁcation of activation phenotypes based on grouping all activators other than IFN-γ and LPS/microorganisms into a common alternative activation group. This classiﬁcation overlooks important immunological differences in the response to modulators such as IL-4, IL-13, IL-10, glucocorticoids (GC) and TGF. For this reason, in 2002 an extended clas-

siﬁcation was proposed in which M1 polarization included the classical activation, whereas M2 polarization was subdivided into M2a, corresponding to aaM; M2b, corresponding to type II–activated Ms; and M2c, which includes heterogeneous M deactivation stimuli (145). The classiﬁcation took into account the production of IL-10 or IL-12, which by themselves are not enough to categorize the phenotypes. Nevertheless, the classiﬁcation M1/M2 is useful because it avoids using the terms alternative and classical, which can create confusion. In addition, M1 and M2 are mnemonic and provide a clear link to the T cell response. We suggest keeping classical/M1 or alternative/M2 as synonyms for those categories, and use the ligand/ligands to identify the other phenotypes until a consensus is reached in the community. We strongly suggest avoiding the grouping of stimuli other than IL-4 and IL-13 under the M2/alternative group.

CONCLUDING REMARKS We have reviewed recent information regarding the effects of IL-4 and IL-13 and have summarized the main evidence while analyzing the robustness of alternative activation of Ms as an immunological paradigm. IL-4 and IL-13 can be actively produced by both innate and acquired immune system cells. Their in vivo production may not be restricted to the primary inﬂammatory locus. Production of IL-4 by innate cells allows induction of aaMs prior to the secondary immune response. The innate production of IL-4 is important for tissue repair and complements IL-4 production by acquired immune system cells in the control of speciﬁc pathogens. IL-4 and IL-13 pathways have convergent and divergent features, accounting for differences and similarities in the phenotype induced in Ms. Even the identical type II receptor heterodimer, coupled to the same intracellular signaling molecules, responds to IL-4 and IL-13 with different signaling potencies and kinetics. It is the balance between activation of IL-4 type I and type II receptors and IL-13Rα2 signaling www.annualreviews.org • Alternative Activation of Macrophages

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cascades and their relation with other autocrine, paracrine, and cell-cell contact pathways that determines the phenotype of aaMs. IL-4 and IL-13 modify the function of Ms at various levels, with some mechanisms strictly dependent on direct signaling and others also requiring de novo gene expression. Gene expression studies have demonstrated that the effect of IL-4 or IL-13 in Ms is not restricted to isolated genes, but instead involves speciﬁc and distinguishable gene signatures. In all, the number of well-characterized speciﬁc alternative activation markers does not exceed a dozen. Although the general functions and behavior of murine and human aaMs are thought to be preserved, the genes required for such functions are to some extent different. IL-4 and IL-13 induce varied effects on human and murine Ms. The functional signiﬁcance of genes induced by IL-4 and IL-13 is difﬁcult to interpret owing to the restricted information available. However, these cells clearly play a key role in the attraction of cells to inﬂammatory foci, suppression of Th1 responses, sampling of the microenvironment by endocytosis, and orchestration of tissue repair. Their role in pathogen killing is less clear. The combination of cytoskeletal changes induced by IL-4 and the receptor repertoire of IL-4-treated Ms makes possible an altered phagocytic and secretory capacity in response to viruses, bacteria, fungi, and helminths, not always with positive consequences for the M or host.

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FUTURE ISSUES The paradigm of alternative activation of Ms now emerges with greater clarity. The development of novel transgenic mice, highthroughput gene expression and proteindetection techniques, infection models, and other advances have improved our understand-

ing of the Th2 response, the cells that participate in it, and their cross talk, as well as the effector functions of aaMs. Human and murine Ms, although having functions in common, clearly express different genes in response to both IL-4 and IL-13. Given the feasibility and versatility of mouse models, most of our knowledge derives from them and not from human studies. Considering the differences between species, we must investigate human alternative M activation in more depth, both in vitro and in vivo. Validation of speciﬁc markers, gene silencing, and transfection of human primary Ms may become powerful tools for this purpose. Initiation of the Th1 response, the link between microbial PAMPs and production of proinﬂammatory cytokines, and the killing capacity of caMs are more or less well understood. However, little is known about the extracellular parasite PAMPs, their recognition by APCs, and the link to initiation of the Th2 response and cytokine production. Similarly, many of the genes and functions of aaMs, especially their antiparasitic properties, are still unidentiﬁed. Further studies are required to understand host defense against such parasites. Accumulating evidence suggests that Ms play major roles in pathology, and several of their products and effector functions make them ideal therapeutic targets. We have reviewed a number of diseases that result from dysregulation of the Th2 response. Similarly, many diseases arise from dysregulation of the Th1 response. To date, the therapeutic interventions that target Ms rather than their products, e.g., TNF, are limited (146), although a number of M speciﬁc drugs are emerging. Cell therapy is also a future possibility, and studies are beginning to assess the beneﬁcial effects of ex vivo M activation and transfusion, with encouraging results (147, 148).

SUMMARY POINTS 1. The ﬁnding of a M classical activation, induced by IFN-γ in response to infection with unrelated intracellular pathogens, represents one of the turning points in M biology

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and innate immunology, given that it demonstrated the presence of a stimulus-dependent but antigen-nonspeciﬁc defense program, true for the diverse forms of M responses found hitherto.

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2. The discovery that IL-4 induced MHC class II and MRC1 in Ms led to the original deﬁnition of alternative activation. A current view has replaced the “on/off ” model of M activation in pathogen defense with a more complex one in which Ms exist in resting, classically, or alternatively activated forms. 3. The concepts of classical and alternative M activation involving IFN-γ and IL-4 refer to the acquisition of resistance against speciﬁc pathogens in response to clearly discernible arms of the acquired immunity: Th1 and Th2 responses. 4. The pathways of IL-4 and IL-13 have convergent and divergent features, which account for differences and similarities in the phenotype induced in Ms. It is the balance between activation of IL-4 type I and type II receptors and IL-13Rα2 signaling cascades in relation to other autocrine, paracrine, and cell-cell contact–induced pathways that ﬁnally determines the phenotype of aaMs. 5. IL-4 and IL-13 can be produced by both innate and acquired immune system cells. Their in vivo production may not be restricted to the primary inﬂammatory locus. 6. Innate production of IL-4 is likely to be important for tissue repair and to complement IL-4 production by acquired immune cells in the control of speciﬁc pathogens. 7. Gene expression studies have demonstrated that the effect of IL-4 or IL-13 in Ms is not restricted to isolated genes, but instead involves speciﬁc and distinguishable gene signatures. These gene signatures depend on the genetic background, maturation, and activation state of cells. 8. Although the general functions and behavior of murine and human aaMs are thought to be preserved, the genes required for such functions are to some extent different. 9. Phagocytic receptors expressed by aaMs differ from those expressed by caMs. Although not fully demonstrated, a common dual feature of these receptors seems to be pathogen and self recognition, leading to increased ligand endocytosis, but also interaction with T cells and negative immune modulation. 10. aaMs play a key role in the attraction of other cells to inﬂammatory foci, suppression of Th1 responses, sampling by endocytosis of the microenvironment, and orchestration of tissue repair. 11. There is an unaccounted degree of divergence between species, still to be determined. 12. Conﬁrmation of aaM markers in studies regarding Th2 responses has become a necessary readout to link cytokine production to the development of immune effector functions. 13. Presence of alternatively activated macrophages has been conﬁrmed in several helminthic and allergic disease models.

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DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We would like to thank all the colleagues who provided information for this review. Fernando O. Martinez Estrada and Laura Helming were supported by grants from the MRC, United Kingdom.

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LITERATURE CITED 1. Metchnikoff E. 1905. Immunity in the Infectious Diseases. New York: Macmillan 2. Mackaness GB. 1964. The immunological basis of acquired cellular resistance. J. Exp. Med. 120:105–20 3. North RJ. 1973. Cellular mediators of anti Listeria immunity as an enlarged population of short lived, replicating T cells. Kinetics of their production. J. Exp. Med. 138:342–55 4. Bloom BR, Bennett B. 1970. Macrophages and delayed-type hypersensitivity. Semin. Hematol. 7:215–24 5. David JR. 1973. Lymphocyte mediators and cellular hypersensitivity. N. Engl. J. Med. 288:143–49 6. Nathan CF, Murray HW, Wiebe ME, Rubin BY. 1983. Identiﬁcation of interferon-γ as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670– 89 7. Howard M, Paul WE. 1983. Regulation of B-cell growth and differentiation by soluble factors. Annu. Rev. Immunol. 1:307–33 8. Mosmann TR, Coffman RL. 1989. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145–73 9. McKenzie AN, Culpepper JA, de Waal Malefyt R, Briere F, Punnonen J, et al. 1993. Interleukin 13, a T-cell-derived cytokine that regulates human monocyte and B-cell function. Proc. Natl. Acad. Sci. USA 90:3735–39 10. Abramson SL, Gallin JI. 1990. IL-4 inhibits superoxide production by human mononuclear phagocytes. J. Immunol. 144:625–30 11. Standiford TJ, Strieter RM, Chensue SW, Westwick J, Kasahara K, Kunkel SL. 1990. IL-4 inhibits the expression of IL-8 from stimulated human monocytes. J. Immunol. 145:1435–39 12. Cao H, Wolff RG, Meltzer MS, Crawford RM. 1989. Differential regulation of class II MHC determinants on macrophages by IFN-γ and IL-4. J. Immunol. 143:3524–31 13. Wirth JJ, Kierszenbaum F, Zlotnik A. 1989. Effects of IL-4 on macrophage functions: increased uptake and killing of a protozoan parasite (Trypanosoma cruzi ). Immunology 66:296–301 14. Stein M, Keshav S, Harris N, Gordon S. 1992. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J. Exp. Med. 176:287–92 15. Mantovani A, Sica A, Locati M. 2005. Macrophage polarization comes of age. Immunity 23:344–46 16. Martinez FO, Sica A, Mantovani A, Locati M. 2008. Macrophage activation and polarization. Front. Biosci. 13:453–61 17. Mosser DM. 2003. The many faces of macrophage activation. J. Leukoc. Biol. 73:209–12 18. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. 1999. The IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17:701–38 19. Miyajima A, Kitamura T, Harada N, Yokota T, Arai K. 1992. Cytokine receptors and signal transduction. Annu. Rev. Immunol. 10:295–331 20. Livnah O, Stura EA, Johnson DL, Middleton SA, Mulcahy LS, et al. 1996. Functional mimicry of a ˚ Science 273:464–71 protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A. 21. Moy FJ, Diblasio E, Wilhelm J, Powers R. 2001. Solution structure of human IL-13 and implication for receptor binding. J. Mol. Biol. 310:219–30 476

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134. Kopf M, Brombacher F, Kohler G, Kienzle G, Widmann KH, et al. 1996. IL-4-deﬁcient Balb/c mice resist infection with Leishmania major. J. Exp. Med. 184:1127–36 135. Mohrs M, Ledermann B, Kohler G, Dorfmuller A, Gessner A, Brombacher F. 1999. Differences between IL-4- and IL-4 receptor α-deﬁcient mice in chronic leishmaniasis reveal a protective role for IL-13 receptor signaling. J. Immunol. 162:7302–8 136. Reyes JL, Terrazas LI. 2007. The divergent roles of alternatively activated macrophages in helminthic infections. Parasite Immunol. 29:609–19 137. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. 2002. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23:549–55 138. Sica A, Saccani A, Mantovani A. 2002. Tumor-associated macrophages: a molecular perspective. Int. Immunopharmacol. 2:1045–54 139. Biswas SK, Gangi L, Paul S, Schioppa T, Saccani A, et al. 2006. A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-κB and enhanced IRF-3/STAT1 activation). Blood 107:2112–22 140. Saccani A, Schioppa T, Porta C, Biswas SK, Nebuloni M, et al. 2006. p50 nuclear factor-κB overexpression in tumor-associated macrophages inhibits M1 inﬂammatory responses and antitumor resistance. Cancer Res. 66:11432–40 141. George J, Mulkins M, Shaish A, Casey S, Schatzman R, et al. 2000. Interleukin (IL)-4 deﬁciency does not inﬂuence fatty streak formation in C57BL/6 mice. Atherosclerosis 153:403–11 142. Kleemann R, Zadelaar S, Kooistra T. 2008. Cytokines and atherosclerosis: a comprehensive review of studies in mice. Cardiovasc. Res. 79:360–76 143. Romagnani S. 2006. Regulation of the T cell response. Clin. Exp. Allergy 36:1357–66 144. Goerdt S, Orfanos CE. 1999. Other functions, other genes: alternative activation of antigen-presenting cells. Immunity 10:137–42 145. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. 2004. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 25:677–86 146. Gordon S, ed. 2003. The Macrophage As Therapeutic Target. Berlin: Springer 147. Wang Y, Wang YP, Zheng G, Lee VW, Ouyang L, et al. 2007. Ex vivo programmed macrophages ameliorate experimental chronic inﬂammatory renal disease. Kidney Int. 72:290–99 148. Frenkel O, Shani E, Ben-Bassat I, Brok-Simoni F, Rozenfeld-Granot G, et al. 2002. Activated macrophages for treating skin ulceration: gene expression in human monocytes after hypo-osmotic shock. Clin. Exp. Immunol. 128:59–66 149. Zlotnik A, Yoshie O, Nomiyama H. 2006. The chemokine and chemokine receptor superfamilies and their molecular evolution. Genome Biol. 7:243 150. Walter MR, Cook WJ, Zhao BG, Cameron RP Jr, Ealick SE, et al. 1992. Crystal structure of recombinant human interleukin-4. J. Biol. Chem. 267:20371–76 151. Frazer KA, Ueda Y, Zhu Y, Gifford VR, Garofalo MR, et al. 1997. Computational and biological analysis of 680 kb of DNA sequence from the human 5q31 cytokine gene cluster region. Genome Res. 7:495–512 152. Brusselle GG, Kips JC, Tavernier JH, Van Der Heyden JG, Cuvelier CA, et al. 1994. Attenuation of allergic airway inﬂammation in IL-4 deﬁcient mice. Clin. Exp. Allergy 24:73–80 153. Hogan SP, Mould A, Kikutani H, Ramsay AJ, Foster PS. 1997. Aeroallergen-induced eosinophilic inﬂammation, lung damage, and airways hyperreactivity in mice can occur independently of IL-4 and allergen-speciﬁc immunoglobulins. J. Clin. Invest. 99:1329–39 154. Kolodsick JE, Toews GB, Jakubzick C, Hogaboam C, Moore TA, et al. 2004. Protection from ﬂuorescein isothiocyanate-induced ﬁbrosis in IL-13-deﬁcient, but not IL-4-deﬁcient, mice results from impaired collagen synthesis by ﬁbroblasts. J. Immunol. 172:4068–76 155. Miyata S, Matsuyama T, Kodama T, Nishioka Y, Kuribayashi K, et al. 1999. STAT6 deﬁciency in a mouse model of allergen-induced airways inﬂammation abolishes eosinophilia but induces inﬁltration of CD8+ T cells. Clin. Exp. Allergy 29:114–23 156. Flynn RJ, Irwin JA, Olivier M, Sekiya M, Dalton JP, Mulcahy G. 2007. Alternative activation of ruminant macrophages by Fasciola hepatica. Vet. Immunol. Immunopathol. 120:31–40

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157. Donnelly S, Stack CM, O’Neill SM, Sayed AA, Williams DL, Dalton JP. 2008. Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages. FASEB J. 22:4022–32 158. Rodriguez-Sosa M, Rivera-Montoya I, Espinoza A, Romero-Grijalva M, Lopez-Flores R, et al. 2006. Acute cysticercosis favours rapid and more severe lesions caused by Leishmania major and Leishmania mexicana infection, a role for alternatively activated macrophages. Cell. Immunol. 242:61–71 159. Dzik JM, Golos B, Jagielska E, Zielinski Z, Walajtys-Rode E. 2004. A nonclassical type of alveolar macrophage response to Trichinella spiralis infection. Parasite Immunol. 26:197–205 160. Siracusa MC, Reece JJ, Urban JF Jr, Scott AL. 2008. Dynamics of lung macrophage activation in response to helminth infection. J. Leukoc. Biol. 84:1422–33 161. Bazzone LE, Smith PM, Rutitzky LI, Shainheit MG, Urban JF, et al. 2008. Co-infection with the intestinal nematode Heligmosomoides polygyrus markedly reduces hepatic egg-induced immunopathology and proinﬂammatory cytokines in mouse models of severe schistosomiasis. Infect. Immun. 76:5164–72 162. Persaud R, Wang A, Reardon C, McKay DM. 2007. Characterization of the immuno-regulatory response to the tapeworm Hymenolepis diminuta in the nonpermissive mouse host. Int. J. Parasitol. 37:393–403 163. Kuroda E, Yoshida Y, En Shan B, Yamashita U. 2001. Suppression of macrophage interleukin-12 and tumour necrosis factor-α production in mice infected with Toxocara canis. Parasite Immunol. 23:305–11 164. Deschoolmeester ML, Martinez-Pomares L, Gordon S, Else KJ. 2008. The mannose receptor binds Trichuris muris excretory/secretory proteins but is not essential for protective immunity. Immunology. In press

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IL-17 and Th17 Cells Thomas Korn,1 Estelle Bettelli,2 Mohamed Oukka,3 and Vijay K. Kuchroo2 Annu. Rev. Immunol. 2009.27. Downloaded from arjournals.annualreviews.org by University of Bergen UNIVERSITETSBIBLIOTEKET on 01/13/09. For personal use only.

1

Technical University Munich, Department of Neurology, 81675 Munich, Germany; email: [email protected]

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Center for Neurologic Diseases, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; email: [email protected], [email protected]

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Center for Neurologic Diseases, Brigham & Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139; email: [email protected]

Annu. Rev. Immunol. 2009. 27:485–517

Key Words

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

T cell differentiation, effector T cells, regulatory T cells, cytokines, tissue inﬂammation, autoimmunity

This article’s doi: 10.1146/annurev.immunol.021908.132710 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0485$20.00

Abstract CD4+ T cells, upon activation and expansion, develop into different T helper cell subsets with different cytokine proﬁles and distinct effector functions. Until recently, T cells were divided into Th1 or Th2 cells, depending on the cytokines they produce. A third subset of IL17-producing effector T helper cells, called Th17 cells, has now been discovered and characterized. Here, we summarize the current information on the differentiation and effector functions of the Th17 lineage. Th17 cells produce IL-17, IL-17F, and IL-22, thereby inducing a massive tissue reaction owing to the broad distribution of the IL-17 and IL-22 receptors. Th17 cells also secrete IL-21 to communicate with the cells of the immune system. The differentiation factors (TGF-β plus IL-6 or IL-21), the growth and stabilization factor (IL-23), and the transcription factors (STAT3, RORγt, and RORα) involved in the development of Th17 cells have just been identiﬁed. The participation of TGF-β in the differentiation of Th17 cells places the Th17 lineage in close relationship with CD4+ CD25+ Foxp3+ regulatory T cells (Tregs), as TGF-β also induces differentiation of naive T cells into Foxp3+ Tregs in the peripheral immune compartment. The investigation of the differentiation, effector function, and regulation of Th17 cells has opened up a new framework for understanding T cell differentiation. Furthermore, we now appreciate the importance of Th17 cells in clearing pathogens during host defense reactions and in inducing tissue inﬂammation in autoimmune disease.

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INTRODUCTION +

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The differentiation of naive CD4 T cells into effector T helper cells is initiated by engagement of their T cell receptor (TCR) (signal 1) and costimulatory molecules (signal 2) in the presence of speciﬁc cytokines produced by the innate immune system upon encounter of particular pathogens: IFN-γ and IL-12 initiate the differentiation of Th1 cells that are characterized by high production of IFN-γ and are indispensable for clearing intracellular pathogens. In contrast, IL-4 triggers the differentiation of Th2 cells. Th2 cells are key in organizing host defense against extracellular pathogens and in helping B cells to produce antibodies. The initial source of the differentiation factors for both Th1 and Th2 cells are cells of the innate immune system responding to microbial antigens, parasitic antigens, or allergens; however, the effector cytokines that are subsequently produced by Th1 and Th2 cells (i.e., IFN-γ and IL-4) can potentially feed back to amplify Th1 and Th2 cells and further enhance differentiation of the respective T cell subset. Moreover, IFN-γ and IL-4 antagonize each other on different levels, and thus Th1 and Th2 development is considered mutually exclusive. Thus, even though the cytokine proﬁle may initially not be entirely polarized, with differentiating T cells producing a combination of both Th1 and Th2 cytokines, chronic stimulation leads to unequivocal, terminally differentiated phenotypes. Over the years, this Th1/Th2 paradigm of T helper cell differentiation, which was ﬁrst introduced by Mosmann & Coffman (1) about 25 years ago, helped explain many phenomena in adaptive immunity. Recently, the Th1/Th2 paradigm has been expanded, following the discovery of a third subset of effector Th cells that produce IL-17 (Th17) and exhibit effector functions distinct from Th1 and Th2 cells. The primary function of Th17 cells appears to be the clearance of pathogens that are not adequately handled by Th1 or Th2 cells. However, Th17 cells are potent inducers of tissue inﬂammation and have been associated with the pathogenesis of many experimental autoimmune diseases and

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human inﬂammatory conditions. In the past ﬁve years, we have witnessed an accumulation of information on this new T cell subset: The cytokines for its differentiation and expansion have been identiﬁed (2–9) and the key transcription factors that are involved in its generation have been elucidated (10, 11), ﬁrmly establishing Th17 cells as an independent T helper cell lineage in human and mouse. Here, we review the ﬁndings that led to the identiﬁcation of Th17 cells and integrate the current knowledge on their differentiation and effector functions. The requirement of TGFβ in combination with other cytokines for the differentiation of Th17 cells and other T cell subsets is a unique new concept that opens up new views on the generation of T helper cell subsets in the peripheral immune compartment and underscores potential developmental relationships between various T cell subsets that were not anticipated. In addition, many questions of lineage stability versus plasticity of differentiated Th17 cells in vivo are as yet unresolved. Most importantly, although we have learned much about the differentiation and stabilization of Th17 cells, there is still uncertainty about their effector function in vivo. Given that Th17 cells secrete not only IL-17 but also IL17F, IL-21, and IL-22, these cytokines most likely cooperate to induce tissue inﬂammation, and Th17-driven effector functions may be different in different tissues. Conversely, Th17 cells are not the only source of IL-17, but rather other cells of the innate immune system can produce IL-17, IL-17F, and IL-22. Moreover, we do not know whether Th17 cells have to cooperate with other effector T helper cells such as Th1 cells to display their full effector functions, and ﬁnally, we have only limited information on the termination of Th17 responses, speciﬁcally whether Th17 responses can be effectively controlled by Tregs.

INITIAL IDENTIFICATION OF Th17 CELLS Dysregulated Th1 responses have been associated with organ-speciﬁc autoimmunity.

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IFN-γ expression in the target tissues correlates with clinical signs in experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). Self-reactive Th1 clones derived in vitro are capable of adoptively transferring EAE in naive recipients. T-bet- and STAT4-deﬁcient mice are resistant to EAE, and targeting IL-12 with polyclonal antibodies to IL-12 is an efﬁcient therapy for EAE and CIA. These ﬁndings led to the idea that IFNγ-producing Th1 cells with speciﬁcity for selfantigens are autopathogenic and required for the induction of organ-speciﬁc autoimmunity. However, the concept that organ-speciﬁc autoimmunity is a Th1-driven condition was challenged when it became clear that IFN-γ- and IFN-γ receptor–deﬁcient mice, as well as mice that lack other molecules involved in Th1 differentiation such as IL-12p35, IL-12 receptorβ2, and IL-18, were not protected from EAE, but rather developed more severe disease (12– 16). This raised the question of whether another subset of T cells, different from Th1 cells, might be required for the induction of EAE and other organ-speciﬁc autoimmune diseases. In 2000, a novel cytokine chain, p19, was discovered by a computational sequence screen looking for homologs of the IL-6 subfamily of proteins (17). This p19 chain forms heterodimers with the p40 chain of IL-12, and this novel cytokine was named IL-23; thus, all approaches that targeted the p40 chain of IL-12 would affect both IL-12 and IL-23. By creating IL-23p19 knockout (KO) mice and comparing them with IL-12p35-deﬁcient mice, Cua and colleagues (8) at the DNAX Research Institute demonstrated that IL-23 and not IL-12 was crucial for the induction of EAE. In the follow-up study (9), they showed that IL-23 expands/generates IL-17-producing T cells that are capable of inducing EAE when adoptively transferred into naive wild-type mice. These IL-17-producing T cells were dramatically reduced in the central nervous system (CNS) of IL-23p19-deﬁcient mice. On the basis of this and other studies, investigators proposed that IL-17-producing T cells are a distinct T

helper cell subset, which was named Th17 cells (9, 18, 19). However, in view of the fact that the IL-23 receptor is not expressed on naive T cells, and, in our hands, IL-23 was not able to generate de novo IL-17-producing T cells from sorted naive T cells (3), this raised the issue of whether IL-23 indeed is the differentiation factor of Th17 cells, and, if not, then what are the factors needed for the differentiation of Th17 cells.

DIFFERENTIATION OF Th17 CELLS Th17 cells were established as an independent subset of T helper cells by the identiﬁcation of differentiation factors and transcription factors that are unique to Th17 cells. In 2006, three independent studies found that a combination of the immunoregulatory cytokine TGF-β and the proinﬂammatory and pleiotropic cytokine IL-6 is required to induce IL-17 in naive T cells (2–4) (Figure 1). We fortuitously identiﬁed TGF-β and IL-6 as the differentiation factors for Th17 cells while looking for the factors that inhibited the TGF-β-driven conversion of naive T cells into Foxp3+ Tregs in vitro and in vivo. Using naive T cells from reporter mice that expressed the green ﬂuorescent protein (GFP) targeted into the Foxp3 locus, the transcription factor for Tregs, we screened a panel of cytokines and found not only that IL-6 is extremely potent in inhibiting the TGF-β-driven induction of Foxp3, but also that TGF-β together with IL-6 induces massive amounts of IL-17 from naive T cells. Given that we started with sorted naive (CD4+ CD62Lhigh CD25− Foxp3− ) T cells, we demonstrated that TGF-β plus IL-6 together can differentiate naive T cells into the Th17 lineage. Interestingly, two cytokines with opposing effects have to cooperate to induce the differentiation of Th17 cells, suggesting that signaling molecules and transcription factors involved downstream of the TGF-β and IL-6 receptors work together to induce Th17 differentiation (see below). To support the role of TGF-β and IL-6 in Th17 differentiation www.annualreviews.org • Th17 Cells

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Figure 1 Differentiation of Th17 cells in mice and humans. (a) Factors required to induce the development of Th17 cells in mice starting from naive T cells (light blue) or activated CD4+ T cells. In humans, different Th17 differentiation factors were initially reported. However, when rigorously sorting for naive T cells as a starting population and controlling hidden sources of TGF-β in the culture conditions, identical factors seem to induce Th17 cells in mouse and human (for details see text). IL-23 might be required to induce further effector molecules in committed Th17 cells (purple) to establish their terminally differentiated effector phenotype (orange). (b) The sources of Th17 differentiation factors have partially been revealed in vivo. Moreover, IL-21 feeds back on developing Th17 cells, amplifying their frequency, as do IFN-γ and IL-4 in the differentiation of Th1 and Th2 cells, respectively.

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further, we found that TGF-β transgenic mice, which overexpress TGF-β under the IL-2 promoter, had higher frequencies of Th17 cells and developed more severe EAE when immunized with myelin autoantigen in complete Freund’s adjuvant (CFA). Because immunization with CFA induces IL-6 from innate immune cells, we hypothesized that this CFA-driven IL-6 acted together with the transgenically expressed TGF-β to induce Th17 cells in vivo. These data suggested that IL-6 plays a pivotal role in dictating whether an immune response is dominated by Foxp3+ Tregs or Th17 cells. On the basis of this observation, we predicted that IL-6-deﬁcient mice would lack the ability to generate Th17 cells and that their repertoire would be dominated by Tregs. To test this prediction, we retested IL-6 KO mice that had been described to be resistant to EAE (3, 20–23) and antigen-induced arthritis (24). We showed that Il6−/− mice had a defect in the generation of Th17 cells and that the immune response in these mice is indeed dominated by Foxp3+ Tregs. However, upon depletion of Tregs, IL-6 KO mice became susceptible to EAE owing to the appearance of a pathogenic Th17 response, raising the possibility that in the absence of IL-6, another factor could induce Th17 cells (5). To identify this factor, we rescreened a panel of cytokines that, like IL6, suppressed the TGF-β-induced expression of Foxp3 and induced IL-17 production. In this screen, we identiﬁed IL-21, an IL-2 family member, that both suppressed the TGFβ-induced expression of Foxp3 and, together with TGF-β, induced IL-17 in naive T cells (5). Because Th17 cells themselves are a major source of IL-21, an autocrine ampliﬁcation loop was proposed by which Th17 cells enhance their own differentiation and precursor frequency (5–7, 25) (Figure 1). As far as human T cells are concerned, the situation for the differentiation of Th17 cells has been confusing. In 2007, several studies claimed that TGF-β was dispensable for the differentiation of human Th17 cells (26, 27). Whereas naive mouse CD4+ T cells differen-

tiate into Th17 cells upon exposure to TGF-β plus IL-6 or TGF-β plus IL-21, the combinations of IL-1β plus IL-6 (26) or IL-1β plus IL23 (27) were proposed to be the differentiation factors for human Th17 cells. Thus, a rather disturbing problem was raised by the claim that human Th17 cells could develop in the absence of TGF-β, suggesting that the requirements for human and mouse Th17 differentiation are essentially different. In these previous human studies, naive T cells were selected from human peripheral blood mononuclear cells according to expression of CD45RA. The differentiation was performed in the presence of serum, and contamination with platelets (an important source of TGF-β), even if no exogenous TGF-β was added, could not be excluded owing to the experimental protocols used to isolate human T cells from peripheral blood. Thus, these results were difﬁcult to interpret until human T cell differentiation cultures were rigorously sorted for naive T cells and sources of TGF-β in serum and platelets were controlled. A series of three new reports tackled these problems, proving that TGF-β is indeed essential for the differentiation of human Th17 cells from naive T cells (28–30) as well. TGF-β is absolutely required to induce RORc (the human homolog of RORγt), but its expression and function are inhibited by excess TGF-β. Only when additional cytokines such as IL-6 plus IL-23 or IL21 are present is RORc relieved from inhibition, and then naive T cells can begin transcribing IL-17 (29). Thus, at a molecular level, the differentiation conditions of mouse and human Th17 cells do not appear to be different (Figure 1).

ROLE OF TGF-β TGF-β is a regulatory cytokine with pleiotropic functions in T cell development, homeostasis, and tolerance (31). However, when T cells are deﬁcient in a functional receptor for TGF-β and thus cannot respond to TGF-β, Th17 cells are not generated and mice are protected from EAE (32). Conversely, as discussed above, transgenic overexpression www.annualreviews.org • Th17 Cells

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of TGF-β by T cells resulted in more severe EAE with enhanced Th17 generation when the TGF-β transgenic mice were immunized with myelin oligodendrocyte glycoprotein in CFA (3). Given that TGF-β is produced by multiple lineages of leukocytes and stromal cells, it has been very difﬁcult to deﬁne the sources of TGF-β that are relevant for the generation of Th17 cells in vivo. In a recent study, Flavell and coworkers (33) generated mice with a disrupted TGF-β1 gene only in T cells. These mice developed a lethal inﬂammatory disorder characterized by enhanced Th1 and Th2 responses, indicating that Treg-derived TGF-β is required to control exaggerated Th1 and Th2 responses. Interestingly, ablation of TGF-β production from T cells also resulted in a defect in the generation of Th17 cells and the development of EAE (33). This study suggested that an autocrine or paracrine source of TGF-β was important for Th17 differentiation in vivo. Thus, T cells and even Tregs themselves (2) may be the source of TGF-β for the differentiation of Th17 cells; in support of this idea, it was shown that transfer of naive T cells together with Tregs into recipient mice promoted the induction of IL-17 but not of IFN-γ in the cotransferred conventional T cells (34). TGF-β is produced in its latent form and needs to be activated. This is achieved either by proteolytic degradation or by conformational changes of latency-associated protein (LAP), which is associated with TGF-β in a heterotetrameric form and constrains the binding of the TGF-β homodimer to its receptor. For example, binding of LAP to thrombospondin on vascular endothelium or to integrins αv β6 on epithelial cells or αv β8 on dendritic cells (DCs) results in processing LAP and activation of TGF-β in vivo. In fact, when αv β8 is conditionally deleted in DCs, the mice suffer from a lymphoproliferative syndrome and tissue inﬂammation that is reminiscent of TGF-β deﬁciency in T cells (35). Thus, it is believed that DCs, though not primary producers of TGF-β, are essential to raising the levels of active TGF-β in the local environment.

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It remains to be determined whether other effector cytokines produced by Th17 cells are differentially regulated by TGF-β. For example, IL-22, which is also produced by Th17 cells, appears to be less dependent on TGF-β. In an antigen-presenting cell (APC)-free system, in a starting population of naive T cells, maximal amounts of IL-22 were induced by TCR stimulation in the presence of IL-6 alone, and titration of TGF-β increasingly inhibited the secretion of IL-22. However, IL-22 is not exclusively produced by Th17 cells, but rather appears to be produced by activated T cells in general, as well as by IL-2- or IL-12-stimulated natural killer (NK) cells (36). IL-6 together with TGF-β is crucial for the generation of Th17 cells, considering that IL-6 alone cannot induce RORγt (the transcription factor involved in the induction of Th17 cells; see below), and Th17 cells are not induced. TGF-β is absolutely required both for the initial induction of IL-17 in naive CD4+ T cells and for the induction of IL-23R, which makes differentiating Th17 cells responsive to IL-23 and therefore further promotes their maturation. However, high concentrations of TGF-β inhibit the expression of IL-23R, suggesting a biphasic effect. When Th17 cells are induced in an APC-free system by the combination of TGF-β plus IL6 (in the absence of any additional exogenous cytokines), IL-22 production is essentially absent. Th17 cells that have been induced with TGF-β plus IL-6 do not produce IL-22 unless they have been exposed to IL-23. Thus, Th17derived IL-22 is dependent on IL-23 and appears to be a Th17 cytokine that is associated with a terminal differentiation stage of Th17 cells. Collectively, TGF-β is indispensable for the generation of Th17 cells, although high concentrations of TGF-β may inhibit the production of various effector cytokines of Th17 cells and ultimately inhibit the generation of Th17 cells by inducing Foxp3. This scenario might seem confusing at ﬁrst, but it ﬁts with the observation that Th17 cells and induced Tregs share common developmental pathways, where TGF-β has been clearly recognized as a link between these two T cell subsets.

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ROLE OF IL-6 Like TGF-β, IL-6 has a plethora of activities outside the immune and even hematopoietic systems and is clearly a pleiotropic cytokine with multiple effects. IL-6 is produced by cells of the innate immune system such as DCs, monocytes, macrophages, mast cells, B cells, and subsets of activated T cells, but also by tumor cells, ﬁbroblasts, endothelial cells, and keratinocytes (for review, see 37, 38). Production of IL-6 is part of the acute-phase response during infection, and, apart from its originally discovered properties as a myeloma growth factor, IL-6 is also a hepatocyte-stimulating factor, resulting in the production of a number of acutephase proteins by hepatocytes. Depending on the cellular source of IL-6, cytokines such as IL-1, tumor necrosis factor (TNF), plateletderived growth factor (PDGF), IL-3, GMCSF, and IL-17 are important inducers of IL-6 (39, 40). Different approaches led to the identiﬁcation of IL-6 as an essential differentiation factor for Th17 cells. In an APC-free culture system of naive T cells, where supernatant from LPSstimulated DCs was used together with TGFβ, the differentiation of Th17 cells could be completely abolished by addition of an antibody to IL-6 (2). We found that addition of recombinant IL-6 was extremely potent in suppressing the TGF-β-driven induction of Foxp3 in naive T cells and instead resulted in strong induction of IL-17 (3). TGF-β alone via activation of the Smad pathway induces genes involved in downregulation of immune responses, IL-6 alone leads to strong but transient activation of STAT3, yet when TGF-β and IL-6 were added simultaneously, they induced a distinct transcriptional program resulting in the development of Th17 cells. Mechanistically, the interaction of TGF-β and IL-6 in inducing the Th17 transcriptional program is incompletely understood. First, naive T cells express a functional IL-6 receptor composed of IL-6Rα and the signaling subunit gp130, which is constitutively expressed. While TGF-β induces the expres-

sion of IL-6Rα, TCR stimulation as well as exposure to IL-6 lead to downregulation and shedding of the IL-6Rα and thus reduces responsiveness to IL-6. TGF-β is necessary to maintain the responsiveness of T cells to IL-6. Second, engagement of the IL-6 receptor and activation of gp130 lead to activation of STAT3. Activation of STAT3 is necessary but not sufﬁcient to induce RORγt. Deﬁciency of STAT3 in CD4+ T cells abrogates the induction of Th17 cells and decreases the induction of RORγt and RORα (11, 41), two transcription factors involved in Th17 differentiation. Accordingly, mice with conditional deﬁciency of STAT3 in CD4+ T cells lack antigen-speciﬁc Th17 responses and are resistant to EAE (42). IL-6driven STAT3 signaling is required for RORγt function. However, full induction of RORγt is only achieved in the presence of TGF-β. Recent studies suggest the TGF-β promotes the expression of RORγt but represses its function (43). Only the additional presence of IL6 or IL-21 signaling relieves the repression of RORγt and promotes the Th17 transcriptional program (29, 43).

ROLE OF IL-21 IL-21 is a cytokine that was ﬁrst identiﬁed in 2000 by Parrish-Novak and colleagues (44). It is produced by activated T cells and NKT cells, but not by APCs (44). IL-21 belongs to the IL2 family of cytokines and uses the common γ chain (γc ) as part of its receptor consisting of γc plus IL-21R. IL-21 has been described as playing an important part in the expansion of previously activated B cells and in class switching of immunoglobulin isotypes (45). IL-21 is abundantly expressed in certain populations of T cells, e.g., in T follicular helper cells (46) that express high levels of CXCR5 and ICOS (47). IL-21 is also produced by Th2 cells (48), but comparing the Th1, Th2, and Th17 subsets, the largest amounts of IL-21 are produced by Th17 cells (5, 6). IL-6 is a strong inducer of IL21 (49), and the expression of IL-21 is dependent on STAT3 but not on RORγt, as RORγt

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KO mice express normal levels of IL-21 (7). IL-21 together with TGF-β is able to induce the differentiation of Th17 cells. Thus, IL-21 produced by differentiating Th17 cells might act in a positive feedback loop, amplifying the precursor frequency of Th17 cells (5–7). This is intriguing, as autoampliﬁcation loops have been described for both Th1 and Th2 cells, in which the effector cytokines IFN-γ and IL-4 also act as the ampliﬁcation factors for Th1 and Th2 cells, respectively. IL-17 cannot amplify Th17 cells because IL-17 does not act as a growth or differentiation factor for the Th17 lineage. In view of the fact that differentiating Th17 cells produce maximum levels of IL-21, we and others have proposed that IL-21 produced by differentiating Th17 cells might play this role in amplifying the frequency of Th17 cells (5–7). The relative impact of IL-6 and IL-21 in the differentiation of Th17 cells in vivo is not yet entirely clear. Both TGF-β plus IL-6 and TGF-β plus IL-21 induce the expression of RORγt, which is necessary and sufﬁcient to trigger the expression of IL-23R. In turn, IL-23 stabilizes the commitment of developing Th17 cells to the Th17 lineage (7). However, in vivo IL-6 plays a dominant role in Th17 differentiation, and only in the absence of IL-6 and following depletion of Tregs did we observe a role for IL-21 in inducing Th17 cells. Moreover, we proposed that in the absence of inﬂammation, when the levels of IL-6 are not elevated, IL-21 might play a role in maintaining the precursor pool of Th17 cells, as the frequency of Th17 cells in the memory T cell pool was reduced in IL-21R KO mice (5). Thus, IL-21, which is produced by Th17 cells themselves (5, 6, 25), helps to maintain and amplify the pool of Th17 precursors when the supply of IL-6 is limited. Under these circumstances, IL-21 might become important for supporting Th17 responses and tissue inﬂammation. Several studies suggest that this is indeed the case in, for example, autoimmune diabetes or chronic inﬂammatory bowel disease (50–52), in which IL-21 plays an important role in inducing autoimmunity. In contrast, in the presence of massive amounts of IL-6 (as induced by immu-

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nization with autoantigen emulsiﬁed in CFA), IL-21 is dispensable, and Th17 cells can be induced and maintained even in the absence of IL-21 (53, 54). Thus, we proposed that IL-21 constitutes an alternative pathway for inducing Th17 cells that is followed only when there is no induction of IL-6 and when Treg cells have been deleted from the peripheral repertoire (5). Under these circumstances, IL-21 is essential in inducing and expanding Th17 cells. In vivo, however, IL-6 appears to be dominant over IL21, and when excess IL-6 is induced by immunization with myelin antigen in CFA (53, 54), the requirement for IL-21 in the induction of Th17 cells is overcome owing to excessive and continuous IL-6 production by the peripheral immune system. That IL-21 is an alternative pathway is consistent with our observation that although IL-21 is induced by and is downstream of IL-6, IL-6 and TGF-β together are able to induce Th17 cells independently of IL-21 signaling. This interpretation of our data is not at odds with the ﬁnding that Th17 cells and EAE can be induced in Il21−/− mice by immunizing these mice with myelin antigen in CFA (53–55) as in such a case massive amounts of IL-6 are produced, thus overcoming the need for IL-21 to amplify the Th17 response.

TRANSCRIPTION FACTORS INVOLVED IN Th17 DIFFERENTIATION The differentiation of T helper cells is initiated by the combined signals mediated downstream of the TCR and cytokine receptors. Those signals then induce and activate speciﬁc transcription factors responsible for the expression of lineage-speciﬁc genes such as cytokines. The engagement of the TCR is able to induce the expression of T-bet or GATA-3, transcription factors speciﬁcally and respectively expressed in Th1 and Th2 cells. GATA-3, on the one hand, is essential for Th2 cell lineage commitment and induces the expression of IL-4. T-bet, on the other hand, can bind to the IFN-γ promoter and induces the expression of this cytokine. The sustained production of effector cytokines and

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subsequent differentiation of Th cell subsets requires the engagement of speciﬁc cytokines with their receptors. For Th1 cells, the engagement of IFN-γ with its receptor leads to the activation of STAT1 and T-bet. In turn, T-bet increases the production of IFN-γ and induces the expression of the inducible IL-12Rβ2 chain of the IL-12R. IL-12, through STAT4, is then essential for the maintenance of Th1 responses. For Th2 cells, the interaction of IL-4 with its receptor activates STAT6, which together with GATA-3 increases IL-4 production and Th2 commitment. The steroid receptor–type nuclear receptor RORγt, which is a splice variant of RORγ expressed in T cells (56, 57), is selectively expressed in in vitro–differentiated Th17 cells and in IL-17+ T cells present in the lamina propria of naive mice (10). RORγt appears to be required for IL-17 production, as mice reconstituted with the bone marrow of RORγtdeﬁcient mice show an impaired Th17 differentiation (10) and as transduction of naive T cells with a retroviral vector containing RORγt induces the development of IL-17-producing T cells (10). However, although reduced, IL17-producing cells are not absent in RORγtdeﬁcient mice. Another member of the retinoid nuclear receptor family, RORα, is also selectively expressed in Th17 cells—very similarly to RORγt. A recent study reported that RORα could fulﬁll a similar but not identical role to RORγt in the differentiation of Th17 cells, suggesting that Th17 cell differentiation could be dictated by two lineage-speciﬁc transcription factors (41). However, the mechanisms by which RORγt and possibly RORα regulate IL-17 production have not yet been fully elucidated. Although there is a potential RORbinding site in the IL-17 promoter, it is not clear whether RORγt binds directly to this promoter. RORγt and RORα are both strongly induced by IL-6 or IL-21 in the presence of low amounts of TGF-β. The induction of RORγt is dependent on STAT3, which is preferentially activated by IL-6, IL-21, and IL-23 and plays an important role in the regulation of IL-17 production in T cells (7, 11, 58–60).

Mice with conditional deﬁciency of STAT3 in CD4+ T cells have impaired Th17 differentiation, and overexpression of a constitutively active form of STAT3 can increase IL-17 production (11, 42). Thus, STAT3 might affect the expression of IL-17 by increasing the expression of RORγt and RORα, which are upstream of IL-17 (11, 41). However, STAT3 also binds directly to the Il17 and Il21 promoters (25, 58). Therefore, STAT3 and RORγt seem also to cooperate, and competent production of IL-17 depends on the presence of both transcription factors. RORγt likely must cooperate with other as yet unidentiﬁed transcription factors in the induction of Th17 cells. For example, the interferon regulatory factor 4 (IRF4), which was previously associated with the differentiation of the Th1 and Th2 subsets (61, 62), is required for the differentiation of Th17 cells as well (63). IRF4 KO mice failed to mount a Th17 response and were resistant to EAE. Consistent with this observation, IRF4deﬁcient T cells failed to upregulate RORγt upon stimulation in the presence of TGFβ plus IL-6 and could not be differentiated into Th17 cells (63). However, overexpression of RORγt in IRF4-deﬁcient T cells failed to fully restore the induction of IL-17, again suggesting that IRF4 or its downstream targets may have to cooperate with RORγt for full commitment of T cells to the Th17 lineage.

RECIPROCAL RELATIONSHIP BETWEEN Th17 CELLS AND TREGS Th17 and Treg developmental programs of T cells are reciprocally interconnected. This discovery was initially based on the observation that upon TCR stimulation, a naive T cell can be driven to express Foxp3 and become a Treg cell in the presence of TGF-β. However, in the presence of TGF-β plus IL-6 or IL-21, the Treg developmental pathway is abrogated, and instead T cells develop into Th17 cells. Only the combination of TGF-β plus IL-6/IL-21, www.annualreviews.org • Th17 Cells

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but neither of them alone, induced robust production of IL-17 in naive T cells. In vitro, there is a true reciprocity between the Th17 and Treg developmental programs on the single-cell level. However, evidence is still incomplete as to whether this reciprocal developmental decision at the single-cell level is also relevant in vivo. Active TGF-β is a cytokine produced by various cell types, including natural Treg cells (nTreg) and cells of the innate immune system. TGF-β has broad inhibitory effects on the entire immune system (for review, see 31). In addition, TGF-β induces the Treg-speciﬁc transcription factor Foxp3, which is required for the induction and maintenance of induced Treg cells (iTreg) in the peripheral immune compartment (64, 65). However, addition of IL-6 to TGF-β inhibits the generation of Tregs and induces Th17 cells. On the basis of these data, we ﬁrst put forward the idea that there is a reciprocal relationship between Tregs and Th17 cells and that IL-6 plays a pivotal role in dictating whether the immune response is dominated by proinﬂammatory Th17 cells or protective Tregs (3). We propose that the unactivated immune system at a steady state (in the absence of inﬂammatory stimuli) produces TGF-β, which induces the generation of iTregs, which together with nTregs keep activated/effector memory cells in check. IL6, induced during an acute-phase response, inhibits the function of nTregs and prevents the generation of iTregs, but instead induces Th17 cells. Thus, IL-6 plays a pivotal role in dictating the balance between the generation of Tregs and Th17 cells. The mechanism by which IL-6 and IL-21 act as switch factors relies on the control of the Foxp3/RORγt balance (43). In line with this concept, conditional deletion of Foxp3 in adult mice results in an increase in both RORγt and IL-17 expression (33). It is clear now that TGF-β is required for the expression of both Foxp3 and RORγt, although the signaling cascades downstream of the TGF-β receptor might be different for the induction of Foxp3 versus RORγt. For example, whereas Smad4 appears to be essential for the induc-

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tion of Foxp3, it is probably dispensable for the induction of RORγt (66). In the presence of TGF-β, IL-6 and IL-21 play an important role in Th17 differentiation by inhibiting TGFβ-driven Foxp3 expression. Both RORγt and RORα physically associate with Foxp3 to antagonize each other’s functions (43, 67). This association is likely the molecular basis for the reciprocal relationship between Tregs and Th17 cells. Foxp3 binds to RORγt via a motif expressed in exon 2. When Foxp3 lacks exon 2, the binding to RORγt is abolished and the Foxp3-mediated inhibition of RORγt is abrogated (43). Although homodimerization of Foxp3 is not required for its inhibitory effect on RORγt, whether or not Foxp3 needs a functional DNA-binding motif to inhibit RORγt is still unclear (43, 66). Foxp3 may also have to bind both RORγt and additional transcription factors such as Runx1, which also cooperates with RORγt, to fully inhibit the Th17 transcriptional program (68). The reciprocal relationship between Tregs and Th17 cells is further supported by the results obtained in IL-6 KO mice, which show a severe defect in the generation of Th17 cells and increased numbers of Tregs in the peripheral repertoire (3, 5). Observations from other groups have further strengthened the idea of a reciprocal relationship between Tregs and Th17 cells (69, 70): IL-2, which is a growth factor for Tregs, inhibits the generation of Th17 cells and promotes the generation of Tregs (69). Subsequently, Il2−/− and Stat5−/− mice exhibit reduced numbers of Tregs, and Il2−/− mice have an increased frequency of Th17 cells in the peripheral repertoire (69). Moreover, Il2−/− , Il2rα−/− , Il2rβ−/− , and Stat5−/− mice develop multi-organ inﬂammatory diseases that can be prevented by the transfer of Tregs (71–74), suggesting that IL-2 and IL-2 receptor signaling promotes the generation of Tregs but inhibits Th17 cells in the peripheral immune compartment in vivo. Additional evidence for a reciprocal developmental relationship between Foxp3+ Tregs and Th17 cells comes from studying the effects of retinoic acid, a vitamin A metabolite, on

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T cell differentiation. Retinoic acid could drive the generation of Tregs while abrogating the differentiation of Th17 cells, but not of Th1 cells (70). CD103+ lamina propria DCs but not splenic DCs appear to be a relevant source of retinoic acid in vivo and, together with TGFβ, are efﬁcient in inducing Foxp3+ Tregs de novo in vitro (75, 76). At the steady state, the frequency of Foxp3+ Tregs in the lamina propria of the gut is three times as high as in the secondary lymphoid tissue (77), suggesting that the repertoire of antigen-speciﬁc Tregs in the gut is induced and expanded by the local environment and driven in part by retinoic acid produced by CD103+ DCs. Retinoic acid is believed to act directly on naive T cells, enhancing TGF-β signaling while inhibiting IL-6 signaling. Mechanistically, retinoic acid enhances the TGF-β-driven phosphorylation of Smad3 but reduces the TGF-β-induced upregulation of the IL-6Rα subunit on T cells. In the presence of both TGF-β and IL-6, retinoic acid suppresses the upregulation of IRF4 and IL23R, resulting in decreased generation of Th17 cells (78). It is clear that retinoic acid cannot induce Tregs on its own, but rather needs to cooperate with TGF-β to mediate its effects. Although the binding of retinoic acid by its nuclear receptor RARα increases Foxp3 promoter activity (79), the relevant mechanism of retinoic acid in modulating the balance between Treg and Th17 cell development in vivo is not entirely clear. The data of others and our own group suggest that in an inﬂammatory setting, retinoic acid inhibits the generation of Th17 cells rather than enhancing de novo generation of Tregs (70, 78). Collectively, these ﬁndings indicate that a common metabolite like retinoic acid can regulate the balance between proinﬂammatory Th17 cells and anti-inﬂammatory Tregs. Moreover, in microarray screenings that were undertaken to detect genes speciﬁcally upregulated in the Th17 or iTreg lineages, the aryl hydrocarbon receptor (AHR) was detected to be highly expressed in both the Th17 (80) and Treg signatures (81). In functional studies, AHR was identiﬁed to be a ligand-

dependent system to control the generation of Th17 versus iTregs in vitro and in vivo. A nonmetabolizable ligand of AHR, 2,3,7,8tetrachlorodibenzo-p-dioxin, induced the expression of Foxp3, resulting in the generation of functional Tregs, whereas 6-formylindolo[3,2b]carbazole, another ligand of AHR, promoted the expression of Th17 cells (80, 82). Endogenous ligands of the AHR exist; however, their relevance in skewing the immune response toward Th17 cells or Tregs is not known. The question of whether reprogramming of either precommitted iTregs or Th17 cells is possible was recently addressed in a study from Chen Dong’s laboratory (66). iTregs could be reprogrammed to the Th17 phenotype in the presence of TGF-β plus IL-6 up to ﬁve days after differentiation (66). Foxp3 is downregulated upon restimulation in the presence of TGF-β plus IL-6; however, in the presence of retinoic acid, IL-6 was unable to induce IL-17 from Foxp3+ T cells. Even naturally occurring Tregs begin to express IL-17 under inﬂammatory conditions such as, for example, in the target tissue of an autoimmune reaction. We have observed that Foxp3+ T cells begin to express IL-17 in the CNS at the peak of disease during EAE (66; T. Korn and V.K. Kuchroo, unpublished observation). IL-6 may be the most crucial factor in mediating this conversion of Foxp3+ T cells into Th17 cells in vitro and in vivo (66; T. Korn and V.K. Kuchroo, unpublished observation). The reexpression of the Th17 program in Foxp3+ T cells appears to be a two-step process that includes downregulation of Foxp3 and release of RORγt and RORα from Foxp3-mediated inhibition.

ROLE OF IL-23 IL-23, a member of the IL-12 family of cytokines, was ﬁrst described in 2000 as a heterodimer composed of a p19 subunit and the p40 subunit shared with IL-12 (17). The receptor for IL-23 was described as being expressed on activated/memory T cell populations (83). A www.annualreviews.org • Th17 Cells

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ﬁrst clue concerning the role of IL-23 in shaping the T cell immune response came from the analysis of IL-23p19-deﬁcient mice. In 2003, Cua et al. (8) discovered that p19-deﬁcient mice, in contrast to p35-deﬁcient mice, were resistant to the development of EAE and had very few cells capable of secreting IL-17 in the CNS (8, 9). A stronger connection between IL23 and Th17 cells was established when investigators showed that IL-23 promotes the production of IL-17 by activated T cells (84) and that IL-23-expanded T cells transfer EAE and CIA (9, 85). IL-23R is clearly not expressed on naive T cells, and after the identiﬁcation of the factors (IL-6, IL-21, and TGF-β) required for the differentiation of Th17 cells, it became clear that IL-23 was not involved in the initial differentiation of Th17 cells. Yet IL23 appears to be essential for the full and sustained differentiation of Th17 cells given that IL-23p19-deﬁcient mice have limited numbers of Th17 cells and that prolonged culture of Th17 cells in vitro requires the addition of IL23. Similarly to IL-12 for Th1 cells, IL-23 could serve to expand and stabilize Th17 responses. Alternatively, IL-23 may induce proinﬂammatory effector cytokines and suppress antiinﬂammatory cytokines like IL-10 in Th17 cells (86). But the precise function of IL-23 for Th17 cells remains elusive, in part because the timing and consistency of IL-23R expression on T cells have been difﬁcult to investigate. Initial studies proposed that IL-23R could be induced by TGF-β (4). More recently, IL-6 and IL-21 were shown to induce IL-23R in a STAT3dependent manner (7). But IL-23R appears also to be dependent on RORγt, as RORγtdeﬁcient mice have reduced expression of IL23R (6). Thus, combined signals of RORγt and STAT3, induced by IL-6/IL-21 together with low amounts of TGF-β, might be required to promote IL-23R expression and confer IL-23 responsiveness. In addition to its role for Th17 cells, IL23 also has an important role in the regulation of the innate immune response. The development of gut inﬂammation in T and B cell–deﬁcient mice depends on IL-23 in that

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the loss of IL-23 but not IL-12 is associated with a decrease in gut inﬂammation induced by anti-CD40 antibody-activated cells of the innate immune system (87). IL-23 appears to induce IL-17, IL-1, TNF, and IL-6 from cells of the innate immune system (87, 88). Whether IL-23-mediated gut inﬂammation is entirely dependent on IL-17 produced by cells of the innate immune system has not been addressed. Consistent with the importance of IL-23 in preclinical models of inﬂammatory bowel disease (87, 89–91), a genome-wide screen revealed that a particular coding variant of the IL23R gene (rs11209026, c.1142G>A, p.R381Q) conferred strong protection from Crohn’s disease, whereas several variants in the noncoding region of this gene were associated with increased susceptibility to Crohn’s disease (92). Similarly, other genome-wide association studies (93, 94) revealed associations of IL23R gene SNPs [R381Q: rs11209026 (same as in Crohn’s disease) and L310P: rs7530511] with psoriasis, further strengthening the idea that IL-23R may be involved in inducing human autoimmune diseases.

SKEWING OF Th17 RESPONSES BY MICROBIAL AGENTS Although IL-23 is not the differentiation factor of Th17 cells, productive and sustained Th17 responses only develop in the presence of IL-23, as revealed by studies with Il23p19−/− and Il23r−/− mice. Indeed, after initial induction of Th17 cells, the availability of IL-23 apparently becomes the limiting factor that determines whether the Th17 response is sustained during an immune and inﬂammatory response (32). Given that IL-23 is predominantly produced by cells of the innate immune system, including DCs and macrophages in the gut, the signals that induce the production of this cytokine might be critical in determining whether the T cell response is dominated by Th17 cells. When mice were immunized with myelin antigen together with zymosan, a fungal wall constituent, as an adjuvant that engages Toll-like receptor 2 (TLR2) and DC-associated

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C-type lectin-1 (dectin-1) receptors on DCs (95), IL-23 was induced and Th17 cells were preferentially generated in vitro and in vivo. This ﬁnding is consistent with studies on human Th17 cells in which memory T cells reactive against fungal cell wall products were mainly identiﬁed in the CCR6+ CCR4+ Th17 subset (96). Furthermore, a genetic defect in the induction of IL-23 and IL-17 may result in fulminant and persistent fungal infections in humans (97), which is also a feature of the autosomal dominant hyper IgE syndrome owing to STAT3 mutations and subsequent loss of Th17 cells (98), further supporting the role of the IL-23/IL-17 pathway in clearing fungal infections. Thus, IL-23 is an important link between innate immune cells and adaptive Th17 responses. However, the exact role of IL-23 in vivo still awaits further elucidation. Upon stimulation with various microbial agents (pathogen-associated molecular patterns), which activate speciﬁc TLRs or dectin receptors, DCs secrete IL-12p70, IL-23, or IL-27. When one of these cytokine signals becomes dominant, it determines the type of immunity that develops, i.e., whether the immune response is skewed toward Th1 or Th17 cells. Much effort has focused on identifying differential stimulation conditions and the associated signaling pathways that lead to the secretion of one or the other member of the IL-12 family of cytokines by DCs (for review, see 99). Whereas TLR4 stimulation by LPS induces p19, p35, and p40—thus producing both functional IL-12p70 and IL-23—stimulation of TLR2 by peptidoglycan induces large amounts of IL-23 but not p35 (100). Interestingly, TLR2-stimulation by Pam3 CysSerLys4 does not result in high levels of IL-23 (101). The reason for this is unclear. However, peptidoglycan also conveys signals through nucleotide-binding domain leucinerich repeat–containing family proteins (NLR proteins), which enhance NF-κB activation. Indeed, when DCs lack the NLR protein NOD2, they are impaired in their capacity to secrete IL-23 (102). Stimulation of endosomal TLR3 by polyinosinic-polycytidylic acid

(polyI:C) might induce DCs to produce both IL-12p70 and IL-27 (99). IL-27 produced by DCs has direct and STAT1-dependent inhibitory effects on the differentiation of Th17 cells (see below). IL-12 promotes Th1 cells whose IFN-γ production inhibits the generation of Th17 cells as well. Thus, TLR3 stimulation by polyI:C is likely to dampen Th17 responses. Accordingly, EAE is attenuated by administration of polyI:C, although this is primarily attributed to the fact that polyI:C is also able to induce type I interferons and especially IFN-β (103). The induction of IFN-β by polyI:C is TLR3 independent. TLRs target NF-κB but also IRFs that function as transcription factors and—in certain combinations—are able to transactivate p19, p28, p35, p40, and Epstein-Barr virus–induced gene 3 (EBI3) product. For example, c-Rel and IRF5 induce the transcription of p19, p35, and p40 and thus both IL-12p70 and IL-23, whereas IRF1, IRF3, and IRF7 exclusively induce p35 and p28 but not p19 and thus would induce IL-12 and IL-27 and not IL-23 (for review, see 99). TLR signals can be modiﬁed by signals from seven-transmembrane domain G protein– coupled receptors. This is relevant because prostaglandin E2 (PGE2), by triggering the E prostanoid receptors EPR2 and EPR4 on DCs, can shift the balance between IL-12 and IL-23 secretion toward IL-23 (104, 105). C-type lectins, unrelated to TLRs, are also pattern-recognition receptors expressed on DCs. Ligation of these receptors might bias DCs to produce IL-23. Indeed, zymosan and curdlan, which trigger dectin, function to trigger preferential secretion of IL-23 and have been used as adjuvants to skew the T cell response toward Th17 (32, 106). Moreover, the two forms of Candida albicans— the yeast form and tissue-inﬁltrating hyphae form—appear to trigger distinct innate immune responses. Whereas the yeast form induces IL-12p70 by engaging TLR2, TLR4, and dectin-1, the hyphae form of C. albicans induces IL-23 by activating dectin-2 and TLR2, but not TLR4 (96). www.annualreviews.org • Th17 Cells

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EFFECTOR FUNCTIONS OF Th17 CELLS IN HEALTH AND DISEASE

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The concept that Th17 cells are a unique subset of effector T helper cells was ﬁrst developed in organ-speciﬁc autoimmunity, where autoantigen-speciﬁc Th17 cells induce severe autoimmunity. However, an early study on in vitro skewing of CD4 T cells with microbial products showed that lysate from Borrelia burgdorferi was able to induce massive amounts of IL-17 in T cell differentiation cultures (107). It is certainly not the primordial function of Th17 cells to induce autoimmunity, but the Th17 lineage constitutes a branch of the adaptive immune system that has a function in the clearance of speciﬁc types of pathogens that require a massive inﬂammatory response and are not adequately dealt with by Th1 or Th2 immunity. Pathogens as diverse as the Gram-positive Propionibacterium acnes; the Gram-negative Citrobacter rodentium, Klebsiella pneumoniae, Bacteroides spp., and Borrelia spp.; the acid-fast Mycobacterium tuberculosis; and fungi-like Pneumocystis carinii and Candida albicans can all trigger a strong Th17 response (4, 107–112). No speciﬁc surface marker has been identiﬁed for Th17 cells, although expression of RANKL and a certain combination of chemokine receptors has been associated with Th17 cells. In humans, coexpression of CCR4 and CCR6 (96) or expression of CCR2 in the absence of CCR5 (113) appears to deﬁne Th17 cells in the memory cell compartment of peripheral blood mononuclear cells (96). Interestingly, IL-17-producing memory CD4+ T cells contain a large fraction of cells reactive against fungal antigens, suggesting that Th17 cells may be preferentially induced in response to fungal infections and may play an important role in orchestrating host defense against certain fungi, as discussed above (96). On the basis of experimental animal studies in infectious diseases, autoimmune conditions, transplantation reactions, allergy, and tumor, investigators have implicated Th17 cells and Th17-associated cytokines in a variety of

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human diseases as well. However, proof of a pathogenic role for human Th17 cells is not yet available. Nevertheless, good circumstantial evidence suggests that Th17 cells are important in human psoriasis (114), rheumatoid arthritis (115), multiple sclerosis (116), inﬂammatory bowel disease (92), asthma (117, 118), and some bacterial and fungal infections. In psoriasis, T cells extracted from psoriatic skin lesions showed predominantly a Th17 phenotype (119), which is in line with the observation that CCL20/CCR6 signaling is an important pathway for chemoattraction of inﬂammatory cells to epithelial tissues. Moreover, a phase II therapeutic trial with a monoclonal antibody targeting p40 was extremely efﬁcient in reducing affected psoriatic skin area (114). However, because this antibody neutralizes both IL-12 and IL-23, the clinical effects cannot be uniquely attributed to the IL-23/IL17 axis, although studies from psoriasis-like skin disease in mice suggest that IL-23 and IL-22 are more important in lesion formation than IL-12/IFN-γ. Similarly, in rheumatoid arthritis patients, in a two-year prospective study, the cytokine expression of TNF, IL-1, and IL-17 was predictive of joint destruction, whereas IFN-γ was protective (115). Furthermore, direct effector functions of Th17 cells in rheumatoid arthritis have been demonstrated in that RANKL expression on the surface of Th17 cells induces osteoclastogenesis (120–122), promoting cartilage and bone destruction/resorption independently of TNF and IL-1 (123, 124). Chondrocytes and osteoblasts respond vigorously to IL-17 by upregulating a plethora of proinﬂammatory cytokines, chemokines, and proteases (see below), and TNF, IL-1, and IL-6 induced by IL-17 might even feed back on the generation and expansion of further Th17 cells in this speciﬁc microenvironment of the joint. In multiple sclerosis, the role of Th17 cells has been more difﬁcult to explore. IL-17 and IL-6 are among the most highly expressed genes in multiple sclerosis lesions (125), and elevated levels of IL-17 have been reported in

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the serum and cerebrospinal ﬂuid of multiple sclerosis patients (116). In a correlative study, IL-17 and CXCL8 (IL-8), which is a target of IL-17 and a strong neutrophil-chemoattractant (see below), were more elevated in opticospinal forms of multiple sclerosis than in conventional multiple sclerosis patients (126). Interestingly, the level of IL-17 in the cerebrospinal ﬂuid of opticospinal multiple sclerosis patients had a signiﬁcant correlation with the extent of spinal lesions as measured by MRI (126). An in vitro study suggested that human Th17 cells might be well equipped to breach the blood-brain barrier and inﬁltrate the CNS parenchyma (127). In monocyte-derived DCs from peripheral blood of multiple sclerosis patients, the expression of IL-23p19 was increased, and this correlated with an enhanced capacity of these DCs to induce IL-17 production in T cells (128). Together with the recent studies in EAE, this suggests an important role for the IL23/IL-17 axis in the pathogenesis of multiple sclerosis as well. Mechanistically, the effector functions of Th17 cells have been partly characterized (Table 1). The cytokines produced by Th17 cells allow Th17 cells to communicate with a wide variety of immune and nonimmune cells. Whereas IL-21 (which is produced in large amounts by Th17 cells) acts on other immune cells such as B cells and feeds back to further amplify Th17 responses, other cytokines produced by Th17 cells, including IL-17, IL-17F, and IL22, have broad effects on many cell types and induce the production of proinﬂammatory cytokines and chemokines to attract neutrophils to the site of inﬂammation and antimicrobial peptides to strengthen host defense directly (Table 1). Th17 cells were named after their production of the signature cytokine IL-17A. However, they also produce IL-17F, IL-21, IL-22, GM-CSF, and potentially TNF and IL-6. The conditions to induce IL-17A have been well deﬁned, and IL-17A and IL-17F are largely coexpressed in CD4+ T cells (129, 130), yet there may be conditions in which subsets of Th17 cells may only produce IL-17A or IL-17F (131).

On the basis of the comparison of IL-17A versus IL-17F KO mice in various disease conditions, investigators have suggested overlapping but differential functions of IL-17A versus IL17F (131). For example, Dong and colleagues (131) suggested that IL-17A but not IL-17F was required to induce EAE, whereas IL-17F but not IL-17A was required to induce airway neutrophilia upon allergen stimulation and severe immunopathology in the dextran sulfate sodium (DSS)-induced colitis model. Moreover, although the differentiation conditions necessary for Th17 cells to yield maximum expression of IL-17A and IL-17F are similar, there are differences in the conditions for the maximum upregulation of the other cytokines associated with the Th17 phenotype, as has been reported for IL-22 (132) (see above). This suggests that Th17 cells might vary in their effector functions depending on the combination of effector cytokines produced by Th17 cells and the corresponding receptor distribution in the target tissue.

IL-17 AND IL-17R IL-17 is the founding member of the IL-17 family of cytokines, which includes IL-17A (also called IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F (169, 170). IL-17E (or IL-25) is not produced by Th17 cells, but it is produced by Th2 cells (171). IL-25 induces the expression of Th2type cytokines and chemokines such as CCL5 (RANTES) and CCL11 (Eotaxin) and might be involved in Th2-type allergic responses (171). Whereas other members of the IL-17 family map to different chromosomes, Il17a and Il17f on mouse chromosome 1 are syntenic to the human genes on chromosome 6. Besides being produced by Th17 cells, both IL-17A and IL-17F are also produced by a variety of cell types, including γδ T cells, NKT cells, NK cells, neutrophils, and eosinophils (117, 172–176). Thus, IL-17 and IL-17F are effector cytokines that are produced by cells of both the innate and the adaptive immune systems, suggesting a bridging function of this www.annualreviews.org • Th17 Cells

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Table 1

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Functions of Th17-associated effector cytokines

Disease/condition

Cytokine

Overall evaluation

Experimental approaches and mechanisms

References

EAE/multiple sclerosis

IL-23

pathogenic

IL-23p19 and IL-23R KO mice are resistant to EAE

8; M. Oukka, unpublished observation

IL-17

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Inflammatory skin disease in mice and psoriasis in humans

IL-22 IL-21

no effect ?

IL-23

pathogenic

IL-17

IL-22

500

pathogenic

Korn et al.

pathogenic

pathogenic

IL-23R is expressed in macrophages inﬁltrating the CNS and macrophages expressing IL-23R respond to IL-23 by expressing IL-17 and IL-22. IL-17A KO mice (IL-17F not reduced) have milder disease. Independently generated strain of IL-17A KO mice (reduced levels of IL-17F) exhibit signiﬁcantly reduced EAE. IL-17F KO mice (normal levels of IL-17A). EAE marginally reduced in IL-17F KO mice. However, in airway hypersensitivity, neutrophil recruitment is severely reduced in IL-17F KO mice, whereas eosinophilic inﬁltration is increased. Anti-IL-17A antibody treatment attenuates EAE but does not abrogate the disease. IL-17 is highly expressed in chronic multiple sclerosis lesions. IL-17 was found in T cells (CD4+ and CD8+ ) and astrocytes of multiple sclerosis lesions. Human Th17 cells are required to breach the blood-brain barrier, which is due to production of IL-17 and IL-22. IL-22 KO mice are not protected from EAE. IL-21R KO have decreased Th17 frequencies in the memory T cell compartment but are not resistant to EAE after immunization with autoantigen in CFA. IL-23 is overproduced by keratinocytes and DCs in psoriatic skin lesions, and injection of IL-23 induces hyperkeratosis. Variants of IL-23R and IL-12B confer resistance to psoriasis. Genetic data on IL-23 and IL-12 assign importance to both IL-12 and IL-23. IL-23R data clearly identify IL-23 as major pathogenic factor in psoriasis. Efﬁciency of ustekinumab (human anti-IL-12/23 monoclonal antibody) in psoriasis in a phase III clinical trial. Higher frequencies of Th17 cells, but not Th1 cells in psoriatic lesions: IL-17A+ , IL-17A/TNF+ , IL-17A/IFN-γ + . Anti-TNF therapy in psoriasis works by decreasing Th17 cells. IL-15 triggers IL-17 from human T cell blasts and IL-15 gene variants (SNP polymorphisms) are associated with psoriasis. IL-22 promotes proliferation of keratinocytes. IL-22 is required for skin inﬂammation in a model for psoriasis.

133, 134 131

131

135 125 136 127 137 5, 53, 54

132, 138

93, 139, 140

141 142

143

144 145, 146

132, 147, 148 149 (Continued )

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(Continued )

Disease/condition

Inflammatory bowel disease

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Cytokine

Experimental approaches and mechanisms

References

IL-21

not evaluated

Region of human chromosome 4 harboring IL2 and IL21 genes was identiﬁed as a potential disease susceptibility locus in genome-wide association study.

94

IL-23

pathogenic

IL-23p19 KO mice have reduced T cell–dependent colitis. IL-23 is essential for driving T cell–dependent colitis in IL-10 KO mice. IL-23p19 but not IL-12p35 is required for mediating gut inﬂammation in a T cell–independent manner, i.e., in Rag KO mice induced with either Helicobacter hepaticus or anti-CD40 treatment. T cell–independent sources of IL-17 are neutrophils and monocytes/macrophages. Anti-IL-23p19 cures IBD in mice, and anti-p40 treatment in humans is beneﬁcial in Crohn’s disease and is associated with decreased IL-12 and IL-23. IL-23R SNP polymorphisms protect from Crohn’s disease. Th17 cells are more potent in transferring IBD than Th1 cells. IL-17 is produced in healthy gut. Blockade of IL-17A in colitis of IL-10 KO mice is inefﬁcient in reducing disease unless IL-6 is also neutralized. Anti-IL-17A monoclonal antibody treatment aggravates DSS-induced colitis. IL-17A protective in DSS-induced colitis (IL-17A KO mice have increased disease with higher levels of CCL2, CCL5, and CCL7 in the colon tissue). IL-17F increases DSS-induced colitis (IL-17F KO mice are relatively protected). IL-22 can be protective in preserving the epithelial barrier function in C. rodentium infection.

89, 91

IL-23p19 KO mice are protected from CIA.

85

PGE2 promotes IL-23 and enhances IL-17 levels in the joints of CIA animals. IL-17 KO mice have reduced CIA. Vaccination against IL-17A reduces arthritis. TLR-4 KO IL-1Ra KO mice are protected from arthritis, whereas TLR-2 KO IL-1Ra KO are highly susceptible owing to lack of Tregs. Expansion of autoreactive Th17 cells drives RA by autoampliﬁcation loops. Th17 cells through secretion of IL-17A act on osteoblastic cells inducing RANKL. Interaction between RANK on osteoclast precursors and RANKL results in the differentiation of osteoclasts and bone resorption.

104

IL-17

Experimental arthritis/ rheumatoid arthritis

Overall evaluation

?

IL-22

protective

IL-21

?

IL-23

pathogenic

IL-17

pathogenic

150 87, 89

89, 151 152, 153

92 152 154 150

155 131

154

156 157 158–160

122

(Continued ) www.annualreviews.org • Th17 Cells

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(Continued )

Disease/condition

Host defense

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Cytokine

Overall evaluation

IL-22

?

IL-21

pathogenic

IL-23

protective

IL-17

protective

IL-22

protective

IL-21

?

Experimental approaches and mechanisms

161

IL-23p19 KO and IL-17R KO are greatly susceptible to K. pneumoniae. Citrobacter rodentium: Induction of Th17 cells in the absence of IL-23p19 in the gut mucosa, but no protective immunity. IL-17A KO are greatly susceptible to K. pneumoniae: IL-17A promotes neutrophil recruitment in infection with K. pneumoniae. Bordetella pertussis-LPS induces IL-23 in DCs in a TLR4-dependent manner resulting in Th17 expansion. IL-17 enhances bactericidal properties of macrophages. CD4+ T cell–derived IL-17 and IL-17F induce lung neutrophil inﬁltration and contribute to pathogen clearance in a murine model of acute respiratory tract infection with Mycoplasma pneumoniae. IL-17-driven chemokine expression is required to attract protective IFN-γ-producing Th1 cells in Mycobacterium tuberculosis infection. IL-17RA KO mice are more susceptible to Candida albicans infection than wild-type mice, and administration of IL-17A to wild-type mice protects from lethal doses of C. albicans. Th17 cells in memory compartment of humans have high frequency of Candida-speciﬁc TCRs. IL-22 is essential for mucosal immunity against pathogens. Together with IL-17 or IL-17F, IL-22 synergistically induces antimicrobial peptides in keratinocytes. Although not required to restrain the pathogen burden in Schistosoma mansoni infection, IL-21 promotes Th2-driven immunopathology. IL-21R KO have less Th2-associated cytokines (IL-4 and IL-13) in the lung draining lymph nodes and less Th2-driven granuloma formation in the liver despite equal parasite loads.

165

type of immunity between innate and adaptive immune responses. The conditions for the induction of IL-17A and IL-17F in CD4+ T cells are similar. Both IL-17A and IL-17F have proinﬂammatory properties (39) and act on a broad range of cell types to induce the ex-

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References

Th17 cells expressing CCR6 migrate to the joints and produce CCL20 to attract other CCR6-expressing cells. Chemoattraction of B cells by Th17 cells: synovial ﬂuid Th17 but not Th1 cells express CXCL13. IL-22R expressed on synovial ﬁbroblasts of rheumatoid arthritis patients. IL-22 induces proliferation of synovial ﬁbroblasts. Blockade of IL-21 signaling by administration of IL-21R-Fc fusion protein attenuated CIA.

162 163 164

4 134

166

167

110

112

96 134 130 168

pression of cytokines (TNF, IL-1β, IL-6, GMCSF, G-CSF), chemokines (CXCL1, CXCL8, CXCL10), and metalloproteinases (18, 177– 184). Moreover, human Th17 cells produce CCL20 themselves (27). CCL20 is a ligand for CCR6 and has antimicrobial as well as

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chemoattractive activity (185). Notably, human Th17 cells express CCR6 (96). IL-17A and IL17F are also key cytokines for the recruitment, activation, and migration of neutrophils (169, 186). Interestingly, IL-17 was reported to be a factor that contributes to the formation of germinal centers (GC) of lymph follicles retaining B cells within GCs and enhancing somatic hypermutation, presumably through modulation of chemokine activity (187). In this study, the source of IL-17 was identiﬁed as Th17 cells but not T follicular helper cells. In the current understanding, T follicular helper cells express CXCR5 and thus respond to the lymph follicle– associated chemokine CXCL13 and home to and help establish the light zone of GCs, where they give cognate help to B cells that have undergone immunoglobulin isotype switching and somatic hypermutation in the GC dark zone (188). Thus, T follicular helper cells in the GC light zone induce further differentiation and selection of B cells. Both Th17 and T follicular helper cells are a major source of IL-21, and therefore both may play an important role in setting up productive GC reactions (187). The IL-17 receptors constitute a distinct family of cytokine receptors (186). The IL-17R family includes IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE. Whereas IL-17RA and IL-17RC are the receptors for IL-17A and IL-17F (177, 189, 190), IL-17RB (also called IL-17RH1 and EVI27) is the receptor for IL17E (IL-25) (191) but also binds IL-17B with low afﬁnity (192). IL-17RB signaling promotes Th2-type immunity. However, in a recent study IL-25 needed to engage both IL-17RB and IL17RA to induce IL-5 and IL-13, suggesting that the functional IL-25R complex might be a heteromeric receptor consisting of IL-17RA and IL-17RB subunits in mouse and human (193). Thus, IL-17RA not only conveys proinﬂammatory IL-17 effects, but also contributes in IL25 signaling. The ligands for IL-17RD and IL17RE are not known. IL-17R family members have one transmembrane domain and a large intracellular C terminus. Except for IL-17RA,

alternatively spliced mRNAs appear to exist for all IL-17R family members, and IL-17RB and IL-17RC are predicted to have soluble isoforms with conserved ligand-binding capacity acting as decoys. As mentioned, some members of the IL-17R family can form dimers or oligomers with other chains from the same family. It is unclear whether IL-17R family members can also associate with chains from different cytokine receptor families. Although IL-17 seems to activate mitogen-activated protein kinase (MAPK) pathways and NF-κB via TRAF6 (194, 195) and interact with the membrane proximal adapter Act1 (196, 197), the signaling cascade downstream of the IL-17R complexes is not yet known. IL-17RA is the cognate receptor for IL-17. IL-17RA binds both IL-17A and IL-17F, although it binds to IL-17A with higher afﬁnity (186). IL-17RA is highly expressed on hematopoietic cells, but also—at lower levels— on osteoblasts, ﬁbroblasts, endothelial cells, and epithelial cells. In humans IL-17RA can form a heterodimer with IL-17RC that binds human IL-17A and IL-17F (190). IL-17RC is the cognate receptor for IL-17F (189). In contrast to IL-17RA, IL-17RC is expressed only at low levels on hematopoietic cells, but is highly expressed on nonhematopoietic cells. Whereas human IL-17RC also binds IL-17A, mouse IL17RC appears to be speciﬁc for IL-17F and does not bind mouse IL-17A (189). The best functional data exist for IL-17RA, and most experimental paradigms deal with models of infectious diseases in which IL-17R signaling is protective by initiating granulopoiesis and orchestrating neutrophil trafﬁcking. IL-17RA KO mice suffer from severe deﬁciencies in host defense against Klebsiella (108) and Candida (112). Porphyromonas gingivalis– driven bone destruction is increased in IL17RA KO mice (198). Although the adaptive immune response against Toxoplasma gondii appears to be intact in IL-17RA KO mice, the animals experience an increased mortality owing to deﬁciency of neutrophil recruitment to the site of infection (199). The myelotoxicity of γ irradiation is increased in IL-17RA KO www.annualreviews.org • Th17 Cells

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mice owing to impaired hematopoietic recovery (200). Thus, IL-17 induces a broad tissue response leading to neutrophil trafﬁcking to the site of inﬂammation. Whereas the clearance of some pathogens essentially depends on this response, other pathogens such as certain viruses (201), bacteria such as Pseudomonas aeruginosa (202), and fungi such as Aspergillus fumigatus (203) induce the production of IL-17, but can be controlled and ﬁnally cleared irrespective of a strong neutrophil inﬁltration. In this scenario, IL-17-driven inﬂammation is no longer protective but carries the risk of severe immunopathology and autoimmunity.

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IL-22 AND IL-22R IL-22 is a cytokine of the IL-10 family of cytokines. This family further comprises IL-19, IL-20, IL-24, IL-26, IL-28, and IL-29. IL-22 was cloned from a murine T cell lymphoma line that had been stimulated with IL-9 (204). IL-22 is produced by terminally differentiated Th17 cells and appears to be produced by Th17 cells in response to IL-23 (86). Thus, robust expression of IL-22 in Th17 cells requires the expression and triggering of the IL-23R in those cells. Because high concentrations of TGFβ inhibit the expression of IL-23R (43), IL22 is also repressed by high concentrations of TGF-β. Like the IL-17R, the receptor for IL-22, which is a heterodimer of the speciﬁc IL-22R and the IL-10R2 (205, 206), is widely expressed, including in epithelial and endothelial cells (207). However, in contrast to the IL-17R, the IL-22R is not expressed on immune cells, and thus IL-22 is used by Th17 cells to communicate with tissues but not with other immune cells. Besides associating with IL-10R2, IL-22R can also associate with IL-20R2, and the heterodimeric IL-22R/IL-20R2 complex is a functional receptor for IL-20 and IL-24 (208). Signal transduction through the IL-22R activates MAPK pathways (Erk, Jnk, p38) and STAT3, and activation of STAT1 and STAT5 have also been described (205, 209). STAT3 activation is functionally relevant given that in vivo effects of 504

Korn et al.

IL-22 on liver cells can be abrogated by overexpression of SOCS3 (210). Importantly, a soluble IL-22-binding protein (IL-22BP) has been discovered that lacks transmembrane and signal transduction domains (211). IL-22BP is produced by LPS-stimulated monocytes and neutralizes IL-22-driven STAT3 activation (212). Part of the complex effects of IL-22 may be due to the existence of this endogenous antagonist of IL-22 that appears to be upregulated under inﬂammatory conditions. IL-22 induces antimicrobial agents in keratinocytes (130) and is essential in the immune barrier function of epithelia, as shown in bronchial infection models with Klebsiella (134) and gut infection with Citrobacter (154). Furthermore, IL-22 induces keratosis in mouse models of psoriasis and plays a role in human psoriasis as well (213). IL-22 may promote the breach of the blood-brain barrier in T cell–mediated CNS autoimmunity (127). The sources of IL-22 in these various disease conditions are not always Th17 cells. Whereas IL-22 is derived from non-T cell and non-B cell sources in Citrobacter-induced gut inﬂammation and is indeed secreted by DCs (154), T cells are the most relevant source of IL-22 in Klebsiella infection of the respiratory tract (134). Similarly, Th17 cells are the main producers of IL-22 in EAE. However, because IL-22-deﬁcient mice are not resistant to EAE (137), IL-22 on its own does not deﬁne the pathogenicity of Th17 cells. Rather, a combinatorial expression of effector cytokines likely contributes to the potential pathogenicity of Th17 cells. IL-22 might not always be proinﬂammatory and may even protect from immunopathology under certain circumstances. For example, IL-22 is protective in inﬂammation of the liver (214), the gut (154, 215), and the myocardium (216). In experimental concanavalin A–induced hepatitis in mice, Th17 cells are a vehicle for delivering IL-22 into the site of inﬂammation to limit liver damage (214). Moreover, IL-22 induces LPS-binding protein and thus could also prevent systemic inﬂammation by neutralizing LPS in case of bacterial infection or intestinal bacterial transmigration

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in inﬂammatory bowel disease (217). Thus, IL22 as an effector cytokine of Th17 cells appears to be complex and probably redundant in some aspects. Collectively, the available data point to an important role for IL-22 in epithelial and endothelial barrier function. Interestingly, the dual effect of IL-22 in promoting inﬂammation and tissue repair might be determined on several levels: ﬁrst by various combinations of Th17-derived effector cytokines that are co-produced together with IL-22, and second by the downstream effector molecules of IL-22 that are speciﬁcally and differentially induced in various target tissues.

REGULATION OF Th17 IMMUNITY IL-27 is a member of the IL-12 family of cytokines and is produced by cells of the innate immune system. IL-27 consists of two subunits, p28 and the EBI3 product. The IL-27 receptor is a heterodimer that is composed of a speciﬁc IL-27R subunit (also called WSX-1) and the gp130 subunit that is shared with the IL-6 receptor family (83). IL-27 was initially described as inducing T-bet and enhancing Th1 responses (218), but IL-27 also has dominant antiinﬂammatory properties (219). Thus, analysis of IL-27R-deﬁcient mice revealed increased inﬂammation in an infectious disease model and increased neuroinﬂammation in EAE (220, 221). Lack of IL-27 signaling resulted in an increased Th17 response that could account for enhanced tissue inﬂammation. The inhibition of Th17 responses by IL-27 depends on STAT1 signaling, but did not require T-bet, IFN-γR, or IL-6 receptor signaling (220, 221). Thus, IL27 dampens Th17 responses independently of cross-inhibition as a result of enhanced Th1 commitment of activated T cells. However, IL27 has even broader effects, as lack of IL-27 signaling not only results in enhanced Th17mediated immunopathology, but also in tissue inﬂammation mediated by Th1 cells. These results suggest that there is a more generalized defect in regulation of proinﬂammatory T cells, which is dependent on IL-27. Indeed, three

recent reports suggest (a) that IL-27 together with TGF-β might be the differentiation factor for IL-10-producing T cells that have Tr1like properties and (b) that IL-27R-deﬁcient (Wsx1−/− ) mice had a defect in generating IL10-producing Tr1 cells (222–224). Thus, IL-27 might also be necessary to control exaggerated immunopathology indirectly by inducing Tr1 cells.

CONCLUDING REMARKS: THE CONCEPT OF Th17 IMMUNITY Since the identiﬁcation of Th17 cells, much progress has been made in understanding their development and in deﬁning the type of immunity that is mediated by this subset of effector T helper cells. Speciﬁc pathogens trigger Th17 responses, and productive Th17 responses are required to clear many pathogens, presumably because Th1 or Th2 responses are not protective in these conditions. The molecular mechanisms by which an adaptive immune response is skewed toward a Th17 response appear to rely in part on the ability of speciﬁc pathogenassociated molecular patterns to trigger cells of the innate immune system to produce cytokines, speciﬁcally IL-23, that favor the development of Th17 cells. IL-17 and Th17 responses are so important in these conditions that patients with a genetic defect in the IL23/IL-17 axis develop devastating fungal infections that cannot be controlled even though productive Th1 or Th2 responses are generated in these patients. Thus, like adaptive Th1 and Th2 responses, the development of Th17 immunity requires cues from the innate immune system. However, compared with the differentiation of Th1 and Th2 cells, the requirement of TGF-β for the differentiation of Th17 cells is one of the fundamental differences in the induction of this effector T cell subset. It is very surprising that an immunosuppressive cytokine is required for the induction of a T helper cell subset with highly proinﬂammatory properties. At the functional level, the requirement of TGF-β for the induction of both Foxp3+ Tregs and Th17 cells www.annualreviews.org • Th17 Cells

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might provide a system to efﬁciently balance between tolerance and immunity: In the steady state, TGF-β induces Foxp3 and Tregs, inhibits inﬂammation, and maintains self-tolerance, but once IL-6 is produced by innate immune cells in response to microbial triggers, Treg generation is prevented, and the function of nTregs is suppressed while Th17 cells are induced to produce a strong proinﬂammatory response. At the molecular level, the balance between Tregs and Th17 cells is maintained by the induction of Foxp3 and RORγt and their association with each other. The steps necessary for the terminal commitment of Th17 cells have been identiﬁed based on the discoveries that IL-21 acts in an autoampliﬁcation loop and that IL-23 further matures and expands precommitted Th17 cells. We have proposed a three-step model: (a) TGF-β plus IL-6 induces the differentiation of Th17 cells, (b) IL-21 ampliﬁes the frequency of Th17 cells, and (c) IL-23 terminally differentiates and stabilizes the phenotype of Th17

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cells. Although ﬁndings in preclinical models of chronic inﬂammation and autoimmunity conﬁrm this hypothesis, many questions remain concerning the plasticity of developing Th17 cells. It remains to be determined how far Th17 cells and also Tregs can proceed in their developmental pathway and still be able to be reprogrammed to attain the other phenotype. Another ﬁeld of considerable uncertainty relates to the effector functions of Th17 cells. The effector cytokines of Th17 cells may have differential and sometimes opposing effects in various target tissues. Nevertheless, accumulating data suggest that Th17-mediated immune responses are very important in host defense but also in promoting chronic inﬂammation and autoimmunity. It is envisioned that the IL-23/IL-17 axis will provide important new targets that allow us to dampen Th17-driven immunopathology and promote the generation of Foxp3+ Tregs while at the same time sparing the protective function of Th17 immunity in host defense and tissue repair.

SUMMARY POINTS 1. Besides Th1 and Th2 cells, Th17 cells constitute a third subset of effector T helper cells with distinct effector functions. The differentiation factors (TGF-β plus IL-6 or IL-21) and speciﬁc transcription factors (STAT3, IRF4, RORγt, and RORα) that deﬁne the Th17 transcriptional program have been identiﬁed. 2. Th17 cells are reciprocally related to Foxp3+ Tregs given that TGF-β induces Foxp3 in naive T cells, whereas IL-6 suppresses the TGF-β-driven induction of Foxp3, and TGF-β plus IL-6 together induce RORγt and the Th17 transcriptional program. At the molecular level, the balance between Th17 cells and Foxp3+ Tregs is mediated by the antagonistic interaction of the transcription factors Foxp3 and RORγt. 3. IL-21 is produced by Th17 cells themselves, and IL-21 together with TGF-β is able to induce Th17 differentiation. Thus, IL-21 may be part of a positive feedback loop to amplify the precursor frequency of Th17 cells. 4. IL-23 is not the differentiation factor of Th17 cells. However, IL-23 stabilizes differentiating Th17 cells and leads to the further maturation of Th17 cells, for example by inducing IL-22 in Th17 cells. 5. Th17 cells are important effector cells in host defense against certain pathogens such as Candida albicans and speciﬁc extracellular bacteria. However, the broad receptor distribution of IL-17 and IL-22 results in a massive tissue response upon activation of Th17 cells. This broad response to Th17-related effector cytokines might be the basis for the prominent capability of Th17 cells to induce tissue inﬂammation and autoimmunity.

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FUTURE ISSUES 1. How are the signals of the immunosuppressive cytokine TGF-β and the signals induced by IL-6 or IL-21 integrated to antagonize each other and result in the Th17 transcriptional program? 2. Is there plasticity between committed Th17 cells and Tregs in vivo? 3. What effector cytokine or what combination of Th17-derived effector cytokines mediate the effector functions of Th17 cells in various tissues?

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4. Do Th17 cells have to cooperate with other T helper cell subsets to induce tissue inﬂammation and autoimmunity? 5. How are Th17 cells regulated in vivo?

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS This work was supported by grants from the National Multiple Sclerosis Society, the National Institutes of Health, the Juvenile Diabetes Research Foundation Center for Immunological Tolerance at Harvard, and the Deutsche Forschungsgemeinschaft (KO 2964/2-1). V.K. Kuchroo is the recipient of the Javits Neuroscience Investigator Award from the National Institutes of Health.

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Further

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Immunological and Inﬂammatory Functions of the Interleukin-1 Family Charles A. Dinarello Department of Medicine, Division of Infectious Diseases, University of Colorado Denver, Aurora, Colorado 80045; email: [email protected]

Annu. Rev. Immunol. 2009. 27:519–50

Key Words

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

cytokine, host defense, caspase-1, autoinﬂammatory, inﬂammasome

This article’s doi: 10.1146/annurev.immunol.021908.132612

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0519$20.00

More than any other cytokine family, the interleukin (IL)-1 family is closely linked to the innate immune response. This linkage became evident upon the discovery that the cytoplasmic domain of the IL-1 receptor type I is highly homologous to the cytoplasmic domains of all Toll-like receptors (TLRs). Thus, fundamental inﬂammatory responses such as the induction of cyclooxygenase type 2, increased expression of adhesion molecules, or synthesis of nitric oxide are indistinguishable responses of both IL-1 and TLR ligands. Both families nonspeciﬁcally affect antigen recognition and lymphocyte function. IL-1β is the most studied member of the IL-1 family because of its role in mediating autoinﬂammatory diseases. Although the TLR and IL-1 families evolved to assist in host defense against infection, unlike the TLR family, the IL-1 family also includes members that suppress inﬂammation, both speciﬁcally within the IL-1 family but also nonspeciﬁcally for TLR ligands and the innate immune response.

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INTRODUCTION

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IL-1 decoy receptor (IL-1RII): IL-1 type II receptor that lacks a cytoplasmic domain and thus binds IL-1 but does not transmit a signal Single Ig IL-1-related receptor (SIGIRR): functions as an anti-inﬂammatory receptor suppressing inﬂammation IL-18-binding protein (IL-18BP): a naturally occurring, constitutively secreted protein that neutralizes IL-18; it is not a soluble receptor, has one Ig-like domain, and has limited homology to the third domain of the IL-18Rα Anakinra: the generic name for the recombinant form of the IL-1Ra (brand name Kineret®); natural IL-1Ra is glycosylated, whereas the recombinant anakinra is not Table 1

There are 11 members of the IL-1 family (IL-1F) of ligands; IL-1α, IL-1β, IL-1 receptor antagonist (IL-1Ra), and IL-18 have been thoroughly studied in vitro, in animal models of disease, and in humans. In humans, blocking IL-1 activity, particularly IL-1β, has entered clinical medicine. Although within the IL-1 family of ligands separate gene products induce local and systemic inﬂammation, the family also diverged into genes that protect against runaway inﬂammation and immune responses (Table 1). For example, IL-1Ra is a speciﬁc inhibitor of the activity of both IL-1α and IL-1β, whereas IL-1F5 and IL-1F7 appear to function as nonspeciﬁc inhibitors of inﬂammation and the innate immune response (1, 2). The most recent addition to the IL-1 family is IL-1F11 (IL-33). IL-33 is the ligand for the former orphan receptor ST2 (3). IL-33 plays a role in mast cell functions and drives allergic and Th2 responses. Similar to IL-1α, IL-33 can act via its speciﬁc cell surface receptor (ST2); however, similar to IL-1α, IL-33 is an intracellular cytokine and can function as a DNA-binding nuclear factor (4, 5). IL-1F7 also is found in the nucleus and may act as a nuclear factor (1). Unlike other cytokine families, the IL-1 family exerts control over inﬂammation at both the receptor and nuclear levels. Members of the IL-1 family of receptors contain activators and suppressors of inﬂammation. For example, the IL-1 type II receptor (IL-1RII) functions as a

IL-1 family members

New Name

Other Name

Property

IL-1F1

IL-1α

Agonist

IL-1F2

IL-1β

Agonist

IL-1F3

IL-1Ra

Receptor antagonist

IL-1F4

IL-18; IFN-γ-inducing factor

Agonist

IL-1F5

FIL1δ

Anti-inﬂammatory

IL-1F6

FIL-1ε

Agonist

IL-1F7

IL-1H4, IL-1ζ

Anti-inﬂammatory

IL-1F8

IL-1H2

Agonist

IL-1F9

IL-1ε

Agonist

IL-1F10

IL-1Hy2

Receptor antagonist (?)

IL-1F11

IL-33

Agonist

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decoy receptor (6), the single Ig IL-1-related receptor (SIGIRR) suppresses inﬂammation (7, 8), and the IL-18-binding protein (IL-18BP) is the high-afﬁnity endogenous neutralizer of IL18 activity (9). IL-1Ra binds tightly to IL-1RI and blocks the activity of either IL-1α or IL-1β. IL-1Ra (generically known as anakinra) is approved in several countries for treating the signs, symptoms, and joint destruction of rheumatoid arthritis. Over 100,000 patients with rheumatoid arthritis and related diseases have been treated with IL-1Ra, some for more than 10 years. Blocking IL-1 activity with IL-1Ra results in an arrest in the progressive joint space narrowing that is one of the hallmarks of rheumatoid arthritis. In a placebo-controlled randomized trial, IL-1Ra appears to beneﬁt patients with stroke (10). IL-1Ra is now the standard of therapy for patients with systemic-onset juvenile idiopathic arthritis (11), refractory adult Still’s disease (12), and several systemic and local inﬂammatory diseases. These chronic inﬂammatory diseases are now classiﬁed as autoinﬂammatory diseases. The IL-1 Trap, a bivalent construction of the extracellular domains of IL-1RI and the IL-1R accessory protein (IL-1RAcP) (13), has been approved for the treatment of autoinﬂammatory diseases such as familial cold-induced autoinﬂammatory syndrome (FCAS) and Muckle-Wells syndrome (14). For many years, there has been a great interest in the ability of IL-1β to damage the insulin-producing pancreatic beta cell (15, 16). But recent studies in humans have demonstrated a role for IL-1 in diabetes. In a placebo-controlled, blinded, randomized trial in patients with poorly controlled type 2 diabetes, 13 weeks of IL-1Ra therapy improved glycemic control and the function of the insulin-producing beta cell (17). In a second study, neutralizing monoclonal antibodies to IL-1β also improved glycemic control and beta cell function in type 2 diabetic patients (18). Therefore, destruction of the insulinproducing beta cell appears to be an IL-1βmediated autoinﬂammatory disease (19).

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Another member of the IL-1 family is IL18BP (9), the naturally occurring inhibitor of IL-18 activity. IL-18BP has been administered to patients with rheumatoid arthritis and plaque psoriasis. IL-18BP is currently being tested in patients with macrophage activation syndrome (20).

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THE DIFFERENCE BETWEEN AUTOIMMUNE AND AUTOINFLAMMATORY DISEASES The Discovery of Cryopyrin (NALP3) and the Caspase-1 Inflammasome Autoinﬂammatory disease is a new classiﬁcation for chronic inﬂammation with a distinct therapeutic role for blocking IL-1β. In the 1970s, an astute clinical observation in a rare disease provided us with a greater understanding of chronic inﬂammation. Alan Wanderer studied patients at the National Jewish Hospital in Denver, Colorado, with cold urticaria but observed that some patients actually suffered from an autosomal dominant disease associated with cold weather. He called the syndrome familial cold urticaria. Wanderer and Charles Kirkpatrick studied these patients by exposing them to 30 min of controlled cold temperatures; soon thereafter, tender, erythematoid macular-like skin eruptions were observed. Histologically, the exanthem was characterized by a neutrophilic inﬁltrate. These patients then developed severe constitutional symptoms with chills, fever, fatigue, and joint aches. Leukocytosis (primarily neutrophilia) followed within a few hours. As systemic symptoms developed over days, colleague Patsy Giclas observed elevations in acute-phase reactants. A debilitating disease, cold urticaria led patients to live in warm climates to avoid the cold. They appeared to have an acute infection, with elevated acutephase proteins and leukocytosis, but in fact their symptoms were triggered simply by exposure to cold. Wanderer asked Hal Hoffman, the young immunogeneticist son of Wanderer’s colleague Leonard Hoffman, to identify the gene in this

disorder to understand better the underlying pathogenic mechanisms. Owing to the systemic nature of the disease, Hoffman changed the name of the syndrome from familial cold urticaria to familial cold autoinﬂammatory syndrome (FCAS). Genetic analysis revealed that these patients possessed single nucleotide mutations in the coding region of a particular gene, which he named cold-induced autoinﬂammatory syndrome-1 (CIAS1). This gene coded for an intracellular protein, which Hoffman termed cryopyrin because the patients developed fever following exposure to cold (21). Subsequently, the name for CIAS1 was changed to nucleotidebinding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) because it was a member of the large NOD-like receptor (NLR) family of genes important in the regulation of innate immune functions (22). Because it has become clear that cryopyrin functions in several inﬂammatory diseases unrelated to FCAS, it is commonly called NALP3, which accurately describes its common structural domains. NALP3 associates by oligomerization with other intracellular proteins by protein-protein interactions to form a complex known as the inﬂammasome (23–25). The inﬂammasome functions to convert inactive procaspase-1 to active caspase1, which cleaves the inactive IL-1β precursor to a secreted, active cytokine. Caspase-1 also cleaves the precursors of IL-18, IL-33, and IL1F7. Unlike members of the IL-1 family, the inﬂammasome plays no role in the activity of tumor necrosis factor (TNF)-α. Since the pivotal discovery of NALP3 (cryopyrin), there has been greater understanding of systemic and local inﬂammation.

Characteristics of Autoinflammatory Diseases

Autoinflammatory diseases: a diverse group of inﬂammatory diseases primarily mediated by IL-1β, some of which are due to mutations in the proteins that comprise the inﬂammasome and result in increased secretion of active IL-1β, but most of which have no known mutations in the inﬂammasome IL-1 receptor accessory protein (IL-1RAcP): forms a dimer with either the IL-1RI or the IL-33Rα chain (ST2) and is required for signal transduction for IL-1 and IL-33; it also forms a complex with the IL-1RII and IL-1β, blocking IL-1β activity Type 2 diabetes: adult-onset diabetes in which the beta cells are still present in the pancreatic islet (unlike in type 1) but the peripheral tissues are unable to use insulin (known as insulin resistance) NOD: nucleotidebinding oligomerization domain NLR: NOD-like receptor

Although nearly all autoimmune diseases have an inﬂammatory component, some diseases appear to be primarily inﬂammatory in nature because of their periodicity, strong associations with exogenous triggering events, and lack of associations with class II MHC haplotypes. In comparison, autoimmune diseases exhibit www.annualreviews.org • Functions of the IL-1 Family

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Inflammasome: a complex of intracellular interacting proteins (oligomerization) that initiates the autocatalysis of procaspase-1 into an active enzyme P2X7 receptor: a purinergic receptor that participates in the activation of the inﬂammasome and caspase-1

Table 2

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distinct MHC-associated haplotype susceptibilities, are progressive rather than periodic, and are not strongly associated with environmental stress triggers. Moreover, autoinﬂammatory diseases include diseases that are due to speciﬁc mutations in proteins that regulate IL1β activity and not TNF-α activity. As such, autoinﬂammatory diseases are highly responsive to IL-1β blockade and not TNF-α neutralization, whereas autoimmune diseases are responsive to anti-TNF-α agents. Other therapies such as CTLA4-Ig and anti-IL-12/IL-23 are also effective in treating patients with autoimmune diseases but have no effect in patients with autoinﬂammatory diseases. Table 2 lists autoinﬂammatory diseases that consistently respond rapidly to blocking IL-1β. Table 3 lists the therapeutic options for reducing IL-1 activity.

Autoinflammatory diseases1

Familial Mediterranean fever Familial cold autoinﬂammatory syndrome (FCAS) Muckle-Wells syndrome Neonatal-onset multi-inﬂammatory disease (NOMID) Mevalonic aciduria Hyper IgD syndrome Adult-onset Still’s disease Systemic-onset juvenile idiopathic arthritis Schnitzler’s syndrome Anti-synthetase syndrome TNF receptor–associated periodic syndrome Macrophage activation syndrome2 Behc¸et’s syndrome Normocomplementemic urticarial vasculitis Pericarditis PAPA syndrome Blau’s syndrome Sweet’s syndrome Urate crystal arthritis (gout) Type 2 diabetes3 1

Responsive primarily to IL-1 blockade. IL-18 likely contributes to the macrophage activation syndrome (20). 3 IL-1 blockade with IL-1Ra (17) or anti-IL-1β monoclonal antibody (18) protects the insulin-producing beta cells. Hyperglycemia stimulates IL-1β secretion from the beta cell and accounts for the loss of the beta cell mass in type 2 diabetes (109). 2

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The pathological mechanism of increased secretion of active IL-1β in autoinﬂammatory diseases is usually but not always dysfunctional activation of caspase-1. In general, the release of active IL-1β from blood monocytes is tightly controlled; less than 20% of the total synthetic IL-1β precursor is processed and released. In contrast, monocytes from patients with autoinﬂammatory syndromes release more processed IL-1β, but the increase is modest, that is, less than tenfold greater than that of monocytes from healthy subjects. In circulating human blood monocytes, caspase-1 can be present in an active state (26); as soon as the monocyte is stimulated, cleavage of the precursor takes place, and active IL-1β is secreted. Although it is possible to demonstrate that monocytes from patients with autoinﬂammatory diseases process and release signiﬁcantly more IL-1β than do monocytes from healthy controls (11, 27–29), the basis for the increase varies. Several autoinﬂammatory diseases have a mutation in NALP3, resulting in a single amino acid change in the protein and an active inﬂammasome (21, 22, 28–31); however, the same autoinﬂammatory diseases with the same clinical characteristics do not have mutations in genes known to regulate caspase-1 or the secretion of IL-1β. For example, the secretion of IL-1β appears to require triggering of the P2X7 purinergic receptor by ATP (32) and active phospholipases C and A2b for secretion via secretory lysosomes (33). Regardless of the mechanism of increased IL-1β release, what characterizes these diseases as autoinﬂammatory is the rapid and sustained response to a reduction in IL-1β activity. Autoinﬂammatory diseases are syndromes. They include neonatal-onset multiinﬂammatory disease (NOMID), MuckleWells syndrome, FCAS, hyper IgD syndrome, familial Mediterranean fever, and urate crystal arthritis (gout) (see Table 2). Some of these syndromes are rare, but the clinical manifestations are common to nearly all inﬂammatory as well as infectious diseases. As discussed below, type 2 diabetes mellitus and gout are autoinﬂammatory diseases and are hardly

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Table 3

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Specific therapies for blocking IL-1 activities

Reagent

Composition

Mechanism of action

Specificity

Anakinraa

IL-1Ra

binds to IL-1RI>IL-1α>IL-1β

IL-1α and IL-1β

IL-1Ra trimer

IL-1Ra

binds to IL-1RI>IL-1α>IL-1β

IL-1α and IL-1β

IL-1 Trapb

IL-1RI + IL-1RAcP

neutralizes IL-1β>IL-1α>IL-1Ra

IL-1β (IL-1α)

Soluble

IL-1RIIc

IL-1RII

neutralizes IL-1β>IL-1α

IL-1β

Anti-IL-1β

monoclonal antibody

neutralizes IL-1β

IL-1β

Anti-IL-1RI

monoclonal antibody

blocks IL-1RI

IL-1α and IL-1β

Anti-IL-1RAcP

monoclonal antibody

blocks IL-1RAcP

IL-1α and IL-1β, IL-33

Peptide antagonistd

RYTVELA

blocks IL-1RI

IL-1α and IL-1β

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a

Anakinra is the recombinant (nonglycosylated) form of IL-1Ra. Naturally occurring IL-1Ra is the glycosylated gene product. Bivalent with Fc. c Efﬁcacy of the extracellular IL-1RII is greatly enhanced by the presence of extracellular IL-1RAcP (164, 166). d Inhibits some but not all IL-1 activities (158). b

rare conditions. One important criterion for characterizing a disease as autoinﬂammatory is that upon blocking IL-1 rather than TNFα activity, patients experience a rapid and sustained cessation of symptoms as well as reductions in biochemical, hematological, and functional markers of their disease. Given that treatment with the IL-1 Trap or monoclonal anti-IL-1β antibodies are equally effective in treating autoinﬂammatory diseases, the culprit in these diseases is IL-1β and not IL-1α. Another criterion for classiﬁcation of an autoinﬂammatory disease is a reduction in disease severity with the use of speciﬁc inhibitors of caspase-1, which targets the processing and secretion of IL-1β.

OVERVIEW OF THE IL-1 FAMILY IN INFLAMMATORY RESPONSES Unlike IL-2 and other cytokines that directly affect lymphocyte function, differentiation, and expansion, most members of the IL-1 family primarily do not directly affect lymphocyte function. As such, the effect of IL-1 family members on immune functions is indirect. For example, the ability of IL-1β to induce gene expression and synthesis of cyclooxygenase type 2 (COX-2), type 2 phospholipase A, and inducible nitric oxide synthase (iNOS) accounts for prostaglandin-E2 (PGE2), platelet activating factor, and nitric oxide (NO) production.

As a result, there is fever, lowered pain threshold, vasodilatation, and hypotension. However, these products also affect immune responses. For example, PGE2 is perhaps the most common mechanism for nonspeciﬁc suppression of T cell responses. Another important proinﬂammatory property of IL-1β is its ability to increase the expression of adhesion molecules such as intercellular adhesion molecule-1 on mesenchymal cells and vascular cell adhesion molecule−1 on endothelial cells. Together with the induction of chemokines, these properties of IL-1β promote the inﬁltration of inﬂammatory and immunocompetent cells from the circulation into the extravascular space and then into tissues where tissue remodeling is the end result of chronic IL-1-induced inﬂammation. IL-1β is also an angiogenic factor (34) and plays a role in tumor metastasis and blood vessel formation. In mice deﬁcient in IL-1β, vascular endothelial cell growth factor (VEGF) cannot stimulate formation of blood vessels, and malignant melanoma cells do not spread. IL-1β also acts on bone marrow stem cells for differentiation of the myeloid series of progenitor cells. In humans injected intravenously with low doses of IL-1β of 1–10 ng/kg, there is fever and increased levels of ACTH, blood neutrophils, NO, acute-phase proteins, and several cytokines and chemokines. IL-6 production is particularly sensitive to IL-1β, and a dose of www.annualreviews.org • Functions of the IL-1 Family

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1 ng/kg results in high levels of IL-6 (reviewed in 35). Blocking IL-1 in systemic diseases reduces IL-6 levels (36).

OVERVIEW OF THE IL-1 FAMILY IN IMMUNE RESPONSES IL-1 Family Members as Costimulators of T Cells

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IL-1 (IL-1α or IL-1β) functions as a costimulator of T cell functions, primarily together with an antigen or a mitogen. IL-18 alone does not stimulate IFN-γ but rather acts as an essential coactivator with IL-2, IL-12, or IL-15, although the combination of IL-18 plus IL-12 is perhaps uniquely effective at inducing IFNγ. IL-33 is also an activator of T cells, but in this case the function of IL-33 is to promote a Th2 response (3, 37). IL-1 can act as a growth factor for thymocytes. Human thymic epithelium produces constitutive levels IL-1α and contributes to the proliferation of thymocytes. However, it is unlikely that IL-1 plays an essential role in thymic growth and function given that mice deﬁcient in IL-1α, IL-1β, or the IL-1RI have normal thymic development. As with IL-1α, IL-18 is strongly expressed in thymic epithelial cells.

IL-1 and Th2 Immune Responses In some models, IL-1 contributes to Th2 polarization. For example, in murine models of asthma using airway sensitization to antigens, the responses to antigen challenge are increased airway responsiveness to bronchoconstricting agents, inﬁltration of the lungs by eosinophils, and increased expression of IL4. However, in mice deﬁcient in IL-1RI or treated with neutralizing anti-IL-1β antibodies, the response to inhaled antigen is markedly reduced (38). Using airway sensitization with ovalbumin, eosinophil, and neutrophil inﬁltration following inhalation, antigen challenge was prevented in mice expressing IL-1Ra adenovirus (39). Suppression of IL-5 was also observed.

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IL-1 and B Cell Functions A role for IL-1β in antibody production has been repeatedly reported. For example, mice deﬁcient in IL-1β do not produce antisheep red blood cell antibodies, a T-dependent response (40). However, antibody production by T-independent antigens was normal in mice deﬁcient in both IL-1α and IL-1, as was the proliferative response to anti-CD3. The evidence that IL-1β is a growth factor for B cell proliferation may be due to IL-1-mediated induction of IL-6. In fact, in vitro and in vivo, IL-6 is often under the control of IL-1. A nonopeptide in the IL-1β sequence is a potent adjuvant, and this activity is independent of IL-6 (41).

IL-1 and Th17 During the generation of the Th17 response, IL-1 appears essential given that T cells from mice deﬁcient in IL-1RI fail to induce IL-17 upon antigen challenge (42). Moreover, IL-23 fails to sustain IL-17 in IL-1RI-deﬁcient T cells. IL-23 is believed to mediate celiac disease, an autoimmune disease in which 95% of patients are positive for HLA-DQ2. Production of IL-23 in monocytes from these patients by beta-glucan is IL-1β dependent (43). The combination of IL-23 plus either IL-1α or IL1β is synergistic in the induction of IL-17. Even TNF-α enhancement of IL-23-induced IL-17 is IL-1 dependent. In some studies in mice, TGF-β and IL-6 are required for differentiation of Th17 T cells. In a recent study, IL-6 was reported to induce Th17 cells when costimulated with TGF-β (44). In that study, IL-1β and TNF-α were not effective. Although supernatants from LPS-stimulated bone marrow dendritic cells induced IL-17 production from naive T cells, an IL-6 receptor–blocking antibody had no effect (44). TGF-β secretion from LPS-stimulated dendritic cells is low or absent. The addition of exogenous mixtures of recombinant cytokines often results in production of IL-17. For example, the combination of IL-6 and TGF-β induces IL-17, and this combination maintained the activation of STAT3

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(44). Here the role of STAT3 activation is relevant for the induction of the transcription factor retinoic acid–related orphan receptor gamma. Another STAT3-activating cytokine is IL-23, which sustains IL-17-producing cells. Therefore, there is likely a cascade of IL-1β-induced IL-6 and IL-23 for Th17 induction, as both IL-6 and IL-23 are IL-1β dependent. However, there is also the exception that the addition of exogenous IL-1β can result in the suppression of IL-6 phosphorylation of STAT3 (45). Speciﬁc cytokine blockade or a cytokine deﬁciency state reveals the functional consequence of a cytokine in immune responses. Because a highly speciﬁc antibody that prevents the activity of IL-6 had no effect on the ability of LPS-stimulated dendritic cell supernatants to induce IL-17, the data suggest that IL-6 is not the naturally occurring cytokine from antigen-presenting cells that accounts for the polarization of CD4+ cells into a Th17 population. In human cord blood T cells, IL-1β induced polarization of Th17 cells, and this effect was enhanced by IL-6 but suppressed by TGF-β (45). Others also report the antagonism of IL1β-induced IL-17 in human cells by TGF-β (46). That TGF-β suppressed the response is hardly unexpected, as antagonism between IL1β (or IL-18) and TGF-β is often reported (47, 48), particularly in T cells (reviewed in 35). In the mouse, however, TGF-β enhances the differentiation to Th17. Monocytes can prime CD4+ T cells for IL-17 production via IL-1β and IL-6 secretion, but not via IL-12 (45). Although there are several reports that IL-1β can synergize with IL-6 in models of T cell activation and angiogenesis, IL-1β can also antagonize the action of IL-6 by suppressing IL-6 phosphorylation of STAT3 as well as by inducing SOCS3 (49). The requirement of IL-1β in the generation of Th17 cells is consistent with mice deﬁcient in IL-1Ra. Lacking this naturally occurring antagonist of IL-1 activity, IL-1Ra-deﬁcient mice exhibit unopposed IL-1 activity and spontaneously develop a rheumatoid arthritis–like disease, but mice deﬁcient in IL-17 do not (50).

Furthermore, IL-1 activation of the cosignaling receptor OX40 induces IL-17. Taken together, the data indicate that IL-1 is upstream of both OX40 activation and IL-17 production. These ﬁndings are consistent with the observation that mice deﬁcient in both IL-1α and IL-1β do not develop experimental autoimmune encephalomyelitis (EAE) (51). Several studies show that IL-1 and IL-17 act synergistically on the induction of NO, PGE, and cartilage breakdown. Not unexpectedly given that IL-1 induces IL-17, IL-17 in turn stimulates the production and release of IL-1β from primary human blood monocytes. In general, proinﬂammatory cytokines induce each other.

IL-18 Is Both a Th1 and Th2 Cytokine Because IL-18 participates with IL-12 and IL15 in the production of IFN-γ, IL-18 contributes to the Th1 response; however, there is a well-described role for IL-18 in the Th2 response (52). For example, IL-18 induces IL-4, resulting in increased numbers of IL-4-positive cells from activated natural killer (NK) T cells in the absence of IL-12. In general, the presence of IL-12 shifts the activity of IL-18 toward IFN-γ and the Th1 response, whereas IL-18 in the absence of IL-12 drives the Th2 response. Combined overexpression of IL-18 and caspase-1 results in the development of spontaneous atopic dermatitis with increased expression of Th2 cytokines and IgE (53). In transgenic mice overexpressing IL-18, serum levels of IgE, IgG1, IL-4, and IFN-γ were significantly increased (54). Therefore, high IL-18 production can polarize toward both Th1 and Th2 responses. Although IL-18 shares may of the same proinﬂammatory properties with IL1β, IL-18 can also counter the effects of IL-1β (55).

DEFICIENCY IN IL-1 FAMILY MEMBERS Mice deﬁcient in IL-1RI, IL-1RAcP, IL-1α, or IL-1β or doubly deﬁcient in IL-1α and IL-1β www.annualreviews.org • Functions of the IL-1 Family

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exhibit no phenotypical differences from samestrain wild-type mice, and they live in routine animal facilities. One can conclude that IL-1α or IL-1β, which play important roles in disease, are not essential for normal embryonic development, postnatal growth, homeostasis, reproduction, or resistance to routine microbial ﬂora. Also, these mice do not exhibit evidence of spontaneous carcinogenesis, their life span appears normal, and lymphoid architecture is also normal. In contrast, in models of local or systemic inﬂammation, a deﬁciency in IL-1 activity usually results in reduced disease severity but increased susceptibility to live infections. However, mice deﬁcient in IL-1Ra exhibit reduced reproduction, have stunted growth, and develop spontaneous diseases such as rheumatoid arthritis–like polyarthropathy (56), a fatal arteritis (57), and tumors in response to chemical carcinogens (58). Thus, one can conclude that without natural IL-1Ra, IL-1-induced inﬂammation is unchecked.

PRO- AND ANTI-INFLAMMATORY FUNCTIONS WITHIN THE IL-1 FAMILY The IL-1 family also includes members that suppress inﬂammation, both speciﬁcally within the IL-1 family but also nonspecifically for TLR ligands and other cytokines. The suppression can be highly speciﬁc. For example, the IL-1 receptor antagonist, a bona ﬁde member of the family, binds to the IL-1 receptor type I with a greater afﬁnity than IL-1 itself and reduces IL-1α and IL-1β responses. The soluble form of the IL-1RAcP is constitutively produced by the liver and forms a complex with the soluble IL-1RII, which binds and neutralizes IL-1β. The IL-18-binding protein binds and neutralizes IL-18 owing to an afﬁnity for IL18 that is greater than the IL-18 receptor. IL-1 family member seven is a nonspeciﬁc anti-inﬂammatory cytokine. IL-1F7 suppresses IL-1, TNF-α, and TLR ligand activities. SIGIRR is also a nonspeciﬁc inhibitor of inﬂammation. IL-33, the ligand for the orphan receptor ST2, can shift a Th1 response induced by IL-18 into a Th2 response. Thus, the IL-1 family of ligands and receptors evolved with the dual role of defending the host via the innate immune response as well as speciﬁcally and nonspeciﬁcally reducing inﬂammation.

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Similarly, mice deﬁcient in IL-18 exhibit reduced responses in several models of disease. For example, IL-18-deﬁcient mice are resistant to endotoxin lethality, collagen-induced arthritis, Fas ligand–mediated hepatitis, graft-versushost disease, ConA-induced hepatitis, and allograft rejection. However, as discussed below, mice deﬁcient in IL-18 or IL-18Rα become obese with increasing age and develop nearly all the parameters of the metabolic syndrome observed in humans (59).

THE DIVERSITY OF THE IL-1 FAMILY Table 1 lists the current members of the IL1 family and their primary function. In this review, I retain the terms IL-1α, IL-1β, IL18, and IL-33 as well as IL-1Ra. With the exception of IL-18 and IL-33, the members of the IL-1 family are located on the long arm of chromosome 2. From the intron-exon organization, some members represent gene duplications. In the case of IL-1F5 and IL-1F10, the duplication of the IL-1Ra gene has taken place (60). IL-1F9 is also related to IL-1Ra but does not function as a receptor antagonist but rather as an agonist. IL-33 (IL-1F11) is closely related to IL-18. However, as described below, the activity of an individual IL-1 family member is not determined by its structure but rather by its ability to transmit an agonist signal or act as a receptor antagonist. Two members of the IL-1 family (IL-1F5 and IL-1F7) appear to function as broad anti-inﬂammatory cytokines. Equally important to the diversity of the IL-1 family is the role of the IL-1 family of receptors. Whereas those receptors transmitting an inﬂammatory signal are well studied, those transmitting an anti-inﬂammatory signal are of considerable importance in regulating inﬂammation.

IL-1α (IL-1F1) IL-1α as an autocrine growth factor. Calpain, a calcium-activated cysteine protease associated with the plasma membrane, can cleave

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the IL-1α precursor into a mature molecule. However, even under conditions of cell stimulation, human blood monocytes do not process or readily secrete mature IL-1α. IL-1α is not commonly found in the circulation or in body ﬂuids, except that the cytokine may be released from dying cells (61). One distinguishing property of IL-1α is that its precursor form is active. Most cell lines, including tumor cell lines, contain constitutive levels of IL-1α (4, 62, 63). The concept that IL-1α acts as an autocrine growth factor assumes that the intracellular IL-1α precursor regulates normal cellular differentiation, particularly in epithelial and ectodermal cells. In support of the concept, an antisense oligonucleotide to IL-1α reduces senescence in endothelial cells (64). In ﬁbroblasts, the constitutive IL-1α precursor binds to HAX-1, a nonreceptor substrate for tyrosine kinases in hematopoietic cells. In ﬁbroblasts, the IL-1α HAX-1 complex translocates to the nucleus (65). The complex seems to function because suppression of HAX-1 results in a failure of IL-1α to bind to DNA, resulting in reduced production of IL-6 and procollagen (65). Although some accept the concept that IL-1α acts as an autocrine growth factor in ﬁbroblasts or endothelial cells in vitro, the data must be interpreted carefully given that mice deﬁcient in IL-1α show no demonstrable defects in growth and development, including skin, fur, epithelium, or gastrointestinal function (66). However, mice deﬁcient in IL-1α still retain the propiece, which functions as a nuclear factor. In fact, in another study, the N-terminal propiece of IL-1α bound HAX-1 (67). At present, there is no absolute IL-1α-deﬁcient mouse model. Is there a role for intracellular precursor IL1α in normal cell function? The IL-1α precursor is expressed constitutively in all epithelial cells, and large amounts of the intracellular form of the IL-1Ra (icIL-1Ra) are also present in these same cells (68). This form of the IL1Ra also binds to the IL-1RI and prevents signal transduction. In fact, icIL-1Ra is produced in the same cells expressing the IL-1α precursor

and is thought to compete with the intracellular pool of precursor IL-1α for nuclear binding sites. Membrane IL-1α. Precursor IL-1α can be found on the surface of several cells, particularly on monocytes and B lymphocytes, where it is referred to as membrane IL-1α (69). Membrane IL-1α is biologically active (70); its biological activities are neutralized by antibodies to IL-1α but not against IL-1β. Membrane IL-1α plays an important role in inﬂammation, as mice deﬁcient in IL-1α exhibit reduced inﬂammation in models in which cell death and the release of intracellular IL-1α do not take place (71). Biological functions of constitutive IL-1α. Primary cells contain constitutive levels of the IL-1α precursor and not IL-1β (72). Not surprisingly, because the IL-1α precursor is biologically active and found in normal epithelial cells, including thymic epithelium, contents from necrotic cells will contain biologically active IL-1α (61). It is well known that contents of necrotic cells are inﬂammatory, and hence their activity is due to IL-1α present in either the cytosol or the membrane fraction (4). Even if a cell contains the IL-1β precursor, any biological activities of its contents would be due to IL-1α because the IL-1β precursor is inactive (72). Furthermore, epithelial cells do not contain caspase-1, and therefore processing of the IL-1β or IL-18 precursor cannot yield the biologically active forms of either cytokine. Constitutively expressed IL-1α is critical for several IFN-γ activities. Using the WISH epithelial cell line, what were considered to be intrinsic IFN-γ activities depended largely on constitutively expressed IL-1α. IFN-γ activities were inhibited in antibodies to IL-1α, but not to IL1β (63). Others report that antibodies to IL-1α and not IL-1β prevent the biological activities of necrotic cells (61). Studies in IL-1α-deficient mice. Mice deﬁcient in IL-1α are born healthy and develop normally. Following subcutaneous injection of

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turpentine, which induces a local inﬂammatory response, wild-type and IL-1α-deﬁcient mice develop fever and acute-phase proteins, whereas IL-1β-deﬁcient mice do not (66). In addition, although the induction of glucocorticoids after turpentine injection was suppressed in IL-1β-deﬁcient mice, this suppression was not observed in IL-1α-deﬁcient mice. However, expression of IL-1β mRNA in the brain decreased 1.5-fold in IL-1α-deﬁcient mice, whereas expression of IL-1α mRNA decreased more than 30-fold in IL-1β-deﬁcient mice. These data suggest that IL-1β exerts greater control over production of IL-1α than does IL1α over the production of IL-1β. In caspase1-deﬁcient mice, IL-1α production is also reduced (73), suggesting that production of IL-1α is under the control of IL-1β. The effects of IL-1 on atherogenesis by a high-cholesterol diet was evaluated in mice deﬁcient in either IL-1α or IL-1β (71). Serum amyloid A protein, a marker of inﬂammation in atherogenesis, was markedly lower in IL1α-deﬁcient mice compared with wild-type or IL-1β-deﬁcient mice. The beneﬁcial effect of IL-1α deﬁciency was due to bone marrow cells. Transplantation of bone marrow cells from IL1α-deﬁcient mice resulted in a reduction in aortic lesion size twice that observed in mice transplanted with IL-1β-deﬁcient bone marrow cells. Therefore, IL-1α appears to play a greater role in the pathogenesis of lipidmediated atherogenesis than does IL-1β, and this may be due to an effect of membrane IL-1α.

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IL-1β (IL-1F2) Dissociation between transcription and translation. Non-TLR ligands such as the complement component C5a, hypoxia, adherence to surfaces, or clotting of blood induce the synthesis of large amounts of IL-1β mRNA in monocytic cells without signiﬁcant translation into the IL-1β protein. The IL-1β mRNA assembles into large polyribosomes, but without signiﬁcant elongation of the peptide (74), and most of the IL-1β mRNA is degraded. In 528

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IL-1β, IL-18, and IL-1F7, an instability element in the coding region accounts for the failure to translate the mRNA into protein (75). However, adding TLR ligands or IL-1 itself to monocytes with high levels of IL-1β mRNA results in augmented translation (76). Regulation of IL-1β production. Unlike most cytokine promoters, IL-1β regulatory regions are distributed over several thousand base pairs upstream from the transcriptional start site. In addition to a cAMP response element, there are NF-κB-like and activating protein-1 sites. IL-1β gene regulation has been reviewed in detail (77). The primary sources of IL-1β are the blood monocyte, tissue macrophages, and dendritic cells. B lymphocytes and NK cells also produce IL-1β. Fibroblasts and epithelial cells generally do not produce the cytokine, however. Circulating blood monocytes or bone marrow aspirates from healthy humans do not constitutively express mRNA for IL-1β. In contrast, several malignant tumors express IL-1β as part of their neoplastic nature, particularly acute myelogenous leukemia, melanoma, multiple myeloma, and juvenile myelogenous leukemia, each of which exhibits constitutive expression of IL-1β. Although nearly all microbial products induce IL-1β via TLR ligands, IL-1 induces itself both in vivo and in monocytes in vitro (78). Other studies supporting this concept have been reported (27, 79, 80). Following LPS stimulation, IL-1β mRNA levels rise rapidly within 15 min but begin to decline after 4 h owing to mRNA half-life or the action of microRNA. However, using IL-1 itself as a stimulant, IL-1β mRNA levels are sustained for over 24 h compared with microbial stimulants (81). Raising intracellular cAMP levels with histamine enhances IL-1-induced IL-1 gene expression and protein synthesis. As shown in Figure 1, the IL-1β precursor accumulates in the cytosol, and processing by caspase-1 is triggered by ATP activating the P2X7 receptor. In freshly cultured human blood monocytes, endogenous levels of ATP rise and are sufﬁcient for triggering the assembly of the inﬂammasome (26).

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Active IL-1β

1 IL-1β

IL-1RI

IL-1RAcP

Secretion of IL-1β 9

ATP trigger

4

Active IL-1β

5a K+ eﬄux

Ca2+ entry

Ca2+ 8

P2X7 R K channel Caspase-1

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MyD88

Processing 7 of IL-1β

Secretory lysosome

9 Secretion of IL-1β

K+ 5b 7 Processing of IL-1β Caspase-1

3 Translation of IL-1β precursor 2 Transcription IL-1β mRNA

Assembly of 6 inﬂammasome

ASC

Procaspase-1 CARD CARD Procaspase-1 CARD CARD FIIND

PYR PYR NACHT NAD

LRR

NALP3 (cryopyrin) Cardinal NALP3 inﬂammasome

Figure 1 Steps in the synthesis and secretion of IL-1β induced by IL-1β. Primary blood monocytes or tissue macrophages are activated by IL-1β (78) (step 1). The formation of the IL-1 receptor complex heterodimer results in approximation of the Toll IL-1 receptor (TIR) domains (red ) and the recruitment of MyD88. The transcription (step 2) and translation into the IL-1β precursor (step 3) are separate events. Translation takes place in the cytosol, not in the endoplasmic reticulum. The activated monocyte/macrophage releases ATP into the extracellular space (26). Upon activation of the P2X7 receptor by ATP (step 4), there is a rapid efﬂux of potassium from the cell (step 5a) resulting in a fall in intracellular levels of potassium (step 5b). The fall in intracellular potassium levels triggers the assembly of the components of the NALP3 inﬂammasome (step 6). Given that NALP3 (cryopyrin) does not contain a caspase-activating recruiting domain (CARD), the adaptor protein termed apoptosis-associated speck-like protein containing a C-terminal CARD (ASC) is required together with CARDINAL for oligomerization. The assembled components of the inﬂammasome initiate the processing of procaspase-1, resulting in the formation of the active caspase-1. Active caspase-1 processes the IL-1β precursor (step 7) in the cytosol or in the secretory lysosome, resulting in the generation of the carboxy-terminal mature IL-1β. An inﬂux of calcium into the cell (step 8) with an increase in intracellular calcium levels (33, 169) provides a mechanism by which mature IL-1β is released from the cell (step 9). The rise in intracellular calcium activates phosphatidylcholine-speciﬁc phospholipase C and calcium-dependent phospholipase A(2), which facilitate the secretion of IL-1β (step 9) with exocytosis of the lysosomal contents (33). Other pathways exist for processed IL-1β to exit the cell (see text).

Processing and secretion of IL-1β via the caspase-1 inflammasome. Regardless of the stimulus, in monocytes and macrophages speciﬁc inhibitors of caspase-1 reduce the secretion of mature IL-1β, and precursor IL-1β accumulates mostly inside the cell. The ratelimiting step in the processing and secretion of IL-1β takes place with activation of the inﬂam-

masome. As shown in Figure 1, several intracellular proteins form a complex with NALP3. As discussed above, NALP3 was initially discovered in patients with FCAS, a genetic disease characterized by constitutional symptoms, fevers, and elevated acute-phase proteins following exposure to cold (21). Assembly of the inﬂammasome components with inactive www.annualreviews.org • Functions of the IL-1 Family

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ASC: apoptosisassociated speck-like protein containing a C-terminal CARD

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procaspase-1 takes place following a fall in intracellular potassium (Figure 1). ATP activation of the P2X7 receptor opens the potassium channel, and simultaneously as potassium levels fall caspase-1 is activated by the inﬂammasome (33, 82–84). NALP3 is unique among the NLRPs in that there is no caspase-activating recruiting domain (CARD). Therefore, the adaptor protein termed apoptosis-associated specklike protein containing a C-terminal CARD (ASC) provides the linkage of the pyrin domain (PYR) on NALP3 to the CARD domain on procaspase-1 (see Figure 1). Another protein (CARDINAL) is also part of the oligomerization of the inﬂammasome. The cleavage of the IL-1β precursor by active caspase-1 can take place in the specialized secretory lysosomes or in the cytoplasm. However, more than one pathway seems available for processed IL-1β to exit the cell. These pathways include exocytosis of the secretory lysosomes (33, 82), shedding of plasma membrane microvesicles, direct release via transporters, or multivesicular bodies containing exosomes (85). In general, the release of processed IL1β takes place before there is signiﬁcant release of lactate dehydrogenase (86), although in vitro cell death eventually takes place. Pyroptosis is a caspase-1-dependent form of cell death and is employed by certain bacteria using Ipaf, a member of the NLR family of intracellular receptors (87). An increase in intracellular calcium is also required for the mature IL-1β to exit the cell, and it is phospholipase C dependent (33). The function of the caspase-1 inﬂammasome is primarily to convert inactive procaspase-1 into the active enzyme, and this step requires activation of the P2X7 receptor by ATP. However, in freshly obtained human blood mononuclear cells from healthy subjects, the p10 subunit of active caspase-1 is present even in the absence of stimulation (26), and, during the subsequent incubation, extracellular levels of ATP increase (26). Upon differentiation into macrophages, activation of the inﬂammasome requires exogenous ATP (26). But in monocytes from patients with a single amino acid mutation in NALP3, activation of Dinarello

caspase-1 occurs without a requirement for a rapid fall in the level of intracellular potassium (28). Therefore, mutated NALP3 allows for the assembly of the complex of interacting proteins in the presence of normal intracellular levels of potassium. Although this process is often studied using LPS-induced synthesis of the IL-1β precursor (88), LPS likely does not play a role in autoinﬂammatory diseases. However, spontaneous secretion of IL-1β from monocytes of patients is due to endogenous IL-1β stimulation. In patients with NOMID, there is a decrease in steady-state levels of procaspase-1 mRNA with IL-1Ra treatment (27), suggesting that IL-1β stimulates its own production and processing (see Figure 1). Thus, any disease process that includes an increase in the steadystate levels of procaspase-1 mRNA, components of the inﬂammasome, or the IL-1β precursor explains the autoinﬂammatory nature of the disease. Type 2 diabetes appears to be an example of an autoinﬂammatory disease in which glucose induces IL-1β production from the insulin-producing beta cell, and IL-1β induces the beta cell to produce its own IL-1β (19). P2X7 and the activation of the inflammasome. Patients with classic autoinﬂammatory diseases such as familial Mediterranean fever or NOMID have nearly identical clinical parameters, secrete more IL-1β, and respond dramatically to IL-1 receptor blockade, yet they have no mutation in NALP3. It is therefore possible that mutations in P2X7 itself or regulation of the other genes controlling potassium channels (11) may account for dysfunctional secretion of IL-1β. For example, monocytes from patients with rheumatoid arthritis are more sensitive to release of IL-1β following ATP activation of the P2X7 receptor compared with monocytes from healthy controls (89). However, monocytes from subjects with a P2X7 Glu496Ala loss-of-function polymorphism secrete significantly less IL-1β (90). Monocytes from subjects homozygous for this polymorphism also release signiﬁcantly less IL-18 (91). Another P2X7 receptor polymorphism is associated with

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increased mortality in patients undergoing allogenic stem cell transplantation (92). Bacteremia was documented in 68% of patients with this polymorphism compared with 18% in wildtype control patients (92). Mice deﬁcient in P2X7 receptors exhibit markedly reduced inﬂammation, pain, and IL1β-mediated IL-6 production (93). In addition to a fall in intracellular potassium, ATP triggers formation of peroxynitrite, which is required for caspase-1 activation, based on the observation that peroxynitrite scavengers prevent IL-1β secretion (94). Pannex-1, a mammalian protein that functions as a hemichannel for the uptake of dyes, is required for caspase-1 processing and release of IL-1β via the P2X7 receptor (95). Pannexin-1 can also function in LPS-induced IL-1β synthesis in the absence of TLR4 (96). P2X7 receptor activity is also regulated by regeneration and tolerance factor (97).

Inactive IL-1β precursor (31 kDa) Active, mature IL-1β (18 kDa) N terminus

Y V H D A P V ............ C terminus 116

Inactive IL-18 precursor (22 kDa) Active, mature IL-18 (17 kDa) N terminus

E S D Y F G K L ............ C terminus 37

Inactive IL-33 precursor (30 kDa) Active, mature IL-33 (18 kDa) N terminus

A L H D S S 114

Inactive IL-1F7 precursor (25 kDa) IL-1F7 (22 kDa) N terminus 18aa E

Noncaspase-1 processing of IL-1β. Figure 2 illustrates the caspase-1 cleavage sites of the IL-1β, IL-18, IL-33, and IL-1F7 precursors. However, for each of these cytokines, noncaspase-1 mechanisms also exist and generate active forms. For example, sterile inﬂammation induces fever, elevated IL-6, and increased production of hepatic acute-phase proteins. These responses are absent in mice deﬁcient in IL-1β but present in mice deﬁcient in caspase-1 (98). Sterile inﬂammation is often associated with neutrophilic inﬁltration, and neutrophils produce IL-1β. Because neutrophils are short-lived cells, dying within hours after emigration, release of the IL-1β precursor from intracellular stores is not unexpected. Therefore, processing of the IL-1β precursor extracellularly into an active cytokine has been reported for the common neutrophil protease proteinase-3 (99). Proteinase-3 also contributes to the processing of IL-18 (100). Other proteases such as elastase, matrix metalloprotease 9, and granzyme A process the IL-1β precursor extracellularly. In addition, a mast cell chymase generates active IL-1β. Mice with a targeted IKKβ deletion in myeloid cells are more susceptible to LPS-

I ............ C terminus

K D

E

P Q C ............ C terminus

Figure 2 Four substrates for caspase-1. Caspase-1 cleaves the IL-1β, IL-18, IL-33, and IL-1F7 precursors at the aspartic acid in P1 position. Shown are the amino acid lengths of the mature cytokines. The true N terminus for IL-1F7 may not be at the caspase-1 site. Caspase-1 site cleavage was reported as forming a 22-kDa peptide, but no amino acid sequence is known (126).

induced shock than are control mice (79), and markedly elevated levels of IL-1β are found in the circulation associated with prominent neutrophilia (79). The elevated levels of IL-1β are lethal because blockade with IL-1Ra protects these mice from death. The source of the IL1β in these mice is the neutrophil. When incubated with proteinase-3, cleavage of the IL-1β precursor is observed with molecular weights of 25 and 15 kDa (79). In fact, the products of proteinase-3 cleavage of the IL-1β precursor are biologically active. In other studies, a molecular weight of active IL-1β at 23–25 kDa has been observed (83). Therefore, in inﬂammatory conditions such as urate crystal arthritis, which is characterized by a prominent neutrophilic inﬁltration, proteinase-3 cleavage of extracellular IL-1β precursor likely takes place. Mice deﬁcient in caspase-1 are not protected against urate-induced inﬂammation. Although IL-1Ra www.annualreviews.org • Functions of the IL-1 Family

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is effective in treating gout, IL-1Ra would be equally effective in any diseases with extracellular processing of the precursor (101). The importance of extracellular processing of the IL1β precursor by serine proteases may explain, in part, the anti-inﬂammatory properties of alpha1-antitrypsin (102).

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Effects in mice deficient in IL-1β. After ten years of continuous breeding, mice deﬁcient in IL-1β exhibit no spontaneous disease. However, upon challenge, IL-1β-deﬁcient mice exhibit speciﬁc differences from their wild-type controls. The most dramatic is the response to local inﬂammation followed by a subcutaneous injection of turpentine. Within the ﬁrst 24 h, IL-1β-deﬁcient mice injected with turpentine do not manifest an acute-phase response, do Table 4

not develop anorexia, and have no circulating IL-6 and no fever (98, 103). These ﬁndings are consistent with those reported in the same model using anti-IL-1R type I antibodies in wild-type mice (103). IL-1β-deﬁcient mice also have reduced inﬂammation following zymosaninduced peritonitis (104). Not unexpectedly, given their lack of endogenous IL-1β, IL-1βdeﬁcient mice have elevated febrile responses to IL-1β and IL-1α. In contrast, IL-1β-deﬁcient mice have nearly the same responses to LPS as do wild-type mice (105). However, unlike wild-type mice, IL-1β-deﬁcient mice injected with LPS have little or no expression of leptin mRNA or protein (106). As shown in Table 4, the data demonstrate that in the mouse, IL-1β is critical for local and systemic inﬂammation.

Responses in IL-1β-deficient mice

Disease model

532

Effects

LPS-induced fever

fever similar to wild-type or ↑ fever

LPS-induced leptin

↓ circulating leptin

LPS-induced hypoglycemia

normoglycemia

LPS-induced shock lung

no effect on neutrophil inﬁltration

LPS-induced coagulopathy

plasminogen activator inhibitor unchanged

disseminated Candida albicans

↓ peritoneal neutrophils, ↓ superoxide production

Zymosan peritonitis

↓ inﬂammation, ↓ mortality, and ↓ IL-6 and chemokines

Turpentine-induced inﬂammation

↓ fever, ↓ IL-6, ↓ SAA, ↓ cortisone, ↓ COX-2

C-protein-induced myositis

↓ disease severity

IL-1α-induced fever

↑ fever, ↑ cytokines

B16 melanoma

↓ hepatic metastasis, ↓ VCAM-1

Brain ischemia-reperfusion

↓ neuronal death

Model of myasthenia gravis

↓ disease development

Systemic lupus erythematosus

↓ anti-dsDNA, ↓ disease severity

Collagen-induced arthritis

↓ disease severity, ↓ CD40 ligand, ↓ OX40

Fas-expressing tumors

↓ neutrophil inﬁltration

Turpentine-induced coagulopathy

↓ plasminogen activator inhibitor

Contact hypersensitivity

↓ Langerhans cell activation

Steady-state levels of p65 (NF-κB)

↓ levels and nuclear translocation

Delayed-type hypersensitivity

↓ sensitized CD4+ T cells, ↓ proliferation to antigen

Airway hypersensitivity to antigen

↓ bronchoconstriction, ↓ IgG1 and IgE

Carcinogen-induced tumors

↓ tumors, ↓ neutrophil inﬁltration

Injury-induced astrogliosis

↓ glial ﬁbrillary acid protein

Meniscus model of osteoarthritis

↓ damage to cartilage

VEGF-mediated neovascularization

↓ hypoxia-inducible factor-1α, ↓ VEGF, ↓ VEGFR-2

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Differences in carcinogenesis between IL1α and IL-1β-deficient mice. Mice deﬁcient in IL-1β were compared with mice deﬁcient in IL-1α in the microenvironment after chemical carcinogenesis (58). On the one hand, in IL-1β-deﬁcient mice tumors developed slower or did not develop in some mice. A deﬁciency in IL-1α, on the other hand, did not impair tumor development compared with wild-type mice injected with the same carcinogen. In IL1Ra-deﬁcient mice, tumor development was the most rapid. A leukocyte inﬁltrate was found at the site of carcinogen injection. The neutrophilic inﬁltrate was almost absent in IL1β-deﬁcient mice, whereas in IL-1Ra-deﬁcient mice, a dense neutrophilic inﬁltrate was observed. In wild-type mice, the leukocytic inﬁltrate was sparse, and the inﬁltrate that was observed in IL-1α-deﬁcient mice was similar to that of control mice. These ﬁndings may reﬂect the fact that IL-1β is secreted into the microenvironment, resulting in the emigration of monocytes and neutrophils, whereas IL-1α that remains cell associated is less likely to affect the microenvironment. The relationship of neutrophil-derived oxygen radicals and carcinogenesis is well established.

IL-1Ra (IL-1F3) The structural analysis of the IL-1RI/IL1Ra complex. X-ray crystallography reveals that IL-1β has two sites of binding to IL-1RI. IL-1Ra also has two binding sites, which are similar to those of IL-1β. However, one of the binding sites of IL-1Ra binds to IL-1RI with a high afﬁnity such that the second binding site is not available to recruit the IL-1RAcP. After binding of IL-1Ra to IL-1RI-bearing cells, there was no phosphorylation of the epidermal growth factor receptor, a well-established and sensitive assessment of IL-1 signal transduction. Overwhelming evidence that IL-1Ra is a pure receptor antagonist comes from studies of intravenous injection of IL-1Ra into healthy humans. At doses 1,000,000-fold greater than that of IL-1α or IL-1β, IL-1Ra had no agonist activity in humans.

Studies using IL-1Ra in animals. Because IL-1Ra exhibits no species speciﬁcity, a large body of data exists revealing a role for IL-1 in animal models of disease. The effects of blocking IL-1 activity in various animal models have been reviewed previously (107). IL-1Ra (anakinra) treatment of autoinflammatory diseases. Table 2 lists several autoinﬂammatory diseases that have been treated with anakinra. In most cases, the responses have been rapid and sustained with daily injections, but disease activity returns upon cessation (12, 27, 108). IL-1β is highly injurious to the insulinproducing pancreatic beta cell (16). High glucose concentrations induce the IL-1β production from human beta cells, resulting in impaired insulin secretion, decreased cell proliferation, and beta cell death (109). In a placebo-controlled trial in patients with type 2 diabetes, anakinra treatment for 13 weeks resulted in signiﬁcantly lower levels of glycated hemoglobin level compared with the placebo group (17). The blockade of IL-1 was also associated with lower levels of C-reactive protein and IL-6. The beneﬁt of blocking IL-1β to protect the insulin-producing beta cells has been conﬁrmed in type 2 diabetic patients treated with a monoclonal antibody to IL-1β (18). Mice deficient in IL-1Ra. Mice deﬁcient in IL-1Ra have low litter numbers and exhibit growth retardation in adult life (110). These animals also have elevated basal concentrations of plasma IL-6 and exhibit higher levels of hepatic acute-phase proteins compared with those of wild-type control mice. The most dramatic phenotype has been observed in IL-1Ra-deﬁcient mice crossed into a BALB/c background (56). In these mice, a chronic inﬂammatory polyarthropathy develops spontaneously. The joints show prominent synovial and periarticular inﬁltration of inﬂammatory cells, osteoclast activation, and bone erosion. The histological pattern is similar to that of humans with rheumatoid arthritis. There are elevated levels of rheumatoid factor and antidouble-stranded DNA antibodies. Steady-state www.annualreviews.org • Functions of the IL-1 Family

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levels of COX-2, IL-1RI, IL-1β, IL-6, and TNF-α mRNA in the affected joints are also increased. IL-17 is required for IL-1Ra-deﬁcient mice to develop the arthritis (50). The onset of this autoimmune process also requires a genetic background favoring the Th2 response, which produces antibodies rather than cytotoxic T cells in response to antigens. The immunologic stimulus likely occurs when either an endogenous antigen or an antigen from the intestinal ﬂora triggers a Th2 response, which, in the absence of endogenous IL-1Ra, results in unopposed IL-1 activities.

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IL-18 (IL-1F4) The discovery of IL-18 was made as an IFN-γinducing cytokine in endotoxemic mice (111); without IL-12 or IL-15, IL-18 does not induce IFN-γ. A role for IL-18 in the Th17 response is unclear at present. Mice injected with the combination of IL-18 plus IL-12 develop high levels of IFN-γ and die with hypoglycemia, intestinal inﬂammation, and inanition (112). In leptindeﬁcient mice, IL-18 plus IL-12 induce acute pancreatitis (113). Several human diseases are associated with elevated production of IFN-γ and IL-18. Diseases such as systemic lupus erythematosus, macrophage activation syndrome, rheumatoid arthritis, type 1 diabetes, Crohn’s disease, psoriasis, and graft-versus-host disease are thought to be mediated, in part, by IL-18. Like IL-1β, IL-18 is initially synthesized as an inactive precursor (24 kDa) and requires caspase-1 cleavage for processing into a mature molecule of 18 kDa (Figure 2). Following an injection of endotoxin, caspase-1-deﬁcient mice have signiﬁcantly lower levels of circulating IFN-γ compared with wild-type mice. IL-12-induced IFN-γ is also caspase-1 dependent (114). In general, processing of the IL-18 precursor is caspase-1 dependent, but exceptions exist. For example, Fas ligand stimulation results in release of biologically active IL18 in caspase-1-deﬁcient murine macrophages (115). As with IL-1β processing, processing of the IL-18 precursor can be accomplished by proteinase-3 (100). 534

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IL-18, hyperphagia, and the metabolic syndrome. There is another side to the biology of IL-18. Whereas there is no constitutive gene expression for IL-1β in freshly obtained human peripheral blood mononuclear cells (PBMCs), the same cells express constitutive mRNA for IL-18 (116). In Western blot analysis from the same cells, the IL-18 precursor was present, but the IL-1β precursor was not. Similar observations have also been made in mice (116). These ﬁndings suggest that IL-18 may act as a regulator of homeostasis. Starting at age 16 weeks, IL-18-deﬁcient mice overeat, become obese, and exhibit lipid abnormalities, increased atherosclerosis, insulin resistance, and diabetes mellitus reminiscent of the metabolic syndrome (59). The higher body weight is attributed to enhanced food intake, which starts to diverge from those of wild-type animals at a relatively early age and reaches values of 30–40% higher than that of wild-type mice. Others have observed similar ﬁndings (117). A striking ﬁnding was a doubling or more in adipose tissue in the deﬁcient animals that was accompanied by fat deposition in the arterial walls. The insulin resistance was corrected by exogenous recombinant IL18. Mice deﬁcient in IL-18 respond normally to a challenge with exogenous leptin, suggesting that a unique mechanism is responsible for the higher food intake in the IL-18-deﬁcient animals. IL-18-binding protein. The discovery of the IL-18BP took place during the search for the soluble receptors for IL-18 in human urine (9). IL-18BP is a constitutively secreted protein, with exceptional afﬁnity to IL-18 (400 pM) (see Figure 3). There is limited amino acid sequence homology between IL-18BP and cell surface IL-18 receptors; IL-18BP lacks a transmembrane domain and contains only one Ig-like domain (118). IL-18BP is a speciﬁc inhibitor of IL-18, neutralizing IL-18 activities. Present in the serum of healthy humans at a 20fold molar excess compared with IL-18 (119), IL-18BP may contribute to a default mechanism by which a Th1 response to foreign

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organisms is blunted in order to reduce triggering an autoimmune response to a routine infection. A clinical preparation of human IL18BP has been shown to be safe in patients (120). Natural neutralization of human IL-18 by IL-18BP takes place during a common viral infection. In Molluscum contagiosum, characterized by raised but bland eruptions, there are large numbers of viral particles in the epithelial cells of the skin, but histologically there are few inﬂammatory or immunologically active cells in or near the lesions. Clearly, the virus fails to elicit an inﬂammatory or immunological response. Amino acid similarity exists between human IL-18BP and a gene found in various members of the poxviruses, the greatest of which is found to be expressed by Molluscum contagiosum (121). The ability of viral IL-18BP to reduce the activity of mammalian IL-18 likely explains the lack of inﬂammatory and immune cells in the infected and provides further evidence for the natural ability of IL-18BP to interfere with IL-18 activity.

IL-1F5 IL-1F5 shares 47% amino acid identity with IL1Ra and is expressed in human monocytes activated by LPS. From the gene sequence, the predicted amino acid sequence of IL-1F5 does not have a leader peptide for secretion, which is in sharp contrast to the IL-1Ra (IL-1F3). IL-1F5 failed to exhibit agonist activity using induction of IL-6 from ﬁbroblasts, a well-described biological property of IL-1α and IL-1β (122). Furthermore, IL-1F5 did not block the IL1α- or IL-1β-induced IL-6- or IL-18-induced production of IFN-γ (122). Therefore, IL-1F5 possesses neither IL-1- or IL-18-like agonist activities nor the property to act as a receptor antagonist for IL-1, despite its close amino acid identity to IL-1Ra. It appears, however, that IL-1F5 functions as an anti-inﬂammatory member of the IL-1 family. IL-1F5 induces IL-4, which can reduce IL-1 activity. The anti-inﬂammatory effects of IL-1F5 include downregulating the re-

IL-18

IL-18BP IL-18Rα

Neutralization

IL-18Rβ

NF-κB

Approximation of cytoplasmic Toll domains

IκB MyD88

Recruitment of kinases

IRAK 1–4

IKKα

TRAF-6

MAPK

IKKβ p38 MAP

Nucleus

Figure 3 IL-18 signal transduction. The afﬁnity of mature IL-18 for the IL-18Rα is low (Kd = 50 nM) but increases 100-fold with a complex of IL-18Rβ. Signal transduction in IL-18 involves MyD88 and IL-1 receptor–associated kinases (IRAKs). TNF receptor–associated factor (TRAF)-6 is also a part of IL-18 signaling. TRAF-6 is required for the phosphorylation of mitogen-activated protein kinase (MAPK) p38.

sponses to IL-1β as well as LPS; in mice deﬁcient in IL-4, IL-1F5 is less effective as an anti-inﬂammatory cytokine (2). The ability of IL-1F5 to dampen inﬂammation is via its interaction with the SIGIRR, also known as Toll IL-1 receptor-8 (TIR8). SIGIRR is an orphan member of the IL-1 family of receptors, and in mice deﬁcient in SIGIRR there is more inﬂammation compared with wild-type control mice (2).

IL-1F6 IL-1F6 is a proinﬂammatory member of the IL-1 family. The cytokine binds to the IL1R-related protein (IL-1Rrp2) as its ligandbinding chain and recruits IL-1RAcP to form the heterodimer. High levels of IL-1F6 are found in mouse embryonic tissues rich in epithelial cells (123). In humans, IL-1F6 is observed in keratinocytes but not in ﬁbroblasts, but it is thought to contribute to the inﬂammation of psoriasis. Upon forming the heterodimer with IL-1Rrp2 and IL-1RAcP, IL1F6 activates NF-κB similar to that of IL-1β (124). In addition to NF-κB activation, IL-1F6 www.annualreviews.org • Functions of the IL-1 Family

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also activates mitogen-activated protein kinase (MAPK), JNK, and ERK1/2 (124).

IL-1F7

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IL-1F7 is an anti-inflammatory cytokine. Discovered by computational cloning, IL-1F7 is a seldom-studied member of the IL-1 family. IL-1F7’s closest structural relative is IL-18. There are ﬁve splice variants of IL-1F7, and IL1F7b contains all but one exon (125). With the sole exception of IL-1Ra, each member of the IL-1 family, including IL-1F7, is ﬁrst synthesized as a precursor without a leader or signal peptide. Although the recombinant form of IL1F7 is processed by caspase-1 in vitro (126), it is unclear whether such processing results in secretion of the carboxyl terminal peptide in vivo. When IL-1F7 is overexpressed in cell lines under various promoters, immunoprecipitates reveal the cytokine complexed with the IL-18 receptor α chain (IL-18Rα) (126, 127). A second study employed recombinant IL-1F7. In Biacore studies, IL-18Rα was immobilized to the matrix, and binding of IL-1F7 was observed, although with a lower afﬁnity than that of IL18 (126). Compared with IL-18, recombinant IL-1F7 does not induce IFN-γ in whole human blood cultures, in PBMCs, or in various cell lines in the presence of IL-12 (126, 128). Therefore, recombinant IL-1F7 lacks true agonist activity; an accessory receptor chain has not been described. Nevertheless, when injected directly into murine tumors, adenovirus expressing the IL-1F7 precursor reduces tumor growth (129). Although recombinant IL-1F7 binds to immobilized IL-18Rα chain, IL-1F7 is also not a receptor antagonist for IL-18 activity (128). The naturally occurring IL-18BP binds IL18 with a high afﬁnity, thus neutralizing IL-18 activity in vitro and in vivo (9). Recombinant IL-1F7 also binds to the IL-18BP (128), and this binding may be due to the similarities of IL-18BP to the third domain of the IL-18Rα chain (130). At low concentrations of IL-18BP, the presence of recombinant IL-1F7 further reduces the inhibition of IL-18 (128), perhaps 536

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by forming a complex of IL-18BP with the IL18Rβ chain and thus depriving the IL-18Rα of its coreceptor. Support for this mechanism comes from mice with collagen-induced arthritis and treated with IL-18BP. Increasing the concentration of IL-18BP can worsen inﬂammation (131). As discussed below, overexpression of IL-1F7 inhibits IL-1β, TNF-α, and TLR production of cytokines. One explanation is that high levels of IL-18BP bind IL-1F7 and thus prevent its interaction with IL-18Rα. In fact, in mice deﬁcient in IL-18Rα, there is increased inﬂammation, whereas in mice deﬁcient in IL-18 itself, there is less inﬂammation (132). In mice with EAE, a deﬁciency in the IL-18Rα results in the opposite outcome compared with mice deﬁcient in IL-18 (133). The two studies concluded that the IL-18Rα chain may bind another ligand in addition to IL-18 and that this ligand may be IL-1F7. Such a mechanism of action would likely require a coreceptor, and one candidate would be SIGIRR (7, 8). In stable transfectants of human IL-1F7b in mouse RAW macrophages stimulated with LPS, levels of TNF-α, IL-1α, and IL-6 as well as the chemokine CXCL2 (MIP-2) were substantially reduced (30–98%) compared with LPS-stimulated stable transfectants with the empty plasmid (1, 134). Reductions in proinﬂammatory cytokines (IL-1α, IL-1β, IL-6, IL-8, TNF-α) were demonstrated in human THP-1 monocytes/macrophages transfected with IL-1F7 and stimulated with LPS (134); similar reductions were also observed in transfected human epithelial A549 cells stimulated with IL-1β (134). To prove causality, endogenous IL-1F7 was reduced by siRNA in TLR agonist-stimulated PBMCs from seven donors. With reduced levels of IL-1F7 in these cells, there was a dose-dependent increase up to threefold in 13 proinﬂammatory mediators, among which were IL-1α, IL-1β, IL-6, TNFα, and GM-CSF (134). Intracellularly, IL-1F7 markedly reduced by up to 75% the activation of several kinases (Stat1 through 4, p38MAPK, c-Jun, and Hck). Therefore, it appears that IL-1F7 is a naturally occurring inhibitor of the innate immune response.

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Using two variants of green ﬂuorescent protein fused to human IL-1F7b in stably expressed RAW macrophages, only the postcleavage mature form of the IL-1F7b precursor, but not the N-terminal propiece, speciﬁcally translocated to the nucleus following LPS stimulation (1). Similar to IL-1β, IL-18, and IL-33, the IL1F7 precursor is cleaved by caspase-1 to generate the mature cytokine (126). Indeed, a speciﬁc caspase-1 inhibitor reduced nuclear binding of IL-1F7b. These results demonstrate that IL1F7b translocates to the nucleus after caspase1 processing and may act as a transcriptional modulator reducing the production of LPSstimulated proinﬂammatory cytokines, consistent with IL-1F7b being an anti-inﬂammatory member of the IL-1 family.

IL-1F8 The receptor for IL-1F8 is IL-1Rrp2 and is expressed on human synovial ﬁbroblasts and human articular chondrocytes. In response to stimulation by recombinant IL-1F8, there is increased production of proinﬂammatory mediators (135). Although IL-1F8 steady-state mRNA is constitutive in chondrocytes, the cytokine is inducible by IL-1β in synovial ﬁbroblasts. Circulating levels of IL-1F8 in patients with rheumatoid arthritis, osteoarthritis, or septic shock were similar to those in healthy subjects. It is unclear to what extent IL-1F8 plays a role in joint disease, although constitutive expression in primary chondrocytes may indicate a role for the cytokine in osteoarthritis.

IL-1F9 IL-1F9 is constitutively expressed primarily in the placenta and the squamous epithelium of the esophagus. The three-dimensional folding of IL-1F9 is similar to that of IL-1Ra; nevertheless, IL-1F9 is a proinﬂammatory cytokine binding to IL-1Rrp2 and recruiting the IL1RAcP. IL-1F9 triggers the same kinase cascade and NF-κB as IL-1F6 (see above).

IL-1F10 IL-1F10 shares 37% amino acid identity with the IL-1Ra and a similar three-dimensional structure (136). This cytokine is secreted from cells and is expressed in human skin, spleen, and tonsil. To date, recombinant IL-1F10 has been shown to bind to the isolated extracellular domains of IL-1RI, but it is unclear whether IL-1F10 binds to complete cell surface–bound IL-1 receptors. Although these data suggest that IL-1F10 is likely to be a receptor antagonist (137), compared with IL-1Ra, its role in health and disease remains unclear.

IL-33 (IL-1F11) IL-1F11 is also known as IL-33. Although given the newest name in the IL-1 family, the existence of this member was predicted 16 years ago as the ligand for the orphan receptor T1/ST2. ST2 is also termed the IL-33 receptor α chain (IL-33Rα), and recombinant IL-33 binds to and activates ST2. From studies using soluble forms of ST2 or antibodies to ST2, we know that the ligand (now known as IL-33) drives T helper type 2 (Th2) responses (3). In 1994, the receptor termed ST2 was ﬁrst reported as regulated by the estrogen-inducible transcription factor Fos (138). The ST2 receptor was similar to the IL-1RI and IL-18Rα in that ST2 is composed of three extracellular Ig domains and an intracellular Toll domain. During the search for its cognate ligand, investigators assumed that its ligand would be a member of the IL-1 family. Thus, the properties of recombinant IL33 recapitulate much of the existing data that ST2 promotes Th2 type responses. Like IL-1 (IL-1α and IL-1β) binding to the IL-1RI, IL-33 forms a heterodimeric complex with IL-1RAcP for signal transduction (37, 139). IL-33 gains considerable legitimacy as a member of the IL-1 ligand family because processing of the IL-33 precursor is accomplished by caspase-1 (3), similar to IL-1β and IL-18. Lack of processing of IL-33 into an active cytokine may contribute to the protection afforded caspase-1-deﬁcient mice. www.annualreviews.org • Functions of the IL-1 Family

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However, there is another side to IL-33. Although IL-33 binds to its speciﬁc surface receptor, IL-33 is identical to a nuclear factor dominantly expressed in high endothelial venules (HEV) (5). This nuclear factor is termed NF-HEV. In fact, IL-33’s binding to DNA and acting as a nuclear factor is similar to IL-1α’s functioning as a nuclear factor (4, 140). Thus, IL-33 is one of a growing number of cytokines that both signal via a classic receptor trigger and function as a nuclear factor in regulating transcription. These cytokines presently include IL-33 (5), IL-1α (4), IL-16 (141), and high mobility factor B (142). There was no dearth of studies on ST2 tissue-speciﬁc localization, regulation of its expression, and effects in transgenic mice overexpressing ST2 or on deletion, neutralization, and antibody cross-linking of ST2. Elevated levels of the soluble form of ST2 were present in the circulation of patients with various inﬂammatory diseases, and exogenous administration of pharmacologic doses of soluble ST2 neutralized the putative ligand and reduced inﬂammation (143). Furthermore, several studies suggested that whatever the ligand for this orphan receptor, it was playing a role in allergic-type diseases, given that activation of ST2 was uniquely driving Th2 responses. Structurally, IL-33 is closest to IL-18, rather than IL-1β. The dominant property of IL-33 is the induction of IL-4, IL-5, and IL-13 as well as properties anticipated for a Th2-type cytokine. Diseases thought to be due to increased immunoglobulin production may also be related to IL-33. IL-33 induces the production of IL-6, IL-1β, and PGE from mast cells. Mice injected with human IL-33 exhibit impressive pathological changes in the arterial walls, lungs, and intestinal tissues (3). Of particular relevance to the concept that IL-33 drives a Th2 response, eosinophilic inﬁltration was a prominent ﬁnding in the lung. Although the interpretation of in vivo effects following the administration of an exogenous cytokine should be conservative, the ﬁndings are clearly consistent with IL-33 being a proinﬂammatory ligand

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of the IL-1 receptor family. Even before the ability to test IL-33-mediated activation, others had reported that neutralization of the putative ST2 ligand using soluble ST2 markedly reduced joint inﬂammation, synovial hyperplasia, and joint erosion when given in the therapeutic phase of collagen-induced arthritis in mice (143). Moreover, organs with high numbers of mast cells appear to undergo dramatic changes upon injection of IL-33 into mice. Consistent with a role in allergic diseases, IL-33 induces in vivo the cytokines IL-5 and IL-13, known contributors to allergic diseases. Mice deﬁcient in ST2 do not develop a Th2 response to Schistosoma egg antigen. How IL-33 favors the Th2 response remains unclear. Similar to IL-1β, IL-33 induces IL6, which is an adjuvant for antibody production. IL-33 induction of IL-6 is prevented by a blocking antibody to IL-1RAcP (37). IL-33 initiates signal transduction via activation of NFκB, which is typical of IL-1α, IL-1β, and IL18 (3), but other studies have shown that antibody cross-linking of ST2 does not result in activation of NF-κB but rather of AP-1. There are also studies that indicate that IL-33 has anti-inﬂammatory properties by inhibiting angiotensin II–induced phosphorylation of IκB in cardiac ﬁbroblasts (144). ST2 can sequester TLR adaptor molecules such as MyD88 and Mal (145). One of the more challenging aspects of IL33’s ability to act as a Th2 cytokine is its role as an antagonist in the ApoE-deﬁcient mouse model of atherosclerosis. In this model, arterial wall plaques of mice on a high-fat diet contain IL-33 and ST2. In mice treated with IL33, the atherosclerotic plaques were markedly reduced (146). IL-33 treatment also increased serum IgA and IgE, an expected response for a switch from Th1 to Th2. In mice treated with soluble ST2 to neutralize IL-33, the disease was worse (146). These studies are inconsistent, however, with the role of caspase-1 as required for processing of IL-33 because the soluble ST2 neutralizes mature IL-33. Also, these studies are inconsistent with a role for IL-33 as NF-HEV (5).

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IL-1 Receptor Family The IL-1 receptor family encodes ten members, some of which remain orphan receptors. As shown in Figure 4, most belong to the group containing three IgG-like segments for the extracellular domain of the receptor. Table 5 summarizes the IL-1 receptor family and their functions (147). IL-1R1, IL-1R2, and IL-1R3 are the bona ﬁde receptors for IL-1α and IL1β. IL-1R4, also known as ST2, is no longer an orphan receptor because IL-33 binds to this receptor (3). Before the identiﬁcation of IL-33 as the ligand for ST2, a number of studies had examined the function, distribution, and gene regulation of this receptor, particularly on mast cells (148). IL-1R5 was formerly an orphan receptor termed IL-1R related protein-1 (149), but it was subsequently discovered to be the ligand-binding (α) chain of the IL-18 receptor (150), now termed IL-18Rα. The IL-1Rrp2 is the receptor for the agonists IL-1F6, IL-1F8, and IL-1F9, but it is also the receptor for IL-1F5, which is the antagonist for IL-1F9 (123, 147). IL-1R7, formerly the nonligand-binding chain of the IL-18 receptor termed IL-1RAcPL (151), is now named IL-18Rβ chain. Similar to the IL-1RAcP, the IL-18Rβ is essential for IL-18 signal transduction (151, 152). Two members of the IL-1 receptor family are particularly unique in that they are found on the X chromosome. These are IL1R8 and IL-1R9, both homologous to the Table 5

Ligandbinding (α) chain

IL-1RI IL-18Rα ST2 (IL-33Rα) TIGIRR-1 TIGIRR-2 IL-1Rrp2

Coreceptor (β) chain

Decoy receptor

IL-1RII

IL-1RAcP IL-18Rβ

SIGIRR

Toll-like domain

Figure 4 IL-1 family of receptors.

IL-1RAcP and IL-18Rβ (see Table 5). IL1R9 is highly homologous to IL-1R8 and both are termed three Ig IL-1-related receptors (TIGIRR). Both forms have no known ligands, and receptors are found in the fetal brain. In fact, nonoverlapping deletions and a nonsense mutation in the IL-1r8 were found in patients with cognitive impairment (153) in which expression in the adult hippocampal area may play a role in memory or learning. The cytoplasmic domains of IL-1R8 and IL-1R9 are longer than the other accessory chains. The IL-1R9 may function as a negative receptor, as was shown in cells overexpressing this receptor as well as the IL-1RI and IL-1RAcP in which IL-1β signaling was blocked with a speciﬁc antibody to the IL-1RAcP. In the presence of the antibody,

The IL-1 receptor family

Name

Designation1

Ligands

Coreceptor

IL-1RI

IL-1R1

IL-1α, IL-1β, IL-1Ra

IL-1RAcP (IL-1R3)

IL-1RII

IL-1R2

IL-1β, IL-1β precursor

IL-1RAcP (IL-1R3)

ST2/Fit-1

IL-1 R4 (IL-33Rα)

IL-33

IL-1RAcP (IL-1R3)

IL-18Rα

IL-1R5

IL-18, IL-1F7

IL-18Rβ (IL-1R7)

IL-1Rrp-2

IL-1R6

IL-1F6, IL-1F8, IL-1F9

IL-1RAcP (IL-1R3)

TIGIRR-2/IL-1RAPL

IL-1R8

unknown

unknown

TIGIRR-1

IL-1R9

unknown

unknown

SIGIRR

TIR8

unknown

unknown

1

IL-1 receptor family members IL-1R3 (IL-1RAcP) and IL-1F7 (IL-18Rβ) are coreceptors and require binding to an IL-1 family ligand plus a ligand-binding chain.

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Inhibitory receptor

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IL-1β-induced luciferase was suppressed, suggesting that a possible complex of the type I receptor with IL-1β plus IL-1R9 results in a negative signal (154).

Single Ig IL-1 Related Receptor

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SIGIRR is a negative regulator of both IL-1α and IL-1β activities but also of TLR agonists. Although there is only a single IgG domain for the extracellular segment, the cytoplasmic domain contains a Toll domain and has the longest cytoplasmic domain of all the members of the IL-1 receptor family (Figure 4). Initially, SIGIRR was thought to be expressed primarily in kidney, lung, and the gastrointestinal tract, primarily in resting epithelial cells. However, SIGIRR is also expressed in human monocytes and mouse macrophages upon the stresses associated with infection and transient hypoxia. Dendritic cells also express SIGIRR (8). In a study of patients resuscitated from cardiac arrest, the peripheral monocytes displayed increased steady-state levels of SIGIRR, which were associated with increased gene expression for the anti-inﬂammatory cytokine IL-10 and decreased expression for TNF-α. In microglia of mice infected with murine leukemia virus, a gene array of 14,000 genes revealed that only three genes were differentially expressed between the virulent and nonvirulent strains of the viruses, and one of these genes coded for SIGIRR (155). With overexpression of SIGIRR in the form of a chimeric molecule with IL-1RAcP, suppression of IL-1-driven NF-κB was observed. SIGIRR inhibition of IL-1-driven NF-κB does not require the presence of the extracellular domain of SIGIRR. The complex of IL-1 with IL-1RAcP activates NF-κB, but in the presence of SIGIRR there is suppression of the response. In another study, inhibition of IL-1 activity required the extracellular single Ig domain of SIGIRR (156). The mechanism for suppression of the IL-1 responses in cells overexpressing SIGIRR is one of competitive inhibition of MyD88, such that there is no activa-

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tion of IL-1 receptor–associated kinase (IRAK) or TNF receptor-associated factor (TRAF)-6 (156). The inhibition of MyD88, IRAK, and TRAF-6 by SIGIRR is not due to the formation of a complex with IL-1RAcP or IL-1RI. Overexpression of SIGIRR results in inhibition of LPS and IL-1 activities, whereas mice deﬁcient in SIGIRR exhibit heightened inﬂammation. Mice deﬁcient in SIGIRR develop a more severe disease in response to LPS or chronic colitis (7, 8). Overexpression of SIGIRR in 293 cells inhibits both IL-1- and IL-18-induced NF-κB reporter activity but not IFN-γ activity (7). Splenocytes from mice deﬁcient in SIGIRR produce several-fold greater levels of the mouse chemokines KC and CXCL2 (MIP2) and CXCL10 (IP-10) following LPS. In a model of live Pseudomonas keratitis, a blocking antibody to SIGIRR resulted in increased bacterial counts and increased steady-state mRNA levels of IL-1β, IL-18, and IL-18 (157). Overexpression of SIGIRR in macrophages resulted in decreased mRNA levels for IL1RI, IFN-γ, IL-12, and IL-18. This SIGIRR downregulates the responses to IL-1 and TLR4.

IL-1 Receptor Type I In crystallization studies, IL-1RI undergoes a conformational change when binding IL-1β and allows the IL-1RAcP to form the heterodimer (Figure 5). The cytoplasmic domain of IL-1RI is unique in that it contains homology to the Drosophila Toll protein, termed the TIR domain. The TIR domain is also found in the cytoplasmic domains of each TLR. The TIR domains of IL-1RI and also of the coreceptor IL-1RAcP are necessary for signal transduction. Although most cells express IL1RI constitutively, expression of IL-1RAcP is not constitutive in some cells. A small synthetic peptide (RYTVELA) derived from the sequence of the third domain of the IL-1RAcP was tested for blocking IL-1 activity. This peptide, known as 101.10, blocks the functions of the IL-1RI in human, mouse, and rat cells

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(158) and has no effect in mice deﬁcient in IL1RI. Active at 1 nM, 101.10 reduced inﬂammatory colitis in the rat and contact dermatitis. The location of the peptide is the site of interactive loops between IL-1RAcP and the IL1RI. When present, the peptide functions as an allosteric, noncompetitive antagonist, inhibiting some but not all functions of the IL-1RI (158). This property of allosteric antagonism has been observed for the leukocyte function– associated antigen-1 (LFA-1) integrin where a peptide binds to an allosteric site and inhibits only some of the properties of LFA-1 (159).

Studies in IL-1RI and IL-1RAcP-Deficient Mice The most proximal (5 ) promoter region of the IL-1RI lacks a TATA or CAAT box and bears a striking similarity to the promoters of housekeeping genes rather than highly regulated genes (160). Phorbol esters, PGE2, dexamethasone, epidermal growth factor, IL-2, and IL-4 increase surface expression of IL-1RI. Part of the immunosuppressive properties of TGF-β may be due to downregulation of the IL-1RI on T cells. Mice deﬁcient in IL-1RI develop normally and exhibit no particular phenotype. However, IL-1RI-deﬁcient mice exhibit an attenuated inﬂammatory response to sterile abscesses compared with wild-type mice. Delayed-type hypersensitivity responses are also reduced in IL-1RI-deﬁcient mice. IL-17 production requires IL-1RI on T cells (42). Not unexpectedly, IL-1RI-deﬁcient mice are susceptible to infection with Listeria monocytogenes. Lymphocytes from IL-1RI-deﬁcient mice with major cutaneous leishmanial infection produce more IL-4 and IL-10, but less IFN-γ, than those from wild-type mice. Cells deﬁcient in IL-1RAcP have normal binding of IL-1α and IL-1Ra to the IL-1RI but a 70% reduction in binding of IL-1β (161). In these cells, there is no biological response to IL-1α despite binding of IL-1α to the type I receptor.

sIL-1RII 14

1 IL-1RI

IL-1β

IL-1RII + IL-1RAcP 15

IL-1RII

IL-1RII + IL-1RAcP

2 13

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3 Approximation of cytoplasmic Toll domains

4 MyD88

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*IRAK-1

*IRAK-2 TRAF-6 6

8

TRAF-6

*

*TAK1 *IKKβ 9

p38 MAP

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JNK

*IκB 10

NF-κB 11

Nucleus

Figure 5 IL-1 signal transduction and decoy receptors. IL-1β binding to the IL-1RI (1) recruits the IL-1RAcP (2) to form a heterodimeric receptor (1+2). The cytoplasmic Toll domains on each receptor chain approximate (3). MyD88 and Tollip are recruited (4). MyD88 binding to the cytoplasmic domains triggers the phosphorylations of the IL-1 receptor–associated kinases IRAK-4, IRAK-2, and IRAK-1 (5). TRAF-6 is recruited (6). Phosphorylated IRAK-1 and TRAF-6 migrate to the membrane and associate with TAK1 (TGF-β-activated kinase 1), TAK1-binding protein (TAB)-1, and TAB2 (7). The complex of TAK1, TAB1, TAB2, and TRAF-6 migrates to the cytosol, where TAK1 is phosphorylated following the ubiquitination of TRAF-6 (8). Phosphorylated TAK1 activates IKKβ (9), and phosphorylated IKKβ phosphorylates IκB (10). Phosphorylated IκB degrades, releasing NF-κB, which enters the nucleus (11). In addition to the phosphorylation of IKKβ, TAK1 also activates mitogen-activated protein kinase (MAPK) p38 and JNK (12). On the surface of the cell, IL-1RII, a decoy receptor, may also bind IL-1β (13), but this complex does not recruit IL-1RAcP, and there is no signal. In the extracellular space, the extracellular domains (soluble or sIL-1RII) of the IL-1RII bind IL-1β and neutralize its activity (14). sIL-1RII can also bind IL-1β and form a complex with soluble IL-1RAcP (15) or cell-bound IL-1RAcP (16). In the latter two complexes, IL-1β is not available to bind to IL-1RI and therefore cannot transmit a signal.

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IL-1R Type II

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As reported by Colotta et al. (162), IL-1RII is a decoy receptor. As shown in Figures 4 and 5, the short cytoplasmic domain is unable to initiate signal transduction because there is no TIR domain. Therefore, IL-1RII captures IL1 without signaling. Intracellularly, the IL-1RII associates with the IL-1α precursor (65). Genes in the pox family of viruses encode for a protein with a high homology to the extracellular (soluble) domains of the receptor (sIL-1RII). In humans, sIL-1RII is released from the cell surface by a protease; sIL-1RII has a particularly high afﬁnity for mature IL-1β and therefore functions as a naturally occurring neutralization mechanism for IL-1β. IL-1β binding to the sIL-1RII is nearly irreversible. The IL-1β precursor also preferentially binds to sIL-1RII. A more efﬁcient function of the type II receptor is to form a trimeric complex of the IL-1β with sIL-1RII and the IL-1RAcP chain (163). This mechanism serves

to deprive the cell of both IL-1β as well as a functional receptor accessory chain (reviewed in 164). As shown in Figure 5, sIL-1RAcP forms complexes on the cell surface of IL-1β bound to type II receptors and accounts for the ability of sIL-1RAcP to reduce B lymphocyte activation (165). sIL-1RAcP inhibits IL1-induced NF-κB activity in B cells but not in T cells, whereas IL-1Ra inhibited IL-1 in both cell types (166).

sIL-1RI The administration of the extracellular domain of the type I receptor (sIL-1RI) has been used in models of inﬂammatory and autoimmune disease, where a reduction in disease severity is reported. However, in humans, sIL-1RI acts as a carrier for IL-1α, and disease activity in patients with rheumatoid arthritis worsens (167). In mice, intravenous injection of sIL-1RI alone induced a rapid increase in circulating IL-1α, but not of TNF-α or IL-1β (168).

DISCLOSURE STATEMENT The author is not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

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91. Sluyter R, Dalitz JG, Wiley JS. 2004. P2X7 receptor polymorphism impairs extracellular adenosine 5 -triphosphate-induced interleukin-18 release from human monocytes. Genes Immun. 5:588–91 92. Lee KH, Park SS, Kim I, Kim JH, Ra EK, et al. 2007. P2X7 receptor polymorphism and clinical outcomes in HLA-matched sibling allogeneic hematopoietic stem cell transplantation. Haematologica 92:651–57 93. Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, et al. 2005. Disruption of the P2X7 purinoceptor gene abolishes chronic inﬂammatory and neuropathic pain. Pain 114:386–96 94. Hewinson J, Moore SF, Glover C, Watts AG, MacKenzie AB. 2008. A key role for redox signaling in rapid P2X7 receptor-induced IL-1β processing in human monocytes. J. Immunol. 180:8410–20 95. Pelegrin P, Surprenant A. 2006. Pannexin-1 mediates large pore formation and interleukin-1β release by the ATP-gated P2X7 receptor. EMBO J. 25:5071–82 96. Kanneganti TD, Lamkanﬁ M, Kim YG, Chen G, Park JH, et al. 2007. Pannexin-1-mediated recognition of bacterial molecules activates the cryopyrin inﬂammasome independent of Toll-like receptor signaling. Immunity 26:433–43 97. Derks R, Beaman K. 2004. Regeneration and tolerance factor modulates the effect of adenosine triphosphate-induced interleukin 1β secretion in human macrophages. Hum. Immunol. 65:676–82 98. Fantuzzi G, Ku G, Harding MW, Livingston DL, Sipe JD, et al. 1997. Response to local inﬂammation of IL-1β converting enzyme-deﬁcient mice. J. Immunol. 158:1818–24 99. Coeshott C, Ohnemus C, Pilyavskaya A, Ross S, Wieczorek M, et al. 1999. Converting enzymeindependent release of TNFα and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or puriﬁed proteinase-3. Proc. Natl. Acad. Sci. USA 96:6261–66 100. Sugawara S, Uehara A, Nochi T, Yamaguchi T, Ueda H, et al. 2001. Neutrophil proteinase 3-mediated induction of bioactive IL-18 secretion by human oral epithelial cells. J. Immunol. 167:6568–75 101. So A, De Smedt T, Revaz S, Tschopp J. 2007. A pilot study of IL-1 inhibition by anakinra in acute gout. Arthritis Res. Ther. 9:R28 102. Numanami H, Koyama S, Sato E, Haniuda M, Nelson DK, et al. 2003. Serine protease inhibitors modulate chemotactic cytokine production by human lung ﬁbroblasts in vitro. Am. J. Physiol. Lung Cell. Mol. Physiol. 284:L882–90 103. Zheng H, Fletcher D, Kozak W, Jiang M, Hofmann K, et al. 1995. Resistance to fever induction and impaired acute-phase response in interleukin-1β deﬁcient mice. Immunity 3:9–19 104. Fantuzzi G, Sacco S, Ghezzi P, Dinarello CA. 1997. Physiological and cytokine responses in interleukin1β-deﬁcient mice after zymosan-induced inﬂammation. Am. J. Physiol. 273:R400–6 105. Fantuzzi G, Zheng H, Faggioni R, Benigni F, Ghezzi P, et al. 1996. Effect of endotoxin in IL-1β-deﬁcient mice. J. Immunol. 157:291–96 106. Faggioni R, Fantuzzi G, Fuller J, Dinarello CA, Feingold KR, Grunfeld C. 1998. IL-1β mediates leptin induction during inﬂammation. Am. J. Physiol. 274:R204–8 107. Dinarello CA. 2004. Therapeutic strategies to reduce IL-1 activity in treating local and systemic inﬂammation. Curr. Opin. Pharmacol. 4:378–85 108. Botsios C, Sfriso P, Furlan A, Punzi L, Dinarello CA. 2008. Reistant Behc¸et disease responsive to anakinra. Ann. Intern. Med. 149:284–86 109. Maedler K, Storling J, Sturis J, Zuellig RA, Spinas GA, et al. 2004. Glucose- and interleukin-1β-induced beta-cell apoptosis requires Ca2+ inﬂux and extracellular signal-regulated kinase (ERK) 1/2 activation and is prevented by a sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2 (SUR/Kir6.2) selective potassium channel opener in human islets. Diabetes 53:1706–13 110. Hirsch E, Irikura VM, Paul SM, Hirsh D. 1996. Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice. Proc. Natl. Acad. Sci. USA 93:11008–13 111. Okamura H, Tsutsui H, Komatsu T, Yutsudo M, Hakura A, et al. 1995. Cloning of a new cytokine that induces interferon-γ. Nature 378:88–91 112. Nakamura S, Otani T, Ijiri Y, Motoda R, Kurimoto M, Orita K. 2000. IFN-γ-dependent and -independent mechanisms in adverse effects caused by concomitant administration of IL-18 and IL12. J. Immunol. 164:3330–36 113. Sennello JA, Fayad R, Pini M, Gove ME, Ponemone V, et al. 2008. Interleukin-18, together with interleukin-12, induces severe acute pancreatitis in obese but not in nonobese leptin-deﬁcient mice. Proc. Natl. Acad. Sci. USA 105:8085–90 www.annualreviews.org • Functions of the IL-1 Family

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114. Fantuzzi G, Reed DA, Dinarello CA. 1999. IL-12-induced IFNγ is dependent on caspase-1 processing of the IL-18 precursor. J. Clin. Invest. 104:761–67 115. Tsutsui H, Kayagaki N, Kuida K, Nakano H, Hayashi N, et al. 1999. Caspase-1-independent, Fas/Fas ligand-mediated IL-18 secretion from macrophages causes acute liver injury in mice. Immunity 11:359–67 116. Puren AJ, Fantuzzi G, Dinarello CA. 1999. Gene expression, synthesis and secretion of IL-1β and IL-18 are differentially regulated in human blood mononuclear cells and mouse spleen cells. Proc. Natl. Acad. Sci. USA 96:2256–61 117. Zorrilla EP, Sanchez-Alavez M, Sugama S, Brennan M, Fernandez R, et al. 2007. Interleukin-18 controls energy homeostasis by suppressing appetite and feed efﬁciency. Proc. Natl. Acad. Sci. USA 104:11097–102 118. Kim SH, Azam T, Novick D, Yoon DY, Reznikov LL, et al. 2002. Identiﬁcation of amino acid residues critical for biological activity in human interleukin-18. J. Biol. Chem. 14:14 119. Novick D, Schwartsburd B, Pinkus R, Suissa D, Belzer I, et al. 2001. A novel IL-18BP ELISA shows elevated serum IL-18BP in sepsis and extensive decrease of free IL-18. Cytokine 14:334–42 120. Tak PP, Bacchi M, Bertolino M. 2006. Pharmacokinetics of IL-18 binding protein in healthy volunteers and subjects with rheumatoid arthritis or plaque psoriasis. Eur. J. Drug Metab. Pharmacokinet. 31:109–16 121. Xiang Y, Moss B. 2001. Correspondence of the functional epitopes of poxvirus and human interleukin18-binding proteins. J. Virol. 75:9947–54 122. Barton JL, Herbst R, Bosisio D, Higgins L, Nicklin MJ. 2000. A tissue speciﬁc IL-1 receptor antagonist homolog from the IL-1 cluster lacks IL-1, IL-1ra, IL-18 and IL-18 antagonist activities. Eur. J. Immunol. 30:3299–308 123. Debets R, Timans JC, Homey B, Zurawski S, Sana TR, et al. 2001. Two novel IL-1 family members, IL-1δ and IL-1ε, function as an antagonist and agonist of NF-κB activation through the orphan IL-1 receptor-related protein 2. J. Immunol. 167:1440–46 124. Towne JE, Garka KE, Renshaw BR, Virca GD, Sims JE. 2004. Interleukin (IL)-1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate the pathway leading to NF-κB and MAPKs. J. Biol. Chem. 279:13677–88 125. Busﬁeld SJ, Comrack CA, Yu G, Chickering TW, Smutko JS, et al. 2000. Identiﬁcation and gene organization of three novel members of the IL-1 family on human chromosome 2. Genomics 66:213–16 126. Kumar S, Hanning CR, Brigham-Burke MR, Rieman DJ, Lehr R, et al. 2002. Interleukin-1F7B (IL1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-γ production. Cytokine 18:61–71 127. Pan G, Risser P, Mao W, Baldwin DT, Zhong AW, et al. 2001. IL-1H, an interleukin 1-related protein that binds IL-18 receptor/IL-1Rrp. Cytokine 13:1–7 128. Buﬂer P, Azam T, Gamboni-Robertson F, Reznikov LL, Kumar S, et al. 2002. A complex of the IL1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity. Proc. Natl. Acad. Sci. USA 99:13723–28 129. Gao W, Kumar S, Lotze MT, Hanning C, Robbins PD, Gambotto A. 2003. Innate immunity mediated by the cytokine IL-1 homologue 4 (IL-1H4/IL-1F7) induces IL-12-dependent adaptive and profound antitumor immunity. J. Immunol. 170:107–13 130. Azam T, Novick D, Buﬂer P, Yoon DY, Rubinstein M, et al. 2003. Identiﬁcation of a critical Ig-like domain in IL-18 receptor α and characterization of a functional IL-18 receptor complex. J. Immunol. 171:6574–80 131. Banda NK, Vondracek A, Kraus D, Dinarello CA, Kim SH, et al. 2003. Mechanisms of inhibition of collagen-induced arthritis by murine IL-18 binding protein. J. Immunol. 170:2100–5 132. Lewis EC, Dinarello CA. 2006. Responses of IL-18- and IL-18 receptor-deﬁcient pancreatic islets with convergence of positive and negative signals for the IL-18 receptor. Proc. Natl. Acad. Sci. USA 103:16852– 57 133. Gutcher I, Urich E, Wolter K, Prinz M, Becher B. 2006. Interleukin 18-independent engagement of interleukin 18 receptor-α is required for autoimmune inﬂammation. Nat. Immunol. 7:946–53 134. Nold MF, Nold-Petry CA, Zepp JA, Buﬂer P, Dinarello CA. 2008. The IL-1 family member IL-1F7 reduces innate immunity by inhibiting Toll-like receptor and IL-1 signaling pathways. Cytokine 43:264 (Abstr.)

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135. Magne D, Palmer G, Barton JL, Mezin F, Talabot-Ayer D, et al. 2006. The new IL-1 family member IL1F8 stimulates production of inﬂammatory mediators by synovial ﬁbroblasts and articular chondrocytes. Arthritis Res. Ther. 8:R80 136. Lin H, Ho AS, Haley-Vicente D, Zhang J, Bernal-Fussell J, et al. 2001. Cloning and characterization of IL-1HY2, a novel interleukin-1 family member. J. Biol. Chem. 276:20597–602 137. Bensen JT, Dawson PA, Mychaleckyj JC, Bowden DW. 2001. Identiﬁcation of a novel human cytokine gene in the interleukin gene cluster on chromosome 2q12-14. J. Interferon Cytokine Res. 21:899–904 138. Bergers G, Reikerstorfer A, Braselmann S, Graninger P, Busslinger M. 1994. Alternative promoter usage of the Fos-responsive gene Fit-1 generates mRNA isoforms coding for either secreted or membranebound proteins related to the IL-1 receptor. EMBO J. 13:1176–88 139. Chackerian AA, Oldham ER, Murphy EE, Schmitz J, Pﬂanz S, Kastelein RA. 2007. IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex. J. Immunol. 179:2551–55 140. Stevenson FT, Turck J, Locksley RM, Lovett DH. 1997. The N-terminal propiece of interleukin 1α is a transforming nuclear oncoprotein. Proc. Natl. Acad. Sci. USA 94:508–13 141. Wilson KC, Cruikshank WW, Center DM, Zhang Y. 2002. Prointerleukin-16 contains a functional CcN motif that regulates nuclear localization. Biochemistry 41:14306–12 142. Yang H, Wang H, Tracey KJ. 2001. HMG-1 rediscovered as a cytokine. Shock 15:247–53 143. Leung BP, Xu D, Culshaw S, McInnes IB, Liew FY. 2004. A novel therapy of murine collagen-induced arthritis with soluble T1/ST2. J. Immunol. 173:145–50 144. Sanada S, Hakuno D, Higgins LJ, Schreiter ER, McKenzie AN, Lee RT. 2007. IL-33 and ST2 comprise a critical biomechanically induced and cardioprotective signaling system. J. Clin. Invest. 117:1538–49 145. Gadina M, Jefferies CA. 2007. IL-33: a sheep in wolf’s clothing? Sci. STKE 2007:pe31 146. Miller AM, Xu D, Asquith DL, Denby L, Li Y, et al. 2008. IL-33 reduces the development of atherosclerosis. J. Exp. Med. 205:339–46 147. Boraschi D, Tagliabue A. 2006. The interleukin-1 receptor family. Vitam. Horm. 74:229–54 148. Moritz D, Rodewald H-R, Gheyselinck J, Klemenz R. 1998. The IL-1 receptor-related T1 antigen is expressed on immature and mature mast cells and on fetal blood mast cell progenitors. J. Immunol. 161:4866–74 149. Parnet P, Garka KE, Bonnert TP, Dower SK, Sims JE. 1996. IL-1Rrp is a novel receptor-like molecule similar to the type I interleukin-1 receptor and its homologues T1/ST2 and IL-1R AcP. J. Biol. Chem. 271:3967–70 150. Torigoe K, Ushio S, Okura T, Kobayashi S, Taniai M, et al. 1997. Puriﬁcation and characterization of the human interleukin-18 receptor. J. Biol. Chem. 272:25737–42 151. Born TL, Thomassen E, Bird TA, Sims JE. 1998. Cloning of a novel receptor subunit, AcPL, required for interleukin-18 signaling. J. Biol. Chem. 273:29445–50 152. Kim SH, Reznikov LL, Stuyt RJ, Selzman CH, Fantuzzi G, et al. 2001. Functional reconstitution and regulation of IL-18 activity by the IL- 18Rβ chain. J. Immunol. 166:148–54 153. Carrie A, Jun L, Bienvenu T, Vinet MC, McDonell N, et al. 1999. A new member of the IL-1 receptor family highly expressed in hippocampus and involved in X-linked mental retardation. Nat. Genet. 23:25– 31 154. Sana TR, Debets R, Timans JC, Bazan JF, Kastelein RA. 2000. Computational identiﬁcation, cloning, and characterization of IL-1R9, a novel interleukin-1 receptor-like gene encoded over an unusually large interval of human chromosome Xq22.2–q22.3. Genomics 69:252–62 155. Dimcheff DE, Volkert LG, Li Y, DeLucia AL, Lynch WP. 2006. Gene expression proﬁling of microglia infected by a highly neurovirulent murine leukemia virus: implications for neuropathogenesis. Retrovirology 3:26 156. Qin J, Qian Y, Yao J, Grace C, Li X. 2005. SIGIRR inhibits interleukin-1 receptor- and Toll-like receptor 4-mediated signaling through different mechanisms. J. Biol. Chem. 280:25233–41 157. Huang X, Hazlett LD, Du W, Barrett RP. 2006. SIGIRR promotes resistance against Pseudomonas aeruginosa keratitis by down-regulating type-1 immunity and IL-1R1 and TLR4 signaling. J. Immunol. 177:548–56 158. Quiniou C, Sapieha P, Lahaie I, Hou X, Brault S, et al. 2008. Development of a novel noncompetitive antagonist of IL-1 receptor. J. Immunol. 180:6977–87 www.annualreviews.org • Functions of the IL-1 Family

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159. Anderson ME, Tejo BA, Yakovleva T, Siahaan TJ. 2006. Characterization of binding properties of ICAM-1 peptides to LFA-1: inhibitors of T-cell adhesion. Chem. Biol. Drug Des. 68:20–28 160. Ye K, Dinarello CA, Clark BD. 1993. Identiﬁcation of the promoter region of the human interleukin 1 type I receptor gene: multiple initiation sites, high G+C content, and constitutive expression. Proc. Natl. Acad. Sci. USA 90:2295–99 161. Cullinan EB, Kwee L, Nunes P, Shuster DJ, Ju G, et al. 1998. IL-1 receptor accessory protein is an essential component of the IL-1 receptor. J. Immunol. 161:5614–20 162. Colotta F, Dower SK, Sims JE, Mantovani A. 1994. The type II “decoy” receptor: a novel regulatory pathway for interleukin-1. Immunol. Today 15:562–66 163. Neumann D, Kollewe C, Martin MU, Boraschi D. 2000. The membrane form of the type II IL-1 receptor accounts for inhibitory function. J. Immunol. 165:3350–57 164. Dinarello CA. 2005. The many worlds of reducing interleukin-1. Arthritis Rheum. 52:1960–67 165. Malinowsky D, Lundkvist J, Laye S, Bartfai T. 1998. Interleukin-1 receptor accessory protein interacts with the type II interleukin-1 receptor. FEBS Lett. 429:299–302 166. Smeets RL, Joosten LA, Arntz OJ, Bennink MB, Takahashi N, et al. 2005. Soluble interleukin-1 receptor accessory protein ameliorates collagen-induced arthritis by a different mode of action from that of interleukin-1 receptor antagonist. Arthritis Rheum. 52:2202–11 167. Drevlow BE, Lovis R, Haag MA, Sinacore JM, Jacobs C, et al. 1996. Recombinant human interleukin-1 receptor type I in the treatment of patients with active rheumatoid arthritis. Arthritis Rheum. 39:257–65 168. Netea MG, Kullberg BJ, Boerman OC, Verschueren I, Dinarello CA, Van Der Meer JW. 1999. Soluble murine IL-1 receptor type I induces release of constitutive IL- 1α. J. Immunol. 162:4876–81 169. Kahlenberg JM, Dubyak GR. 2004. Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release. Am. J. Physiol. Cell Physiol. 286:C1100–8

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Contents

Volume 27, 2009

Frontispiece Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Translating Molecular Insights in Autoimmunity into Effective Therapy Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Structural Biology of Shared Cytokine Receptors Xinquan Wang, Patrick Lupardus, Sherry L. LaPorte, and K. Christopher Garcia p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Immunity to Respiratory Viruses Jacob E. Kohlmeier and David L. Woodland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 61 Immune Therapy for Cancer Michael Dougan and Glenn Dranoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 83 Microglial Physiology: Unique Stimuli, Specialized Responses Richard M. Ransohoff and V. Hugh Perry p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p119 The Liver as a Lymphoid Organ Ian Nicholas Crispe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Immune and Inﬂammatory Mechanisms of Atherosclerosis Elena Galkina and Klaus Ley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p165 Primary B Cell Immunodeﬁciencies: Comparisons and Contrasts Mary Ellen Conley, A. Kerry Dobbs, Dana M. Farmer, Sebnem Kilic, Kenneth Paris, Soﬁa Grigoriadou, Elaine Coustan-Smith, Vanessa Howard, and Dario Campana p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 The Inﬂammasomes: Guardians of the Body Fabio Martinon, Annick Mayor, and Jürg Tschopp p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p229 Human Marginal Zone B Cells Jean-Claude Weill, Sandra Weller, and Claude-Agn`es Reynaud p p p p p p p p p p p p p p p p p p p p p p267

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Aire Diane Mathis and Christophe Benoist p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p287 Regulatory Lymphocytes and Intestinal Inﬂammation Ana Izcue, Janine L. Coombes, and Fiona Powrie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p313 The Ins and Outs of Leukocyte Integrin Signaling Clare L. Abram and Clifford A. Lowell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p339

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Recent Advances in the Genetics of Autoimmune Disease Peter K. Gregersen and Lina M. Olsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p363 Cell-Mediated Immune Responses in Tuberculosis Andrea M. Cooper p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p393 Enhancing Immunity Through Autophagy Christian Munz ¨ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p423 Alternative Activation of Macrophages: An Immunologic Functional Perspective Fernando O. Martinez, Laura Helming, and Siamon Gordon p p p p p p p p p p p p p p p p p p p p p p p p451 IL-17 and Th17 Cells Thomas Korn, Estelle Bettelli, Mohamed Oukka, and Vijay K. Kuchroo p p p p p p p p p p p p p p485 Immunological and Inﬂammatory Functions of the Interleukin-1 Family Charles A. Dinarello p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p519 Regulatory T Cells in the Control of Host-Microorganism Interactions Yasmine Belkaid and Kristin Tarbell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p551 T Cell Activation Jennifer E. Smith-Garvin, Gary A. Koretzky, and Martha S. Jordan p p p p p p p p p p p p p p p591 Horror Autoinﬂammaticus: The Molecular Pathophysiology of Autoinﬂammatory Disease Seth L. Masters, Anna Simon, Ivona Aksentijevich, and Daniel L. Kastner p p p p p p p p p621 Blood Monocytes: Development, Heterogeneity, and Relationship with Dendritic Cells Cedric Auffray, Michael H. Sieweke, and Frederic Geissmann p p p p p p p p p p p p p p p p p p p p p p p p669 Regulation and Function of NF-κB Transcription Factors in the Immune System Sivakumar Vallabhapurapu and Michael Karin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p693

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Further

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Regulatory T Cells in the Control of Host-Microorganism Interactions∗ Yasmine Belkaid1 and Kristin Tarbell2 1

Mucosal Immunology Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; email: [email protected]

2

Immune Tolerance Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; email: [email protected]

Annu. Rev. Immunol. 2009. 27:551–89

Key Words

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

infection, Foxp3, dendritic cells, Treg

This article’s doi: 10.1146/annurev.immunol.021908.132723

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0551$20.00 ∗ The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

Each microenvironment requires a speciﬁc set of regulatory elements that are ﬁnely and constantly tuned to maintain local homeostasis. Various populations of regulatory T cells contribute to the maintenance of this equilibrium and establishment of controlled immune responses. In particular, regulatory T cells limit the magnitude of effector responses, which may result in failure to adequately control infection. However, regulatory T cells also help limit collateral tissue damage caused by vigorous antimicrobial immune responses against pathogenic microbes as well as commensals. In this review, we describe various situations in which the balance between regulatory T cells and effector immune functions inﬂuence the outcome of host-microorganism coexistence and discuss current hypotheses and points of polemic associated with the origin, target, and antigen speciﬁcity of both endogenous and induced regulatory T cells during these interactions.

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INTRODUCTION

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Humans have coevolved with microbial partners, and for the most part the interactions established can be considered positive or neutral. This is clearly the case of the ﬂora composed of commensals and symbiotes that invade our lung, skin, or gut soon after birth. However, in some more rare instances, interactions with microorganisms can be detrimental and lead to pathological consequences. Despite these different outcomes, the distinction between a commensal and a pathogenic microorganism is not always obvious. For instance, Mycobacterium tuberculosis, present in two billion individuals worldwide, behaves more often as a commensal than a parasite. It is therefore not surprising that many of the strategies we have developed to coexist peacefully with our positive partners can be hijacked or manipulated by potentially harmful microorganisms to ensure their own survival. Indeed, many pathogens have evolved mechanisms to manipulate the regulatory network of the host to their advantage, thereby generating conditions that secure their survival for an extended period of time. In particular, microorganisms induce a large array of regulatory cells to ensure their own survival (1). However, surviving an infection requires the generation of a controlled immune response in the host that recognizes and controls the invading pathogen while limiting collateral damage to self-tissues that may result from an exuberant immune response. This implies that induction of regulatory T cells also arises as a result of the host response to the infectious process in a bid to maintain or restore a homeostatic environment (1). In this review, we discuss the roles and origins of regulatory T cells in the regulation of host-microorganism interaction. Although investigators have long recognized that T cells with suppressive or anergic activity or IL-10-producing T cells could be generated in vivo during infection (2), only recently have we learned that specialized subsets of regulatory T cells also contribute to this regulatory network. Several types of regulatory T cells have been described on the basis of their origin, generation, and mechanism of action, 552

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with two main subsets identiﬁed: naturally occurring Foxp3+ regulatory T cells (referred to here as Treg cells), which develop in the thymus; and inducible regulatory T cells, which develop in the periphery from conventional CD4+ T cells after exposure to signals such as regulatory cytokines, immunosuppressive drugs, or antigen-presenting cells (APCs) conditioned by microbial products (3). Both types of regulatory T cell, by virtue of their capacity to control the intensity of effector responses, play a major role in infections. However, the discovery that expression of forkhead box P3 (Foxp3), a transcription factor that is crucial for the development and function of Foxp3+ Treg cells, can be induced de novo in conventional CD4+ T cells renders the distinction between natural Treg cells and inducible regulatory T cells less obvious. For the purpose of this review, we deﬁne natural Treg cells as the population of regulatory T cells that is present in the host before pathogen exposure and inducible regulatory T cells as those cells that acquire regulatory function in the context of a given infection. Inducible regulatory T cell populations include (a) T regulatory 1 (Tr1) cells, which secrete IL10; (b) transforming growth factor (TGF)-βproducing cells; and (c) inducible Foxp3+ regulatory T cells.

ROLE OF Foxp3+ Treg CELLS IN THE CONTROL OF IMMUNOPATHOLOGY Foxp3-expressing Treg cells were initially described as a unique population of CD4+ T cells that prevent the expansion of self-reactive lymphocytes and subsequent autoimmune disease (reviewed in 4). These cells are classically deﬁned by their constitutive expression of the IL-2 receptor α chain (CD25). These cells also express the cytotoxic T lymphocyte antigen 4 (CTLA4) and the tumor necrosis factor (TNF)-receptor family members GITR (glucocorticoid-induced TNF receptor–related protein) and OX40 (5), CD39, CD73 (6), and high levels of the folate receptor FR4 (7). However, none of these markers is speciﬁc for Treg

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cells, as activated T cells can also express them. The transcription factor Foxp3 is the most deﬁnitive signature of Treg cells in mice (5), but its expression can be transiently upregulated by activated human T cells. Despite extensive studies in various models, the mechanism by which Treg cells limit effector responses in vivo remains poorly understood. Recently performed in vivo imaging indicates that transferred Treg cells form long-lasting interactions with dendritic cells (DCs) soon after they enter the lymph nodes. These interactions impair the ability of DCs subsequently to activate effector T cells, indicating that, in vivo, Treg cells may inhibit T cell responses indirectly by modulating the function of APCs (8). Treg cells may also directly inhibit T cells, either via cell-cell contact or by production of anti-inﬂammatory cytokines (9). TGF-β and IL-10 contribute to Treg cell suppressive activity in vivo (10). CTLA4-expressing Treg cells induce the expression by APCs of the enzyme indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan, and lack of this essential amino acid inhibits T cell activation and promotes T cell apoptosis (11). More recently, adenosine and cAMP have also been shown to contribute to Treg cell suppressive activity (6, 13). Treg cells can also induce apoptosis of T cells via cytokine withdrawal (12). However, in most cases, which mechanisms of suppression are being employed by Treg cells are still largely unclear. During infection, such mechanisms are likely to be redundant and vary according to the site of infection or the degree of inﬂammation. Some of the earliest studies on Treg cells emphasized that they control the extent of immune-mediated pathology. Activated Treg cells efﬁciently control T cells and innate responses in mouse models of colitis, thereby minimizing collateral tissue damage (14). Importantly, Treg cells prevent exuberant responses against the ﬂora and maintain gut integrity (14). A similar scenario probably occurs during chronic infection, whereby Treg cells would be required to monitor the constant immune response by the host and to prevent detrimental tissue damage. For example,

the presence of Treg cells limits pulmonary inﬂammation and lung injury in a mouse model of Pneumocystis pneumonia (15). Treg cell– mediated control of immunopathology may be particularly important for protecting immuneprivileged environments or tissues with highly specialized functions, such as the liver or eyes. In a model in which mice were infected in the eye with herpes simplex virus (HSV), Treg cells protected against the development of virusinduced inﬂammatory lesions (16). Chronic infection with Schistosoma mansoni in mice also illustrates the protective role of Treg cells, as their removal increases damage to the liver (17). Treg cells are also involved in maternal tolerance toward the fetus bearing alloantigens (18). Recent evidence suggests that an imbalance in this regulation can occur in the context of certain infections. Acute maternal infection with Toxoplasma gondii during pregnancy is associated with adverse pregnancy outcomes that correlate with a sharp decrease of Treg cells in the placenta and peripheral tissues compared with uninfected mice. Transfer of Treg cells led to enhanced protection of the pregnancy in toxoplasma-infected mice (19), supporting the idea that a failure in Treg cell function may contribute to the pathogenesis of abortion caused by this parasite. In contrast, removal of Treg cells can sometimes protect against pathology associated with high parasite burden. In a model of cerebral malaria in mice, removal of Treg cells protected against the immunopathology that occurs in response to massive inﬂux of effector cells in the brain (20). This paradoxical effect, removal of Treg cells leading to reduced immunopathology, is explained by the strong reduction of parasites accumulating in the brain of the depleted mice compared with untreated mice (20).

ROLE OF Foxp3+ Treg CELLS IN MICROBIAL PERSISTENCE AND MAINTENANCE OF PROTECTIVE RESPONSES Even when Treg cells successfully preserve homeostasis in the host by controlling excessive

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immune responses, one consequence of such control is enhanced pathogen survival and, in some cases, long-term pathogen persistence. For example, in a resistant mouse model of Leishmania infection, mice remained chronically infected at the site of primary infection. Treg cells that accumulate at the site of infection regulate the function of local effector cells, which prevents efﬁcient elimination of the parasite (21). Similarly in the context of African trypanosomiasis, IL-10-producing Treg cells can limit the pathogenic consequences of the immune response against the microorganism without compromising parasite persistence (22). Treg cells can also favor the persistence of various pathogens (see Table 1) such as Mycobacterium tuberculosis (23, 24), mouse mammary tumor virus (25), or the pathogenic Seoul virus in their reservoir host (26). In some cases, Treg cells can control the ﬁne balance established between the pathogen and its host, mediating an equilibrium that can become mutually beneﬁcial. In other cases, regulatory control is too excessive, allowing the pathogen to replicate without restraint and overwhelm the host, thereby compromising host survival. In a mouse model of malaria, for example, depletion of Treg cells protected mice from death caused by the lethal strain of Plasmodium yoelii by restoring a vigorous effector immune response that eradicated the parasites (27). Following experimental infection with a hypervirulent strain of Mycobacterium tuberculosis, the early Th1 response was rapidly reduced, correlating with the rapid emergence of IL10-producing Foxp3-positive cells (28). Filarial disease caused by infection with a ﬁlarial nematode is associated with a profound systemic suppression of the host immune system (29). Related to this balance of protective versus damaging pathogen-directed immune response is the balance between immune activation and development of autoimmunity. Immune responses in the context of infection can have varying effects on the potential development of autoimmunity. On the one hand, the damage to self-tissue, activation of innate immunity, and crossreaction between self and foreign

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antigens can increase the probability of developing self-speciﬁc immune responses. For example, risk for developing multiple sclerosis, a neuronal autoimmune disease, is altered by migration from one latitude to another, suggesting a link with infection (30). On the other hand, pathogens, via Toll-like receptor (TLR) ligands and other mechanisms, can enhance immunesuppressive cell types such as Treg cells, which can result in infection being negatively correlated with autoimmunity. For example, rates of both allergy and autoimmunity are lower in developing countries with higher rates of infectious disease (31). There is a growing body of literature supporting the idea that microorganism persistence is in some cases necessary for the maintenance of protective immunity. In a situation of chronic infection or high exposure to microbial antigens, such as in the case of plasmodium during transmission season, the generation of potent effector/memory cells could lead to pathogenic consequences. In a model of Leishmania major infection, parasite persistence, as a result of immune suppression by Treg cells, was necessary for the maintenance of protective immunity against the parasite (16, 21). Another example of this entente between the host and the pathogen is provided by the ocular infection of mice with HSV1. When infected with a low dose of virus, Treg cells protected mice from the CD4+ T cell–mediated pathology, a situation that is compatible with the establishment of immunity to reinfection (16). Similarly, in Candida albicans infection in mice, reduction of the number of Treg cells led to better control of the primary infection but enhanced pathology as well as loss of immunity to reinfection. Immunity to reinfection can be recapitulated when Treg cells are transferred back (32). Thus, a role for Treg cells in the maintenance of immunity may be general to other chronic infections in which poor quality effectors are generated and pathogen persistence is required to sustain protective responses. A large number of studies support a role for Treg cells in the control of persistent infection in humans (Table 1). In humans, however,

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Microbial infections that involve Foxp3+ Treg cells

Disease Microorganism

Species

Effect of Treg cells on immunopathology and pathogen load

References

Mouse Mouse Mouse

Control of liver pathology; favors host survival Suppression of antigen-speciﬁc cell proliferation in vitro In resistant strains: Th1 cell responses controlled through IL-10-dependent and -independent mechanisms; favors parasite persistence In susceptible strains: Th2 cell responses controlled; removal of Treg cells transiently exacerbates disease

17, 179, 275, 288, 289 290 21, 181, 182, 199, 243, 291–293

Mouse

Removal of Treg cells leads to enhanced parasite numbers and enhanced pathology Accumulation of Treg cells at cutaneous sites of infection

294

Early burst of TGF-β associated with Treg cell expansion in the blood; correlation between high parasite expansion and Treg cell increase; removal of Treg cells enhances immune responses in vitro; correlation between Treg deﬁcit and low susceptibility to infection Control of effector immune responses; favors uncontrolled expansion of parasite, leading to death of the host Removal of Treg leads to enhanced parasite control and protection against cerebral malaria Parasite expansion by limiting effector responses Th2 cell responses reduced Controls of effector responses; promotes parasite persistence

159, 296

Mouse

Limits effector responses and prevents immunopathology

22

Mouse

Treg cells activated/induced by infection protect against airway inﬂammation in an asthma model

162, 236, 299

Friend virus

Mouse

165, 166, 300

Murine AIDS HSV1

Mouse Mouse

Virus persistence by limiting CD8+ effector T cell functions; in vitro suppression through cell contact Effector responses limited; favors viral expansion Controls CD8+ T cell proliferation and effector functions; favors pathogen expansion; controls eye immunopathology Accumulates in the cornea and suppresses virus-speciﬁc CD4+ and CD8+ effector T cells Controls memory T cell responses to HSV2 Limits immune response in the periphery; favors local immune response; limits local microorganism expansion; controls CD4+ and CD8+ T cell responses in neonates; favors pathogen expansion in neonate Increases in Treg cells in lymphoid organs and mucosal tissues; removal of Treg cells from the blood or lymphoid tissues increases virus-speciﬁc immune responses

Parasitic infections

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Schistosomiasis: Schistosoma mansoni Schistosoma japonicum Leishmania major

Leishmania amazonensis Leishmania braziliensis

Human

Malaria: Plasmodium falciparum

Human

Plasmodium yoelii

Mouse

Plasmodium yoelli (ANKA) Plasmodium berghei Brugia pahangi Litomosoides sigmodontis Trypanosoma congolense Heligmosomoides polygyrus

Mouse

295

27 20 297 298 29, 273

Viral infections

Rabbit HSV2

HIV

Human Mouse

301 16, 95, 251, 254 302 303 81, 304

33, 41, 42, 44–46, 51, 53, 58–60, 305–309 (Continued )

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Disease Microorganism

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Species

Effect of Treg cells on immunopathology and pathogen load

References

HCV

Human and chimpanzee

71, 74, 251, 310

HBV

Human

Suppression of IFN-γ production, expansion, and activation-induced cell death of HCV-speciﬁc T cells after recovery and during persistent infection; negative correlation between percentage of Treg cells and inﬂammation; HCV peptide can stimulate Treg cells from HCV patients in vitro Treg cells accumulate in the liver during chronic severe hepatitis B; their frequency in the blood correlates with viral load; depletion of Treg cells leads to increased antigen-speciﬁc IFN-γ production; Treg cells suppress the proliferation of autologous PBMCs mediated by HBV antigens. Dysfunction of Treg cells during infection; inverse correlation between Foxp3 expression and viral load Treg cells suppress virus-speciﬁc T-cell responses from PBMC Persistent HPV16 infection correlates with high Treg frequencies CD8+ T cell responses controlled; responses to immunodominant epitopes suppressed CD8+ T cell responses controlled, responses to immunodominant epitopes suppressed Viral replication and immune activation in lymphatic tissue correlates with increased Treg cell numbers; frequency of Treg cells inversely correlates with the magnitude of the SIV-speciﬁc CTL responses Suppression of effector responses in infants Loss of Treg in lymph node during pathogenic infection Increased frequency of activated Treg cells in the blood and lymph nodes of chronically infected host

261

Antifungal Th1 cell responses limited; immunopathology controlled Increased frequency of CTLA4+ GITR+ TGF-β+ FOXP3+ Treg cells in the blood and fungi-induced granuloma; increased suppressive activity in vitro Local recruitment of Treg cells to the lung (site of infection); neutrophils controlled through IL-10 and CTLA4 and IDO

251

Controls innate immune responses Accumulation of Tregs at site of infection; suppresses effector responses; higher Treg frequencies correlates with higher bacterial colonization; high frequency from gastric tissue correlates with low pathology in children Controls antibody and T cell responses Controls primary and secondary CD8+ T cell responses Controls pro-inﬂammatory cytokines and lung pathology limited Controls effector responses

251 323–327

Favors microorganism persistence

23

HTLV-1 CMV Human papillomavirus type 16 Vaccinia virus

Human

Mouse

Inﬂuenza virus

Mouse

SIV

Macaque

FIV

Cat

62, 64, 66, 311

312–314 41 315

71, 261 48, 55, 316–318

319 320 251, 321

Fungi infections Candida albicans Paracoccidioides brasiliensis

Human

Aspergillus fumigatus

Mouse

322

227

Bacterial infections Helicobacter hepaticus Helicobacter pylori

Mouse Human

Helicobacter pylori Listeria monocytogenes Pneumocystis carinii Mycobacterium tuberculosis

Mouse Mouse Mouse Mouse

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(Continued )

Disease Microorganism

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Effect of Treg cells on immunopathology and pathogen load

References

Mycobacterium tuberculosis

Human

35, 38, 330–332

Chlamydia trachomatis

Human

Treg cell number increases in the blood and sites of infection in patients with active tuberculosis; frequency of FOXP3+ cells inversely correlates with local Mycobacterium tuberculosis-speciﬁc immunity FOXP3+ cells accumulate in infected conjunctiva; potential role in control of immunopathology

reliable identiﬁcation of Treg cells is complicated by the fact that Foxp3 expression does not always correlate with regulatory properties, and activated T cells can transiently express it. Likewise, CD25 or other Treg cell markers cannot be used to discriminate reliably between Treg cells and highly activated T cells. Furthermore, in the lymphoid tissues of HIV-infected subjects, for example, most Foxp3+ T cells are CD25−/low (33). Because peripheral blood is the most accessible compartment, most human studies that evaluate Treg cell functions or numbers are done using this compartment. However, as in some chronic infections Treg cells accumulate in infected tissues and consequently are reduced in the blood compartment. Despite these caveats, some reports provide convincing evidence for a role of Foxp3-expressing Treg cells in a large number of human infections (reviewed in 34). In pulmonary tuberculosis patients, the frequencies of Treg cells are signiﬁcantly higher in the blood and at the site of infection compared with normal individuals (35–38). Furthermore, the frequency of Treg cells in pleural ﬂuid inversely correlates with local Mycobacterium tuberculosis–speciﬁc immunity (37). These Treg cells display an activation phenotype, and in vitro the addition of Treg cells back to cultures signiﬁcantly suppresses the antigen-speciﬁc production of interferon (IFN)-γ by effector T cells (35, 36). Conversely, removal of Treg cells signiﬁcantly enhances effector responses against bacteria by αβ and γδ T cells (31, 32, 35). Some data suggest that differences in susceptibility to infection may rely on a functional deﬁcit of Treg cells. Previous interethnic com-

333

parative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups (296). This lower susceptibility is not associated with classic malaria-resistance genes, and analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as to nonmalaria antigens, revealed higher immune reactivity in Fulani. Microarray analysis on RNA from Treg cells indicated obvious differences between the two ethnic groups, with 23% of genes, including TGF-β, TGF-β receptors, CTLA4, and Foxp3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of low Treg activity, Fulani showed lower serum levels of TGF-β (296). Furthermore, the proliferative response of Fulani to malaria antigens was not affected by the depletion of Treg cells, whereas that of Mossi was signiﬁcantly increased. The results suggest that the higher resistance to infection in a deﬁned ethnic group may derive from a functional deﬁcit of Treg cells (296).

Treg CELLS AND VIRAL INFECTIONS Several studies suggest that Treg cells may limit the ability of adaptive T cell response to control HIV and SIV replication (39, 40). In particular, anti-HIV responses were increased after in vitro removal of Treg cells from peripheral leukocytes (41–45) and from lymph nodes of HIV-infected patients (46). Similarly anti-SIV responses were increased after removal of Treg

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cells from lymph node of SIV-infected rhesus macaques (47, 48). Several groups have shown that Treg cell frequencies are decreased in the peripheral blood of HIV-infected patients (49– 53). This observation could suggest that Treg cells, just as with conventional T cells, are progressively lost during HIV infection. Furthermore, cells from infected individuals that show strong HIV-speciﬁc Treg cell function in vitro had signiﬁcantly lower levels of plasma viremia and higher CD4+ -to-CD8+ T cell ratios than did individuals with undetectable Treg cell activity (42). However, the observation that the expression of Treg cells is increased in lymphoid or mucosal tissues from HIV-infected patients and SIV-infected macaques suggests that the redistribution of Treg cells in infected tissues could account for the decreased frequency of Treg cells in the blood (48, 54–57). Importantly, Treg cells puriﬁed from the lymph node of HIV patients maintain their suppressive capacity against antiviral responses (46). The high frequency of Foxp3+ Treg cells was much higher in the duodenal mucosa of patients infected with HIV compared with healthy controls (56). These ﬁndings suggest that Treg cells, by suppressing virus-speciﬁc immunity, may contribute to uncontrolled viral replication, therefore playing a detrimental role in HIV infection. Indeed, in humanized mice infected with HIV, depletion of Treg cells significantly increased viral control (58). However, there is a strong negative correlation between the level of T cell activation and the frequency of Treg cells, which is most prominent during the early chronic stage of the disease, suggesting a role for these cells in the control of immune activation (59). Furthermore, the preservation of Treg cells in the peripheral blood mononuclear cells (PBMCs) of HIV-infected elite suppressors (ES) correlates with low CD4+ T cell activation, supporting the idea that it may be one mechanism associated with the nonprogressive nature of HIV infection in ES (60). Thus, although there is a consensus on the capacity of Treg cells to suppress immune responses during HIV, the overall consequence

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on the development of the disease remains uncertain and is likely to be tightly dependent on the stage of the infection or the site targeted. Treg cells also appear to play a role in the control of chronic viral hepatitis (61). Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most common causes of liver disease worldwide, and failure to control infection with either virus results in an immune-mediated acute and chronic necroinﬂammatory liver disease. In patients with chronic infection with HBV, the frequencies of Foxp3+ Treg cells is highly increased both in the periphery and in the liver and correlates with viral load (62–66). More recently, a positive correlation was shown between IL-10-producing Foxp3+ Treg cells expanded in response to viral antigen and viral load (67). Furthermore, antigen-speciﬁc suppression of effector responses in vitro suggests that the expansion of antigen-speciﬁc Treg cells during this infection may contribute to the associated liver pathology (62). HCV-associated liver disease also seems to involve Treg cells, which could impede immune defense against the virus. Individuals who are chronically infected with HCV have a higher number of Treg cells in the blood compared with that of a person whose HCV infection spontaneously resolved or with that of a healthy subject (68–70). Depletion of Treg cells enhances antigen-speciﬁc CD8+ T responses in vitro (71, 72). During HCV infection, Treg cells display enhanced suppressive function when assessed in vitro during both the acute and chronic stages of the infection, compared with uninfected donors or with patients with spontaneous resolution (70). Treg cells also accumulate in the liver of patients with chronic HCV (62, 73). The inverse correlation between the HCV-speciﬁc TGF-β response by Treg cells and liver damage could support the idea that Treg cells also have a role in controlling chronic inﬂammatory responses and liver damage in HCV carriers (68, 74). However, in another study the high inﬁltrate of Foxp3+ T cells in the liver of HCVinfected patients was comparable between mild

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and severe ﬁbrosis (73). Interestingly, patients who are chronically infected with HCV and go on to develop autoimmunity have fewer peripheral Treg cells (75). However, the link between chronic infection, autoimmune disorders, and dysregulation of Treg cell function requires further analysis.

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ROLES OF Treg CELLS DURING ACUTE INFECTIONS Most available studies both in human and experimental models support the idea that a limitation in Treg cell function or number is usually associated with enhanced immune responses and subsequent better control of the infection (1). The overwhelming evidence for this function comes from models of chronic infections (Table 1). In the context of acute infection, a role for these cells is often unclear or controversial. In some infections, such as those due to Trypanosoma cruzi or murine malaria, conﬂicting reports suggest or refute a role for Treg cells in controlling effector responses (76, 77). Some of the conﬂicting results may reﬂect an inefﬁcient approach to targeting Treg cells. For instance, during malaria infection, the anti-CD25 treatment commonly used to deplete Treg cells leads to incomplete depletion (78). Furthermore, in an inﬂammatory setting, effector cells can express CD25, but in deﬁned tissues such as the gut, most Treg cells do not express this marker. The recent development of mice in which Foxp3-expressing cells can be selectively depleted will give us a better understanding of the situation in which Treg cells play a nonredundant role (79, 80). This will also allow a better understanding of the situation in which Treg cells may be overwhelmed or destabilized by exuberant inﬂammation. A recent study provides evidence that in some acute infections Treg cells may have a paradoxical role (81). Following delivery via the natural route, HSV type 2 infection (HSV2) replicates initially in the vaginal mucosa and rapidly disseminates into the central nervous system, resulting in fatal paralysis. The pres-

ence of plasmacytoid DCs and IFN-γ production by natural killer (NK) cells and by CD4+ T cells delay the onset of death by a few days, revealing the existence of a transient viral control (81). During HSV2 infection, as previously described during several experimental viral infections, Foxp3+ Treg cells expand, accumulate at the primary site of infection, and display enhanced suppressive function. As expected, their elimination leads to enhanced immune responses in the regional lymph node (82). However, paradoxically this enhanced immune response is associated with exacerbated viral load in vaginal mucosa, earlier dissemination in the central nervous system, and accelerated fatal outcome. Treg cell depletion also favors early viral replication in the liver during lymphocytic choriomeningitis virus infection (81). Uncontrolled chemokine production in the regional lymph node together with their selective reduction in the vaginal mucosa may account for the impaired or delayed migration to the infected site. Control of chemokines by Treg cells during infection has been previously reported but was associated with a distinct outcome. In a model of HSV type 1 (HSV1), the mechanism of Treg cell control of stromal keratitis involved suppressed antiviral immunity and impaired expression of the molecule required for T cell migration to lesion sites (16). The ﬁnding that Treg cells can facilitate the arrival of innate cells and therefore direct appropriate immune responses is an intriguing concept. What remains to be established is if this paradoxical role for Treg cells is associated with speciﬁc microenvironments and inﬂammatory settings, or if it represents a more general function. Infection in certain sites could be particularly dangerous, and Treg cells may become, in some circumstances, an active partner of protective responses. Another possibility is that such functions of Treg cells might be associated with high microbial replication or inﬂammation. Thus, in highly dynamic infection, Treg cells may be important to restrain exuberant immune responses in lymphoid organs and favor timely recruitment of innate cells.

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REGULATORY T CELLS INDUCED BY MICROORGANISMS All persistent microorganisms obey the same principle: The immune system constitutes their ecological niche, and they have coevolved with their host to learn how to manipulate APC function in order to dictate an immune response appropriate to ensure their survival. For instance, microorganisms induce a large array of regulatory cells to ensure their own survival (1).

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IL-10-Producing Regulatory T Cells The role of IL-10 as an immunoregulatory cytokine in infection has been documented primarily in the context of chronic infections (83). IL-10 can inhibit the immune responses (by both Th1 cells and Th2 cells) to many pathogens in experimental models (84–86) and in human infectious diseases, such as tuberculosis, malaria, HCV, ﬁlariasis, leishmaniasis, and schistosomiasis (87–92). The most remarkable example of this control is illustrated by its crucial role during acute infection of mice with Toxoplasma gondii. In this model, IL-10 production by T cells is the key regulator of effector cell responses, as IL-10-deﬁcient mice can control parasite number but succumb to lethal immunopathology driven by unrestrained effector cell responses (93). During Th2 cell– dominated helminth infection, Th2 cells are a major source of IL-10 (83). Besides T cells, numerous other cell types can also produce IL10, including B cells, NK cells, macrophages, and DCs (reviewed in 83). In acute Plasmodium yoelii infection, a subset of regulatory DCs with the phenotype CD11clo CD45RBhi and inducing IL-10-secreting T cells becomes the predominant DC population in the spleen (94). IL10 can also be produced by natural Treg cells and, in some cases, is associated with their function; however, in most cases, other cells are the relevant source of this cytokine during infection (20, 95–97). In a large number of infections, such as murine malaria, T cells producing IL-10 (Tr1) represent a dominant source of this cytokine (98, 99). Tr1 cells can develop from conventional T cells after encountering 560

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deﬁned signals, such as exposure to deactivated or immature APCs, to antigen, or to IL-10 itself (reviewed in 3, 100). Of note, these conditions prevail during chronic infection in which APC functions are often targeted by the pathogen, and cells of the immune system are chronically exposed to microbial antigens. Consistent with a role for these cells in human disease, Tr1 cell clones can be isolated from patients who are chronically infected with HCV (87). Interestingly, these regulatory clones had similar viral antigen speciﬁcity to protective Th1 cell clones isolated from the same patient (87). Deﬁned microbial products can manipulate DCs in a way that induces Treg cell populations (101). For example, ﬁlamentous hemagglutinin from Bordetella pertussis induces IL-10 production by DCs; these DCs favor the differentiation of naive T cells into Tr1 cells (102). Similarly, Tr1 cells can be generated from naive T cells in the presence of DCs stimulated with phosphatidylserine from Schistosoma mansoni (103). Although Tr1 cells deﬁne a population of T cells that can produce IL-10 and/or TGFβ, some IL-10-producing T cells can also produce IFN-γ. The autocrine regulation by IL-10 of Th1 and Th2 cells was initially described in human clones (104). In the context of an infectious disease, IFN-γ and IL-10 double producers were ﬁrst described in the bronchoalveolar lavage of patients with tuberculosis (105) and in individuals chronically infected with Borrelia burgdorferi (106). Indeed, in many chronic infections, in humans and in experimental animals, the presence of CD4+ T cells that produce high levels of both IL-10 and IFN-γ has been documented (reviewed in 107). During experimental infection with T. gondii and in a model of nonhealing leishmaniasis, CD4+ T cells producing IFN-γ and IL-10 that share many features with Th1 cells are the main source of protective IL-10 (96, 97). These T cells were identiﬁed as activated T-bet+ Th1 cells and were distinct from Th2 cells, Treg cells, or other subsets of inducible regulatory T cells. Unlike IFN-γ production, IL-10 production was transient—observed in only a fraction of the IFN-γ-producing cells—and it was

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produced more rapidly by recently activated T cells than by resting T cells (96). The instability of IL-10 synthesis, which was observed only when the Th1 cells were fully activated, is probably necessary to prevent sustained suppression of effector functions. Thus, in some cases cells with regulatory properties could arise from fully differentiated Th1 cells as a negative feedback loop. It is likely that numerous previous studies of Tr1 cells were in fact incriminating similar populations. These IFN-γ- and IL-10-producing T cells may represent a dominant regulatory response to infections that induce highly polarized Th1 cell responses. The nature of the APC or status of activation required for the imprinting of IL-10 on these Th1 cells remains poorly understood, but some evidence suggests that the cytokines produced by DCs could contribute to this phenotype. For instance, repetitive exposure to IL-12 could induce IL-10 in IFN-γ-producing cells (108). Several recent reports suggest that IL-27 might be an important determinant for the induction of IL-10 on Th1 cells (109–111). A role for this cytokine as a regulatory mediator has recently emerged. IL-27 can limit Th1, Th2, and Th17 cell responses in various models of infection and autoimmunity (112). When splenic DCs were exposed to Treg cells producing TGF-β, these DCs acquired some characteristics of plasmacytoid DCs and released TGF-β and IL-27, which in turn allowed the induction of IL-10producing T cells (111). How IL-27 could contribute to the induction of IL-10 in the gastrointestinal tract and how this pathway could contribute to the maintenance of gut homeostasis remain to be addressed.

Induction of Foxp3+ Treg Cells in the Periphery Although a role for Foxp3+ Treg cells in the maintenance of immune tolerance has been demonstrated in both humans and mice, the origin of these cells is still not completely understood. Early neonatal thymectomy experiments in mice strongly suggested that Treg cells are generated in the thymus. Studies using

Foxp3 reporter mice (113) and transgenic mice that express non-self antigens in thymic tissue (114, 115) have also traced the development of Foxp3+ cells to the thymus. Aside from evidence that Foxp3+ Treg cells arise and mature in the thymus, there is mounting evidence that they can develop extrathymically under certain conditions. Both murine (116, 117) and human (118) naive T cells express Foxp3 and acquire suppressive activity in vitro after T cell receptor (TCR) stimulation in the presence of TGF-β. In vivo, delivery of subimmunogenic doses of antigen (119) as well as endogenous expression of foreign antigen in a lymphopenic environment (120) can also induce peripheral Foxp3+ Treg cell development. Despite a growing body of literature documenting a potential role for these converted cells in the control of autoimmune or inﬂammatory diseases (121), the nature of the APCs involved in this conversion process remains poorly understood. Several reports support the idea that immature DCs may be more efﬁcient than activated DCs at inducing Foxp3+ Treg cell development in the presence of TGF-β. For example, targeting of antigens to immature DCs via the regulatory receptor DEC205 can favor the induction of Foxp3+ T cell development de novo (120). Although virtually all APCs under steady-state conditions may have the capacity to induce antigen-speciﬁc Treg cells, DCs appear to be more efﬁcient at this process than are other APCs (122). Thus, spleen DCs are more potent than DC-depleted APCs for the induction of Treg cells and require lower doses of peptide antigen (122). In the absence of exogenous IL-2, endogenous IL-2 production by T cells favoring Treg cell conversion can be efﬁciently triggered by DCs expressing CD80 and CD86 but not by other APCs (122). However, another study has proposed that B cells are more efﬁcient at inducing Foxp3+ Treg cells than are splenic DCs in the presence of TGF-β (123). This discrepancy is likely associated with the level of activation of the APCs in these different settings. The demonstration that some DC subsets from lymphoid tissues could be more efﬁcient at inducing Treg cells than others

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came from a study showing that CD8+ DCs induce higher conversion than other spleen DC subsets in the presence of TGF-β (124). Several molecules contribute to the induction of Foxp3+ Treg cell development. For instance, the B7-CTLA4 axis is important for induction of these cells (125–127). A role for PD-L1 expressed by DCs in Treg cell induction has been recently reported (124), adding another potential role for this molecule in the control of peripheral tolerance (128). Gut DCs also have an important role in dictating the homing potential of lymphocytes. DCs isolated from the Peyer’s patches, small intestinal lamina propria, and mesenteric lymph nodes (MLNs) promote the expression of the gut-homing receptors α4 β7 integrin and CCR9 by CD4+ and CD8+ T cells (129–132). The molecule CCR9 binds to CCL25 produced by epithelial cells of the small intestine, and α4 β7 integrin binds to mucosal vascular addressin cell-adhesion molecule 1 (MADCAM1), which is expressed by the vascular endothelium of the gastrointestinal tract. It is also becoming clear that nutrient status can impact an individual’s susceptibility to intestinal pathologies (133). In the case of vitamin A and, in particular, its transcriptionally active metabolite retinoic acid, prolonged insufﬁciency not only disrupts the integrity of the intestinal epithelial barrier, but also prevents the proper deployment of effector lymphocytes into the gut-associated lymphoid tissue (GALT) after priming. Indeed, the capacity of GALT DCs to imprint guthoming receptors to lymphocytes is associated with their capacity to release retinoic acid (133– 135). Another effect of retinoic acid on the immune regulation of the gastrointestinal tract is associated with its capacity to enhance the TGF-β-mediated generation of Foxp3+ Treg cells from naive T cells by gut DCs (123, 136– 142). Importantly, retinoic acid can induce the conversion of naive CD4+ T cells puriﬁed from human cord blood into Foxp3+ Treg cells (139). Reciprocally, retinoic acid can inhibit the generation of Th17 cells (138–141), suggesting that it may play an important role in maintaining the balance between effector and regulatory popu-

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lations in the gastrointestinal tract. The mechanism by which retinoic acid produced by DCs can enhance the capacity of TGF-β to induce Foxp3 on naive T cells remains unclear but is likely due to a conjunction of effects on both T cells and DCs. One possible role of retinoic acid is via its capacity to enhance TGF-β signaling; indeed, retinoic acid can increase the expression of the TGF-β receptor subunit (143). Another possibility is associated with retinoic acid’s capacity to suppress effector cytokines that suppress the induction of Foxp3 by T cells (144, 145). Although the capacity of GALT DCs or macrophages to imprint gut-homing receptors and induce Foxp3+ Treg cells is associated with their capacity to release retinoic acid, it remains unclear if these cells are the main producers of this metabolite in the gut. Synthesis of retinoic acid from stored or dietary retinol depends on the direct expression of the appropriate enzymes by GALT DCs. DCs from Peyer’s patches and MLNs express Aldh1a1 and Aldh1a2, respectively (134, 137). DCs from the lamina propria express a large array of this family of enzymes (C.M. Sun and Y. Belkaid, unpublished observation). Supporting the idea of a role for these cells as producers of retinoic acid, Peyer’s patch and MLN DCs can directly convert retinol to retinoic acid in culture (134). However, other cells, including epithelial cells, can express enzymes associated with vitamin A metabolism (146), suggesting that DCs may also acquire retinoic acid from other sources and store it. A recent study demonstrated that monocyte-derived DCs pretreated with retinoic acid can acquire several attributes characteristic of mucosal DCs, such as secretion of TGF-β and IL-6 and the capacity to augment mucosal homing receptor expression and IgA responses in lymphocytes. In this particular study, these gut-derived features acquired by DCs were associated with the capacity of DCs to become carriers and not producers of retinoic acid (146). The precise factors that govern the activation of some of these enzymes as well as how inﬂammation or infections modify the metabolism of vitamin A remain to be

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explored. Another important question is the timing necessary for DCs migrating in the GALT to acquired retinoic acid from epithelial cells and how these processes can be modiﬁed during infection. How retinoic acid contributes to oral tolerance and at the same time protective immunity in the gastrointestinal tract also remains to be addressed. One possibility is that retinoic acid favors the induction of Treg cells in the absence of secondary signals but enhances effector response following exposure to inﬂammatory mediators. Indeed, lamina propria DCs stimulated with ﬂagellin can induce the differentiation of Th17 in a retinoic acid–dependent manner, suggesting that at a physiological dose retinoic acid does not inhibit but rather promotes this pathway (147). This observation is consistent with the fact that the gut, enriched in retinoic acid, is home to a large number of IL17-producing T cells under steady-state conditions (148). The ability of local mediators to imprint homing and regulatory properties is also observed in another deﬁned microenvironment, the skin. Vitamin D3 is generated as an inactive form in the skin in response to sunlight and is converted to the active form, 1,25dihydroxyvitamin D3 (1,25(OH)2 D3 ), by an enzymatic cascade involving 25-hydroxylases and 1-hydroxylase. The D vitamins have many effects on immune cells. 1,25(OH)2 D3 can inhibit the differentiation and maturation of DCs (149–151). Vitamin D3 in combination with dexamethasone can also favor the induction of IL-10-producing cells with strong regulatory properties (152). Vitamin D3 also confers T cell tropism in the skin (153) and enhances the suppressive capacity of Foxp3+ T cells from the regional lymph node when delivered topically (154). Deﬁned microenvironments may have evolved self-containing strategies in which local mediators can imprint homing properties while also favoring the induction or function of Treg cells. Site-speciﬁc cells or factors such as neurons or hormones can also favor the induction of Foxp3+ Treg cells (155, 156). It is therefore tempting to speculate that a link between

homing and regulatory function induction may represent a more general mechanism. Such a strategy could allow the constant generation and migration of Treg cells to deﬁned compartments. These Treg cells are expected to have the prerequisite antigen speciﬁcities (e.g., persistent microorganisms, ﬂora antigens), status of activation, and survival requirement that allow them to regulate a deﬁned microenvironment.

Induction of Foxp3+ Treg Cells During Infections Acute infection of mice with Listeria monocytogenes failed to induce Foxp3 expression by conventional CD4+ T cells (157). Thus, highly inﬂammatory environments that prevail in acute infection may not favor the emergence of Foxp3+ T cells. This hypothesis is supported by the observation that Th1 or Th2 cell–polarizing cytokines can interfere with the induction of these cells (145). However, chronic infections may require an additional layer of regulation, which would be provided by converted Foxp3+ Treg cells. This hypothesis is supported by the observation that during infection, the downstream effects of inﬂammatory responses are also often associated with antiinﬂammatory processes, including TGF-β production. Furthermore, some pathogens target sites in which TGF-β is highly produced, such as the gastrointestinal tract, the skin, and the eye. TGF-β can also be produced by infected cells or by cells with which the microorganisms are in contact, or TGF-β can arise as a result of an inﬂammatory process. One example of the latter is the trypomastigote stage of Trypanosoma cruzi–induced TGF-β and IL-10 secretion by DCs (158). Compelling data in a mouse model of malaria suggest that TGF-β and Treg cells are central regulators of immunopathology and parasite expansion (159). During late infection with Plasmodium yoelii, DCs migrate to the spleen of infected mice and secrete TGFβ, together with IL-10 and prostaglandin E2 (PGE2) (160). After experimental malaria infection of human volunteers, enhanced TGFβ and Foxp3+ Treg cell responses in PBMCs

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correlate with a faster parasitic growth rate (159). Cells with Treg cell characteristics are rapidly induced after blood stage infection and are associated with a decrease in proinﬂammatory cytokines and antigen-speciﬁc responses. Monocytes are a likely source of the early TGFβ production in this infection (159). The serum concentration of TGF-β also correlates with the frequencies of Treg cells in HBV-infected patients (66). Some nematodes can themselves express homologs of TGF-β (161). Compelling experimental data support the idea that Foxp3+ Treg cells can be induced during Heligmosomoides polygyrus infection (162). This pathway may not be limited to the gastrointestinal tract, as we found that the bacillus Calmette-Gu´erin (BCG) can induce new populations of Foxp3+ T cells in vivo that accumulate at the dermal site of infection (R. Blank and Y. Belkaid, unpublished observation). Another means by which microorganisms could lead to Treg cell conversion is associated with the expected enhanced apoptosis at the site of infection. Previous work demonstrated that CD3 antibody treatment transiently depletes large numbers of T cells and subsequently induces long-term immune tolerance (127). Clearance of apoptotic bodies leads to the development of Treg cells (163). A recent study provides evidence that the mechanism underlying this regulatory effect is an enhanced production of TGF-β by macrophages and immature DCs after engulfment of apoptotic T cells (164). This increase in TGF-β induces the development of Treg cells and contributes to immune tolerance (164). Experimental data suggest that Treg cell conversion may occur in the context of chronic infections. Treg cells control friend virus (FV) infection (165, 166), during which a large fraction of myeloid DCs are infected with the virus. These infected DCs have a defect in maturation and establish prorogated contact with naive T cells compared with noninfected DCs (166). The result of this interaction is the generation of a population of Foxp3+ Treg cells from naive T cells (167). Monocytes exposed to Mycobacterium tuberculosis can induce Foxp3

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Treg cells in a PGE2-dependent manner on CD25-negative T cells from healthy tuberculin reactors (37). A single microbial product, mannose-capped lipoarabinomannan (ManLam), previously shown to induce IL-10 and inhibit IL-12 production by DCs, can reproduce this effect (168). HIV infection of T cells in vitro enhances Foxp3 expression by T cells following TCR stimulation in a manner partially dependent on TGF-β (169). HIV infection also enhances Foxp3 expression following addition of exogenous TGF-β (169). APCs may not be the only cell able to direct such a phenotype. Indeed, during infection with Helicobacter pylori, gastric epithelial cells upregulate the PD1 ligand B7-H1, which favors the development of Foxp3-expressing Treg cells (170). Several reports suggest that the activation status of DCs and inﬂammatory mediators modulate the capacity of these cells to induce Treg cells de novo (117, 171–175). Thus, activated DCs are poorer inducers of Foxp3+ Treg cells than of effector responses. This contrasts with the observation that activated DCs are more efﬁcient at inducing the proliferation of Treg cells than are immature DCs (176, 177). This could suggest that in some deﬁned situations Treg cells play a sequential and complementary role. For instance, the endogenous population may preferentially control highly inﬂammatory settings, whereas converted Treg cells may play a more important role downstream of the inﬂammatory response. The relative contributions of induced Treg cells to peripheral tolerance and to the outcome of infections, as well as how pathogens use or interfere with this pathway to favor their own survival, need further exploration. Currently, in the absence of deﬁnitive markers to distinguish endogenous versus converted Foxp3+ Treg cells, these questions remain difﬁcult to answer. However, APC manipulation by microorganisms together with the induction of factors that induce Treg cells support the idea that the induction of Treg cells may be a dominant pathway in the context of chronic infections.

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Antigen Specificities of Regulatory T Cells Although the antigen speciﬁcity of inducible regulatory T cells is associated with microbial antigens, the nature of the antigens recognized by Foxp3+ Treg cells is less obvious. Treg cells are believed to recognize a wide array of selfantigens as a consequence of their development and selection in the thymus (178). During the onset of acute disease, Treg cells could recognize self-antigens that are released by tissue damage; however, during chronic infection Treg cells can recognize microbial antigens (17, 21, 42, 45, 68, 87, 179, 180). In a murine model of Leishmania infection, Treg cells that accumulate at the site of infection are able to recognize parasite-derived antigen (181). In addition, far from being anergic, as in vitro experiments suggested, Treg cells proliferate vigorously when they encounter their cognate microbial antigens (181). Notably, these cells are restricted to the site of infection and are dependent on antigen for their maintenance (181). Such compartmentalization provides a potential explanation for the concept of concomitant immunity in which hosts are immune to reinfection in a secondary site while maintaining a local chronic infection (182). In a natural model of mouse mammary tumor virus infection, the virus caused early and progressive increases in superantigen-speciﬁc Foxp3+ Treg cells in Peyer’s patches if DCs were present (25). In the context of severe tissue damage and enhanced self-antigen presentation, Treg cells that control microorganism-speciﬁc immune responses may also be self-reactive. Because of the recent understanding that some sites or infections can generate new populations of Foxp3+ Treg cells, the origin of the Foxp3+ Treg cells able to recognize microbial antigen will have to be redeﬁned.

MANIPULATION OF FOXP3+ Treg CELLS BY MICROORGANISMS Because Treg cells offer an opportunity for microorganisms to generate conditions favorable to their persistence, microorganisms may also

manipulate Treg induction and survival. For example, DCs infected with FV had prolonged contacts with naive Treg cells during antigen presentation and preferentially expanded endogenous Treg cells (167). In addition to the recognition, through their TCRs, of speciﬁc antigen, whether host or pathogen derived, Treg cells can also respond to microbial products independent of TCR signals. TLR signaling can directly or indirectly control Treg cells (reviewed in 183). Consistent with a direct role for TLRs, human Treg cells express TLR5 at levels comparable with those of APCs. Costimulation with the TLR5 ligand ﬂagellin increases their suppressive capacity and enhances Foxp3 expression (184). This feature could offer certain pathogens an opportunity to enhance immunosuppression. Whereas some interactions with TLRs may increase Treg cells’ suppressive capacity, others limit their function (185). TLR2 signaling temporally abrogates the suppressive phenotype of Treg cells and decreases Foxp3 expression (183, 186). A decrease in total numbers of Treg cells occurs in TLR2-deﬁcient mice, which could be explained by the fact that TLR2 agonists induce Treg cell proliferation (186, 187). An indirect role for TLRs is suggested by the observation that mature DCs are more efﬁcient at inducing the proliferation of transgenic Treg cells than are immature DCs (177). Therefore, microorganism-associated DC maturation, stimulation of TLRs or other pattern-recognition receptors, induction of cytokine production, and release of factors and antigens from pathogen-mediated tissue damage could all favor Treg cell activation and thereby support survival of the pathogen. DCs matured by pathogen-derived TLR ligands will often activate effector T cell populations; these T cells in turn produce cytokines such as IL-2 and IL-15 that are potent growth factors for Treg cells (188, 189). This is likely to occur at later time points in infections. One potential result of tissue damage is release of endogenous TLR ligands that can similarly promote Treg cell expansion. For example, self-DNA/RNA can activate TLR7 and TLR9 (190). Likewise,

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heat shock proteins and the extracellular matrix component hyaluronan can signal through TLR2 and/or TLR4. All these self proteins and nucleic acids are released from necrotic cells as a result of tissue damage. Depending on the particular TLR triggered, this could lead to enhancement or suppression of the Treg cell compartment. Interestingly, in nonobese diabetic (NOD) mice, a mouse model of autoimmune diabetes, infection or injection of TLR ligand– containing adjuvants, including complete Freund’s adjuvant and polyIC, paradoxically prevents the development of diabetes (191, 192). This may be a result of enhanced Treg cell function; in one study, the ability of a bacterial extract to prevent diabetes was TGF-β dependent (193). BCG vaccination also prevented diabetes and was associated with an increase in CD4 T cells with regulatory markers (194). Similar enhancements of Treg populations occur during some infections. Recent reports have suggested that HIV may provide a survival/proliferative signal to Treg cells (57). In an in vitro model, exposure of Treg cells to inactivated HIV induced increased numbers of Treg cells in an HIV gp120dependent manner (57). Increases in Treg cell numbers were not a result of increased resistance to apoptosis, suggesting that these cells may exhibit a survival advantage over effector T cells. This advantage could protect Treg cells from destruction in lymphoid sites where virus replication occurs at high levels such as in GALT. Another means by which HIV may protect itself while favoring regulatory responses is not to infect Treg cells. Some data suggest that the expression of Foxp3 itself may interfere with the suppression of HIV-1 promoter transcription, thereby limiting viral replication in Treg cells. This could selectively prevent cell death of Treg cells and potentially contribute to the general immunosuppression observed in HIV patients (195). This ﬁnding is supported by the observation that puriﬁed CD127− CD4+ lymphocytes, a subset that contains both Tregs and recently activated effector T cells, exhibited relatively lower levels of viral DNA in vivo than

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did CD127+ T cells (196). On the other hand, a recent report suggests that Foxp3 can directly enhance HIV-1 gene expression in human T cells (197). One mechanism by which microorganisms might manipulate Treg function is by the creation of an environment that favors their retention. The integrin αE β7 (also known as CD103), expression of which is positively modulated by TGF-β, favors Treg cell retention at sites of infection by the parasite Leishmania major (198). Of interest, exposure of T cells to parasite-infected DCs also enhances the expression of αE β7 integrin. In the same model of infection, CCR5 expression by Treg cells was required for their migration to the infected site (199). Furthermore, infection of APCs by the parasite favors the production of ligands for CCR5 by the APC (199), suggesting that the pathogen itself manipulates its environment to favor Treg cell recruitment and retention. Similarly, CCR5 is necessary for the migration of Treg cells at sites of pulmonary infection caused by the dimorphic fungus Paracoccidiodes brasiliensis (200). Notably, Treg cells that respond to parasitic antigen are restricted to the site of infection (181), whereas antigenspeciﬁc IFN-γ-producing effector T cells are found at distal sites. Furthermore, within the infected site, the percentage of Treg cells undergoing apoptosis was twice as high as it was for non-Treg cells. These results suggest that one mechanism by which the strong proliferative capacity of Treg cells that accumulate in infected sites is controlled in vivo is through their rapid cell death. Such a mechanism could allow for the compartmentalization of Treg cell function and prevent a general immunosuppression that would be associated with activated Treg cell dissemination.

CONTROL OF Treg CELLS BY GUT FLORA One of the dominant roles of Treg cells is to prevent exuberant responses against gut ﬂora (201). The adult human intestine harbors up to 1014 microorganisms (202). These microﬂora play a

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central role in the maintenance and control of host homeostasis. In addition to promoting development of the immune system and control of metabolic functions, intestinal microﬂora play a major protective role by displacing pathogens and enhancing barrier fortiﬁcation (203, 204). The intestinal tract is in intimate contact with the commensal microﬂora. Nevertheless, how commensals communicate with cells to ensure immune homeostasis is still unclear. New data suggest that some deﬁned components of the gut ﬂora can play a major role in intestinal homeostasis. In particular, gut ﬂora interactions with speciﬁc TLRs can protect against gut injury (205–209) or mediate oral tolerance against dietary antigens (210). Alteration of the structural integrity of TLR signaling components is often associated with profound clinical outcome and susceptibility to various infections or autoimmune disorders (211). During conditions of ﬂoral translocation, peripheral TLR9 signaling is a crucial mediator of polymicrobial sepsis (212). Moreover, in other conditions in which ﬂoral translocation occurs, during irradiation and HIV infection, for example, peripheral TLR4 signals enhance the activation status of both CD4+ and CD8+ T cells (213, 214). However, under most circumstances, the tissues of the gastrointestinal tract are constantly exposed to TLR ligands harbored by the commensal gut ﬂora (215). Tregs are crucial to the integrity of the gastrointestinal tract by preventing exuberant responses against the ﬂora (201). Recent evidence suggests that TLR signaling can impact Treg cell homeostasis (185). Treg cells themselves selectively express TLRs, including TLR2, 4, 5, and 8 (185). Interaction with some of these ligands, such as those binding TLR2, can favor Treg cell expansion both in vitro and in vivo (183, 186). Accordingly, in TLR2-deﬁcient mice Treg cell frequencies in both gut and secondary lymphoid tissues are decreased (183, 186). Mice deﬁcient in TLR9 display increased frequencies of Treg cells within intestinal

effector sites and reduced constitutive IL-17and IFN-γ-producing effector T cells (216). Complementing this, gfDNA–limited lamina propria DCs induced Treg cell conversion in vitro (216). Furthermore, Treg versus effector T cell disequilibrium in Tlr9−/− mice leads to impaired immune responses to oral infection with the pathogen Encephalitozoon cuniculi. Impaired intestinal immune responses were recapitulated in mice treated with antibiotics and were reversible after reconstitution with gut ﬂora DNA (216). Thus, some signals derived from the gut ﬂora act as adjuvants of immune responses for priming intestinal responses against oral pathogens via modulation of the equilibrium between Treg and effector T cells. The structure and composition of the gut ﬂora reﬂect natural selection at both the microbial and host levels. Modiﬁcation of gut ﬂora was shown in areas of inﬂamed gut in inﬂammatory bowel disease patients (217–223). Furthermore, the presence of certain bacteria can aggravate small intestinal immunopathology following oral infection (224). It is tempting to speculate that alteration of Treg homeostasis mediated by TLR signaling, either because of genetic polymorphism or because of changes in gut ﬂora composition, could also have consequences on development of gut inﬂammatory disorders. Indeed, gut ﬂora bacteria are not equal in their capacity to stimulate TLR9 and do so with various levels of efﬁciency that correlate with their frequency of cytosine-guanine dinucleotides (225). Thus, control of Treg ratio and effector T cell function in the gastrointestinal tract is likely to be differentially regulated by speciﬁc gut ﬂora species. An illustration of how the presence of deﬁned bacterial species can inﬂuence the outcome of an infection comes from the observation that mice fed Biﬁdobacterium infantis are protected from the pathogenic effect and translocation of salmonella. Activation of Treg cells by the probiotic microorganism contributed to this protective effect (226).

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CROSS TALK BETWEEN REGULATORY T CELL POPULATIONS

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The distinction between Treg cells and inducible regulatory T cells in vivo is not always clear, particularly in highly inﬂammatory settings. Moreover, regulatory T cells may be able to inﬂuence the emergence or function of one another. This notion was recently suggested in a model of Aspergillus conidia infection in mice (227). In this model, control of allergic immunopathology induced by the fungus required the sequential activity of various populations of regulatory T cells: (a) Early in infection, inﬂammation is controlled by the expansion and local recruitment of Treg cells that can limit innate immune responses through the combined action of IL-10 and CTLA4, which induces the production of IDO by APCs. (b) Subsequently, this control of innate responses, particularly of DCs, leads to the activation and expansion of Tr1 cells that produce both IL-10 and TGF-β. (c) In turn, Tr1 cells can inhibit Th2 cells, which are responsible for the allergic response to the fungus (227). This sequential role for various populations of regulatory T cells may not be an exception but rather the rule, as most infections proceed through various stages and therefore require various layers of regulation.

BYSTANDER EFFECT OF REGULATORY T CELLS INDUCED DURING INFECTIONS In the Context of Coinfections Following activation, Treg cells can suppress unrelated immune responses in a nonantigenspeciﬁc manner either through cell contact or through the regulatory cytokines they produce—a mechanism known as bystander suppression (reviewed in 10). Recent evidence supports the idea that infection-induced Treg cells can play a major role in the outcome of secondary infections, as well as in autoimmune or allergic responses. Some parasitic infections, such as schistosoma infection in humans, can 568

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generate a strongly polarized Th2 cell response, which in turn can negatively modulate Th1 cell responses to unrelated antigens, thereby diminishing the strength of immune responses against secondary infections. Protection against Plasmodium falciparum is associated with the production of IgG1 and IgG3, which is dependent on the provision of help to B cells by Th1 cells. Thus, a highly Th2 cell–polarized environment may account for the increased susceptibility to malaria of individuals coinfected with Schistosoma mansoni compared with noninfected individuals (228). Similarly, prior infection with S. mansoni or exposure to nonviable S. mansoni reduces both the incidence and severity of experimental autoimmune encephalomyelitis (229), as well as the development of insulitis in NOD mice (230) and the induction of colitis by trinibenzesulphonic acid (231), which are all regarded as Th1 cell–associated diseases. Although some of these observations could be the consequence of crossregulation between Th2 cell and Th1 cell responses, experimental and clinical evidence supports the idea that activated Treg cells induced by the prior infection also contribute to this control (232). In a cohort of patients with multiple sclerosis, helminth infections are associated with signiﬁcantly fewer disease exacerbations (relapses) compared with uninfected patients with multiple sclerosis (233). Infection in these patients also correlated with the emergence of myelin-speciﬁc Treg cells producing IL-10 and TGF-β (233). However, these results have to be conﬁrmed in longitudinal studies to determine whether the occurrence of helminth infection directly correlates with the amelioration of symptoms of multiple sclerosis.

Hygiene Hypothesis The hygiene hypothesis concept states that increasing rates of allergy and asthma in Western countries could be a consequence of reduced infectious stresses during early childhood (234). The mechanistic explanations appear to be associated with a “counterregulatory” model involving the induction of various Treg cell

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populations during infection. Experimental work has lent strong support for this hypothesis (235). For example, during gastrointestinal infection, helminth-driven Treg cell suppression of effector function protects against subsequent airway inﬂammation (236). Part of this mechanism has likely evolved as a result of our symbiotic relationship with gut ﬂora. Interestingly, probiotic microorganisms have beneﬁcial effects in the treatment of inﬂammatory bowel diseases through the induction of Treg cell populations (237). Thus, the presence of symbiotic and pathogenic microorganisms in the gut or other peripheral tissues could lead to the maintenance of a pool of activated Treg cells (both natural and inducible) that would maintain host immune homeostasis and enhance the threshold required for immune activation and induction of an immune response (235). The beneﬁt of such deactivation is to decrease the instances of aberrant immune responses, such as allergic and autoimmune disorders. Pathogenic microorganisms may also have evolved to express antigens that crossreact with gut ﬂora antigens. In infections, the removal or modiﬁcation of the gut ﬂora is associated with a modiﬁcation of the phenotype of the host responses (238, 239). So some microorganisms may hijack Treg cells that are induced or activated in the gut to limit pathogenic responses against gut ﬂora to ensure their own survival.

In the Context of Aging Declines in immune function are well described in the elderly. In particular, declines in T cell functions appear to be associated with an increased risk and severity of infection, impaired response to vaccination, and poorer control of cancer. Reactivation or activation of persistent infections occurs with increased frequency in the elderly. Several reports have shown an accumulation of cells with regulatory functions in older mice compared with younger ones (240, 241). Such accumulation of Treg cells correlates with poor responses against tumors (242) and could also lead to disease reactivation. Indeed, in a model of Leishmania infection, Treg

cell accumulation at the primary site of infection eventually leads to disease reactivation in elderly mice (243).

In the Context of Tumors In the gastric mucosa of patients with Helicobacter pylori–induced gastric adenocarcinoma, a higher number of Treg cells could be detected compared with the number in tumorfree patients (244). Interestingly, Treg cells puriﬁed from the gastric tumor could suppress H. pylori–speciﬁc effector cell responses in vitro. So the presence of functional antigen-speciﬁc Treg cells could contribute to bacterial persistence and potentially to gastric tumor progression by suppressing both antibacterial and antitumoral responses through bystander suppression. During chronic infection with Leishmania major, Treg cells that accumulate at the site of infection favor the growth of the B16 melanoma by limiting local antitumoral responses (G. Pothiawala and Y. Belkaid, unpublished observations). The continued presence of Treg cells at sites of infection can upset the homeostasis of the infected organ and cause local immunosuppression, potentially leading to disease reactivation or tumor development. We are just beginning to grasp the importance of counterregulation induced by the infectious process. As discussed below, these concepts have provided the basis for new therapeutic approaches in which microbial molecules could be used to induce Treg cells to control allergic and autoimmune diseases.

TARGETING Treg CELLS TO CONTROL INFECTIONS OR THEIR PATHOLOGICAL CONSEQUENCES In some circumstances, the regulation exerted by Treg cells is excessive, preventing the establishment of protective immune responses, whereas in other circumstances, this control is not sufﬁcient to prevent immunopathology. At both extremes, manipulation of Treg cells offers therapeutic potential.

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Targeting Treg Cells to Control Infections

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The capacity of a host to mount an effective immune response is limited by the preexistence of counterregulatory elements. When Treg cells are activated in the context of an infection, many immune cells will be affected, both at the site of infection and in the draining lymph node. DCs are one important cell type that is downregulated by Treg cells via several mechanisms. Treg cells can more readily form aggregates with DCs compared with other CD4+ (CD25− ) T cells (8, 245). This aggregation has two main consequences: DCs are prevented from making as many synapses with effector T cells (246), and Treg cells can license the DCs, changing their phenotype. Treg cells downregulate costimulatory molecules, cytokine production, and antigen-presenting functions of DCs (247, 248). In a variety of experimental systems, CD80 and CD86 expression, IL-6 expression, and the ability to stimulate T cells are all downmodulated on DCs by Treg cells (249, 250). Cytokines secreted by Treg cells also alter DCs. Treg cells can sometimes produce TGF-β or IL-10, both of which can impart a tolerogenic phenotype on DCs. Together, these changes that Treg cells inﬂict on DCs can result in an inability of DCs to fully induce effector T cells, which soon after an infection could prevent an adequate adaptive immune response to the pathogen. Targeting the molecules involved in Treg cell activity in vivo, such as CTLA4, TGF-β, or IL-10, alone or in combination, has proven effective in controlling many chronic infections (reviewed in 251). Many mechanisms that boost immune responses and favor the control of pathogens also abrogate Treg cell functions, mainly by rendering effector T cells unresponsive to Treg cell suppression (251). Targeting GITR in vivo has had a beneﬁcial outcome in infection models (29, 252). Although the target of such treatment has not been identiﬁed, the primary mechanism might be associated with enhanced effector responses (253). Blockade of other molecules highly expressed on Treg cells,

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such as the folate receptor FR4, to enhance immune responses against pathogens remains to be addressed (7). Recent ﬁndings suggesting a role for adenosine (6) and cAMP (13) in Treg cell suppressive function potentially offer a new means to limit their function. Like naive and effector T cells, Treg cells proliferate and their suppressive functions are boosted by encounters with activating signals, such as activated APCs and some microbial products (such as ﬂagellin) (177, 184). Strategies to manipulate Treg cell function or number clearly have high therapeutic potential. In many infections in mice and humans, depletion of Treg cells (using CD25-speciﬁc antibodies) has resulted in enhanced effector immune responses (42, 68, 182, 254) (Table 1). However, complete ablation of Foxp3 in adult mice leads to the development of autoimmunity (79), suggesting that systemic strategies to target Treg cells may not be applicable in humans as they may run the risk of triggering autoimmune disorders or uncontrolled pathological immune responses. The identiﬁcation of molecules favoring tissuespeciﬁc migration of Treg cells such as CCR4 (255) or unique factors favoring their generation (such as retinoic acid) or survival could allow investigators to devise targeted manipulation of their functions and minimize such risk.

Manipulation of Treg Cells to Establish Protective Memory Responses To date, no vaccines are available against many life-threatening diseases such as malaria, tuberculosis, or HIV. The failure of traditional approaches and the growing understanding that most pathogens thrive in the presence of regulatory responses support the idea that efﬁcient protective immune responses have to be initiated under conditions that prevent the initiation of regulatory responses. Treg cells can control the intensity of secondary responses to infections. In a model of HCV infection in chimpanzees, Treg cells control HCV-speciﬁc effector T cells not only during chronic infection but also after recovery

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(256). Likewise, these cells can hamper the efﬁcacy of vaccines against infectious agents. In studies using a vaccine against Listeria monocytogenes, Treg cells restricted the magnitude of pathogen-speciﬁc CD8+ T cell responses upon secondary challenge with the bacterium or the vaccine (257). Similarly, control of the number of Treg cells prior to DNA vaccination against HSV1 or HBV had an adjuvant effect on the quality and the intensity of the effector responses in both acute and memory stages (258, 259). In a mouse model of vaccination against malaria, depletion of Treg cells during vaccination results in a more durable immunity and better control of parasite burden after challenge compared with vaccine alone (260). Interestingly, such depletion also leads to enhanced T cell responses to subdominant epitopes (260). Treg cell depletion also signiﬁcantly increases CD8+ T cell responses following exposure to inﬂuenza A virus and vaccinia virus (261). In this study, Treg cells selectively suppresses responses to the most immunodominant CD8+ T cell epitopes, therefore inﬂuencing immunodominance hierarchies (261). This point may be particularly important for vaccines against parasitic infections, in which responses to only a few, if any, dominant antigens can be detected. The importance of preventing the induction of regulatory responses during vaccination has been highlighted by recent ﬁndings. Conventional antigen-speciﬁc T cells converted into Treg cells in the periphery under subimmunogenic conditions can be subsequently expanded by the delivery of antigen under immunogenic conditions (119). So, if not done in optimal conditions, vaccination itself can generate its own set of Treg cells. In a mouse model of vaccination against T. gondii, IL-10 produced by CD4+ T cells that were reactivated following secondary challenge is controlled by IFNγ. This production of IL-10 upon secondary exposure to the parasite interferes with the efﬁciency of vaccination and leads to the death of the animal (262). Previous reports clearly show that vaccination with the Leishmania antigen LACK (Leishmania analog of the receptors of

activated C kinase), when used with adjuvant, protects mice against rechallenge (263). Surprisingly, vaccination of mice with LACK antigen without adjuvant can favor the emergence of IL-10-producing Treg cells (264). The presence of these cells predicts vaccination failure. Removal of CD25+ cells abrogated IL-10 production and restores protection by the vaccine (264). After standard hepatitis B surface antigen vaccine immunization, nonresponders demonstrate an increase in antigen-speciﬁc Foxp3+ Treg cells in the blood compared with responders (265). Thus, investigators must address the potential of each microbial antigen to trigger Treg cells following vaccination and also deﬁne adjuvants that prevent Treg cell priming or activation. Another approach to promote protective immune responses in the face of counterregulation is to select a vaccination site where Treg cells are not overrepresented. For example, the skin (dermis) contains the highest percentage of Treg cells in the body (Y. Belkaid, unpublished observations). Thus, in infection with Leishmania major, the site of primary exposure to the pathogen (dermal versus subcutaneous) conditions the efﬁciency of control of secondary infection at a distal site (266). Although preventing Treg cell induction or function as a vaccination strategy may favor the establishment of protective immunity, we need to consider the possibility that in some situations secondary responses also contribute to immunopathology. For example, in a model of vaccination against Borrelia burgdorferi, destructive osteoarthropathy ensues after bacterial challenge (267), a model that may address the mechanism underlying lyme arthritis in humans. In this particular case, the presence of Treg cells prevented the development of arthritis. Thus, the presence of regulatory elements upon secondary exposure to the antigen may prevent exuberant responses in the context of vaccination. Finally, the efﬁciency of protective responses either induced by vaccination or in response to infection can be conditioned by the preexistence of regulatory responses in

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the host. Murine CD4+ T cells speciﬁc for the Leishmania antigen LACK are present in naive mice (268). Importantly, LACK antigen crossreacts with antigens present in the gut ﬂora (268). Thus, regulatory responses prior to pathogen exposure may exist in the host as a consequence of crossreactivity with gut ﬂora antigen. Chronic exposure to a low dose of antigens derived from pathogen could also favor the emergence of a regulatory population that could limit subsequent immune responses. For instance, after BCG injection, Treg cells are recruited to the draining lymph nodes and negatively control antimycobacterial effector responses (269, 270). Treatment of BCGimmunized mice with an anti-CD25 monoclonal antibody induced an increased IFN-γ response against both subdominant and immunodominant regions of the protective immunogen TB10.4 and, following challenge with Mycobacterium tuberculosis, decreased the lung mycobacterial load signiﬁcantly, albeit moderately, compared with the control mice (271).

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Blocking Treg Cell Conversion to Favor Protective Immune Responses Several reports suggest that the status of activation of DCs as well as of inﬂammatory mediators and effector cytokines modulates the capacity of these cells to induce Treg cells de novo (117, 171–175). For example, IL-6 and TGFβ in tandem can direct the production of IL17-secreting T cells (Th17 cell) over Treg cells (117, 171), and Th1 and Th2 cell effector cytokines have an antagonistic effect on Treg cell conversion (145). Using an APC-free system in vitro, investigators have suggested that strong costimulation provided by extensive CD28 signaling can inhibit Foxp3 induction (123). In vivo, efﬁcient induction of Foxp3+ Treg cells is also abolished in the presence of strong costimulation (119). DCs deﬁcient in CD80 and CD86 induce higher expression of Foxp3 on naive T cells than do control DCs (123). Furthermore, activating ex vivo spleen DCs with an agonist anti-CD40 or LPS impaired induction of Foxp3+ Treg cells (272). This point high572

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lights the need to devise adjuvants with the objective of limiting the induction of Treg cells. The challenge is to deﬁne the site-speciﬁc elements responsible for this control. Regulation is often the consequence of multiple complementary strategies, and in a chronic situation many cell types can acquire regulatory properties. Targeting Treg cells may be important to enhance control of the infection, but this approach should be done in conjunction with the neutralization of other factors, such as IL10, which is often produced by other cells. For instance, during murine ﬁlaria infection with Litomosoides sigmodontis, efﬁcient killing of the adult parasite requires depletion of Treg cells as well as CTLA4 blockade on non-Treg T cells (273). Alternatively, in the context of some infections, focusing on simultaneously enhancing the effector immune responses to clear the pathogen may be more important.

Manipulating Treg Cells to Minimize Immunopathology Much of the tissue damage that occurs during an infection results not directly from the pathogen but rather from the immune response to the infection. Therefore, homeostatic mechanisms that dampen inﬂammation after the initial response to the pathogen can be important for preventing tissue damage, and Treg cells normally contribute to this process. It may be possible to manipulate Treg cells to enhance control of immunopathology during infection. The balance between allowing the immune response necessary to clear pathogens and minimizing immunopathology is delicate and may be speciﬁc to each pathogen. Any treatment to enhance Treg cells in the context of infection would likely need to be transient and need to be compatible with the establishment of pathogenspeciﬁc immunity. In a mouse model of colitis, the transfer of Treg cells was sufﬁcient to control established inﬂammatory disease (274). Increasing Treg cell function or number could potentially be achieved by providing a cytokine milieu that favors Treg cell activity or survival, such as

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IL-2 or TGF-β. Enhancing the number or function of Foxp3+ cells can also be achieved in vivo by retroviral transfer of Foxp3 (275). In a mouse model of Schistosome infection, such an approach at the onset of granuloma formation enhanced Foxp3 expression in the granuloma and strongly suppressed granuloma development (275). Several methods that have been studied in the context of autoimmunity could be used to enrich Treg cells at later stages of infection. Some involve injecting agents that expand Treg cells in vivo. For example, IL-2 is an important growth factor for Treg cells (188, 276). Recent studies show that IL-2 complexed with certain antibodies speciﬁc for IL-2 can potently expand Treg cells (277). OKT3, an antibody speciﬁc for CD3, has shown some efﬁcacy for treatment of type 1 diabetes in humans, and some evidence indicates that it induces Treg cells, although they appear to be CD8+ cells rather than the typical CD4+ cells (278). In mouse models, induction of regulatory cells after anti-CD3 treatment has also been observed (279). Anti-CD3 can also induce fever and short-term release of some proinﬂammatory cytokines such as IL6 and TNF-α (280), but this may be beneﬁcial for short-term treatment in the context of infection. Anti-CD3 treatment has also been successfully used in the context of chronic viral infection (282). In vivo administration of CD3 F(ab )2 during HSV1 infection in mice resulted in signiﬁcant reduction in the severity and incidence of the disease. Following treatment, fewer pathogenic CD4+ T cells inﬁltrated the cornea, and there was a lower percentage of cells that could be induced to express IFN-γ. Similar observations were noted in the secondary lymphoid tissues. Additionally, an increase in the frequency of Treg cells was noticed in both the cornea and lymphoid tissues of treated animals compared with the untreated animals. However, use of this reagent was moderately effective in limiting established lesions, suggesting that complementary approaches should be used in more advanced stage of infection (282).

Treg cells could also be expanded or differentiated ex vivo via a variety of stimuli, including DCs, anti-CD3, or cytokines such as TGF-β or IL-10. For example, puriﬁed CD4+ CD25+ T cells proliferate extensively in response to either DCs or anti-CD3 (177, 283). Importantly, DCs can be used both to maintain TCR expression and to impart antigen speciﬁcity (176). Mature DCs expressing high levels of costimulation are more efﬁcient at expanding Treg cells than are immature DCs (176, 177). Cytokine-matured human DCs can expand Foxp3+ Treg cells, even in vivo (284). The demonstration that TCR ligation in the presence of TGF-β can lead to the generation of functional Foxp3+ Treg cells in vitro and in vivo offers great therapeutic potential. We still need to evaluate the relative stability of these converted cells, as the reversion to an effector phenotype against the target antigen could have severe consequences in vivo. Furthermore, enhancing Treg cell numbers or functions in vivo can lead to the reactivation of dormant infections (182) or the suppression of antitumoral responses. Nevertheless, this strategy has been successfully used in the treatment of immunopathology consequent to infection. In vitro–generated antigen-speciﬁc Foxp3+ Treg cells control the severity of HSV-induced ocular immunoinﬂammatory lesions (285). One promising therapeutic approach has emerged from the observation that microbial products can favor the induction of Tr1 cell populations in vivo. IL-10-producing Tr1 can be induced in vitro by DCs stimulated with phosphatidylserine isolated from Schistosoma mansoni (286). Exposure of mice to S. mansoni antigen also prevents development of type 1 diabetes in NOD mice (230), as well as experimental colitis (231). Single microbial molecules can be used as therapeutic agents, as ﬁlamentous hemagglutinin of Bordetella pertussis can efﬁciently treat experimental colitis (287). Similarly, the single microbial product ManLam, derived from Mycobacterium tuberculosis, can induce Foxp3+ Treg cells (168). Maintaining peripheral homeostasis in the face of infection requires not only efﬁcient

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control of immune responses but also the establishment of an immune response ﬁt for the invaded site. Given the wide spectrum of hostpathogen interactions in terms of microorganisms, virulence, duration or target tissue, the roles exerted by Treg cells during infection are expected to be diverse and adaptable. Although

Treg cell populations have taken center stage over the past few years, virtually all cell populations can acquire regulatory properties. The challenge of the next few years will be to decipher the relative dependency and contribution of all these cells to the regulatory responses against infections.

DISCLOSURE STATEMENT

Annu. Rev. Immunol. 2009.27:551-589. Downloaded from arjournals.annualreviews.org by Karolinska Institutet on 03/29/09. For personal use only.

The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

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256. Manigold T, Shin EC, Mizukoshi E, Mihalik K, Murthy KK, et al. 2006. Foxp3+ CD4+ CD25+ T cells control virus-speciﬁc memory T cells in chimpanzees that recovered from hepatitis C. Blood 107:4424–32 257. Kursar M, Bonhagen K, Fensterle J, Kohler A, Hurwitz R, et al. 2002. Regulatory CD4+ CD25+ T cells restrict memory CD8+ T cell responses. J. Exp. Med. 196:1585–92 258. Toka F, Suvas S, Rouse BT. 2004. CD4+ /CD25+ T cells regulate vaccine generated primary and memory CD8+ T cell responses against Herpes Simplex virus. J. Virol. 78:13082–89 259. Furuichi Y, Tokuyama H, Ueha S, Kurachi M, Moriyasu F, Kakimi K. 2005. Depletion of CD25+ CD4+ T cells (Tregs) enhances the HBV-speciﬁc CD8+ T cell response primed by DNA immunization. World J. Gastroenterol. 11:3772–77 260. Moore AC, Gallimore A, Draper SJ, Watkins KR, Gilbert SC, Hill AV. 2005. Anti-CD25 Antibody enhancement of vaccine-induced immunogenicity: increased durable cellular immunity with reduced immunodominance. J. Immunol. 175:7264–73 261. Haeryfar SM, DiPaolo RJ, Tscharke DC, Bennink JR, Yewdell JW. 2005. Regulatory T cells suppress CD8+ T cell responses induced by direct priming and cross-priming and moderate immunodominance disparities. J. Immunol. 174:3344–51 262. Shaw MH, Freeman GJ, Scott MF, Fox BA, Bzik DJ, et al. 2006. Tyk2 negatively regulates adaptive Th1 immunity by mediating IL-10 signaling and promoting IFN-γ-dependent IL-10 reactivation. J. Immunol. 176:7263–71 263. Gurunathan S, Sacks DL, Brown DR, Reiner SL, Charest H, et al. 1997. Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major. J. Exp. Med. 186:1137–47 264. Stober CB, Lange UG, Roberts MT, Alcami A, Blackwell JM. 2005. IL-10 from regulatory T cells determines vaccine efﬁcacy in murine Leishmania major infection. J. Immunol. 175:2517–24 265. Yang Z, Wang L, Niu W, Wu Y, Zhang J, Meng G. 2008. Increased CD4+ CD25+ FoxP3+ regulatory T cells in the blood of nonresponders after standard hepatitis B surface antigen vaccine immunization. Clin. Immunol. 127:265–66 266. Tabbara KS, Peters NC, Afrin F, Mendez S, Bertholet S, et al. 2005. Conditions inﬂuencing the efﬁcacy of vaccination with live organisms against Leishmania major infection. Infect. Immun. 73:4714–22 267. Nardelli DT, Burchill MA, England DM, Torrealba J, Callister SM, Schell RF. 2004. Association of CD4+ CD25+ T cells with prevention of severe destructive arthritis in Borrelia burgdorferi-vaccinated and challenged γ interferon-deﬁcient mice treated with anti-interleukin-17 antibody. Clin. Diagn. Lab. Immunol. 11:1075–84 268. Julia V, McSorley SS, Malherbe L, Breittmayer JP, Girard-Pipau F, et al. 2000. Priming by microbial antigens from the intestinal ﬂora determines the ability of CD4+ T cells to rapidly secrete IL-4 in BALB/c mice infected with Leishmania major. J. Immunol. 165:5637–45 269. Jaron B, Maranghi E, Leclerc C, Majlessi L. 2008. Effect of attenuation of Treg during BCG immunization on anti-mycobacterial Th1 responses and protection against Mycobacterium tuberculosis. PLoS ONE 3:e2833 270. Quinn KM, Rich FJ, Goldsack LM, de Lisle GW, Buddle BM, et al. 2008. Accelerating the secondary immune response by inactivating CD4+ CD25+ T regulatory cells prior to BCG vaccination does not enhance protection against tuberculosis. Eur. J. Immunol. 38:695–705 271. Roy S, Barnes PF, Garg A, Wu S, Cosman D, Vankayalapati R. 2008. NK cells lyse T regulatory cells that expand in response to an intracellular pathogen. J. Immunol. 180:1729–36 272. Wang L, Pino-Lagos K, de Vries VC, Guleria I, Sayegh MH, Noelle RJ. 2008. Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+ CD4+ regulatory T cells. Proc. Natl. Acad. Sci. USA 105:9331–36 273. Taylor MD, Harris A, Babayan SA, Bain O, Culshaw A, et al. 2007. CTLA-4 and CD4+ CD25+ regulatory T cells inhibit protective immunity to ﬁlarial parasites in vivo. J. Immunol. 179:4626–34 274. Mottet C, Uhlig HH, Powrie F. 2003. Cutting edge: cure of colitis by CD4+ CD25+ regulatory T cells. J. Immunol. 170:3939–43 275. Singh KP, Gerard HC, Hudson AP, Reddy TR, Boros DL. 2005. Retroviral Foxp3 gene transfer ameliorates liver granuloma pathology in Schistosoma mansoni infected mice. Immunology 114:410–17

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276. Tang Q, Adams JY, Penaranda C, Melli K, Piaggio E, et al. 2008. Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity 28:687–97 277. Boyman O, Kovar M, Rubinstein MP, Surh CD, Sprent J. 2006. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311:1924–27 278. Ablamunits V, Herold KC. 2008. Generation and function of human regulatory CD8+ T cells induced by a humanized OKT3 monoclonal antibody hOKT3γ1(Ala-Ala). Hum. Immunol. 69:732–36 279. You S, Leforban B, Garcia C, Bach JF, Bluestone JA, Chatenoud L. 2007. Adaptive TGF-β-dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment. Proc. Natl. Acad. Sci. USA 104:6335–40 280. Herold KC, Hagopian W, Auger JA, Poumian-Ruiz E, Taylor L, et al. 2002. Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N. Engl. J. Med. 346:1692–98 281. Deleted in proof 282. Sarangi PP, Kim B, Rouse BT. 2008. Non-mitogenic anti-CD3 F(ab )2 monoclonal antibody: a novel approach to control herpetic stromal keratitis. Invest. Ophthalmol. Vis. Sci. In press 283. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, et al. 2004. In vitro-expanded antigen-speciﬁc regulatory T cells suppress autoimmune diabetes. J. Exp. Med. 199:1455–65 284. Banerjee DK, Dhodapkar MV, Matayeva E, Steinman RM, Dhodapkar KM. 2006. Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokinematured DCs in myeloma patients. Blood 108:2655–61 285. Sehrawat S, Suvas S, Sarangi PP, Suryawanshi A, Rouse BT. 2008. In vitro-generated antigen-speciﬁc CD4+ CD25+ Foxp3+ regulatory T cells control the severity of herpes simplex virus-induced ocular immunoinﬂammatory lesions. J. Virol. 82:6838–51 286. van der Kleij D, Latz E, Brouwers JF, Kruize YC, Schmitz M, et al. 2002. A novel host-parasite lipid crosstalk. Schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and affects immune polarization. J. Biol. Chem. 277:48122–29 287. Braat H, McGuirk P, Ten Kate FJ, Huibregtse I, Dunne P, et al. 2006. Prevention of experimental colitis by parenteral administration of a pathogen-derived immunomodulatory molecule. Gut 56:351–57 288. Taylor JJ, Mohrs M, Pearce EJ. 2006. Regulatory T cell responses develop in parallel to Th responses and control the magnitude and phenotype of the Th effector population. J. Immunol. 176:5839–47 289. Baumgart M, Tompkins F, Leng J, Hesse M. 2006. Naturally occurring CD4+ Foxp3+ regulatory T cells are an essential, IL-10-independent part of the immunoregulatory network in Schistosoma mansoni egg-induced inﬂammation. J. Immunol. 176:5374–87 290. Cai XP, Zhang H, Zhang YC, Wang Y, Su C, et al. 2006. Dynamics of CD4+ CD25+ T cells in spleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum. Acta Biochim. Biophys. Sin. 38:299–304 291. Liu H, Hu B, Xu D, Liew FY. 2003. CD4+ CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-β, and CTLA4. J. Immunol. 171:5012–17 292. Sufﬁa I, Reckling S, Salay G, Belkaid Y. 2005. A role for CD103 in Treg retention at site of Leishmania major infection. J. Immunol. 174:5444–55 293. Aseffa A, Gumy A, Launois P, MacDonald HR, Louis JA, Tacchini-Cottier F. 2002. The early IL4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+ CD25+ T cells. J. Immunol. 169:3232–41 294. Belkaid Y, Blank RB, Sufﬁa I. 2006. Natural regulatory T cells and parasites: a common quest for host homeostasis. Immunol. Rev. 212:287–300 295. Campanelli AP, Roselino AM, Cavassani KA, Pereira MS, Mortara RA, et al. 2006. CD4+ CD25+ T cells in skin lesions of patients with cutaneous leishmaniasis exhibit phenotypic and functional characteristics of natural regulatory T cells. J. Infect. Dis. 193:1313–22 296. Torcia MG, Santarlasci V, Cosmi L, Clemente A, Maggi L, et al. 2008. Functional deﬁcit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria. Proc. Natl. Acad. Sci. USA 105:646–51 297. Long TT, Nakazawa S, Onizuka S, Huaman MC, Kanbara H. 2003. Inﬂuence of CD4+ CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice. Int. J. Parasitol. 33:175–83 www.annualreviews.org • Treg Cells in Host-Microorganism Interactions

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298. Gillan V, Devaney E. 2005. Regulatory T cells modulate Th2 responses induced by Brugia pahangi third-stage larvae. Infect. Immun. 73:4034–42 299. Rausch S, Huehn J, Kirchhoff D, Rzepecka J, Schnoeller C, et al. 2008. Functional analysis of effector and regulatory T cells in a parasitic nematode infection. Infect. Immun. 76:1908–19 300. Zelinskyy G, Kraft AR, Schimmer S, Arndt T, Dittmer U. 2006. Kinetics of CD8+ effector T cell responses and induced CD4+ regulatory T cell responses during Friend retrovirus infection. Eur. J. Immunol. 36:2658–70 301. Beilharz MW, Sammels LM, Paun A, Shaw K, van Eeden P, et al. 2004. Timed ablation of regulatory CD4+ T cells can prevent murine AIDS progression. J. Immunol. 172:4917–25 302. Nesburn AB, Bettahi I, Dasgupta G, Chentouﬁ AA, Zhang X, et al. 2007. Functional Foxp3+ CD4+ CD25Bright+ “natural” regulatory T cells are abundant in rabbit conjunctiva and suppress virus-speciﬁc CD4+ and CD8+ effector T cells during ocular herpes infection. J. Virol. 81:7647–61 303. Diaz GA, Koelle DM. 2006. Human CD4+ CD25 high cells suppress proliferative memory lymphocyte responses to herpes simplex virus type 2. J. Virol. 80:8271–73 304. Fernandez MA, Puttur FK, Wang YM, Howden W, Alexander SI, Jones CA. 2008. T regulatory cells contribute to the attenuated primary CD8+ and CD4+ T cell responses to herpes simplex virus type 2 in neonatal mice. J. Immunol. 180:1556–64 ¨ 305. Epple HJ, Loddenkemper C, Kunkel D, Troger H, Maul J, et al. 2006. Mucosal but not peripheral FOXP3+ regulatory T cells are highly increased in untreated HIV infection and normalize after suppressive HAART. Blood 108(9):3072–78 306. Zhang Z, Jiang Y, Zhang M, Shi W, Liu J, et al. 2008. Relationship of frequency of CD4+ CD25+ Foxp3+ Regulatory T cells with disease progression in antiretroviral-naive HIV-1 infected chinese. Jpn. J. Infect. Dis. 61:391–92 307. Montes M, Lewis DE, Sanchez C, de Castilla DL, Graviss EA, et al. 2006. Foxp3+ regulatory T cells in antiretroviral-naive HIV patients. AIDS 20:1669–71 308. Hoji A, Coro A, Ng HL, Jamieson BD, Yang OO. 2008. Proliferation and foxp3 expression in virusspeciﬁc memory CD8+ T lymphocytes. AIDS Res. Hum. Retroviruses 24:1087–95 309. Lim A, Tan D, Price P, Kamarulzaman A, Tan HY, et al. 2007. Proportions of circulating T cells with a regulatory cell phenotype increase with HIV-associated immune activation and remain high on antiretroviral therapy. AIDS 21:1525–34 310. Li S, Jones KL, Woollard DJ, Dromey J, Paukovics G, et al. 2007. Deﬁning target antigens for CD25+ FOXP3+ IFN-γ− regulatory T cells in chronic hepatitis C virus infection. Immunol. Cell Biol. 85:197–204 311. Feng IC, Koay LB, Sheu MJ, Kuo HT, Sun CS, et al. 2007. HBcAg-speciﬁc CD4+ CD25+ regulatory T cells modulate immune tolerance and acute exacerbation on the natural history of chronic hepatitis B virus infection. J. Biomed. Sci. 14:43–57 312. Yamano Y, Takenouchi N, Li HC, Tomaru U, Yao K, et al. 2005. Virus-induced dysfunction of CD4+ CD25+ T cells in patients with HTLV-I-associated neuroimmunological disease. J. Clin. Invest. 115:1361–68 313. Oh U, Grant C, Grifﬁth C, Fugo K, Takenouchi N, Jacobson S. 2006. Reduced Foxp3 protein expression is associated with inﬂammatory disease during human t lymphotropic virus type 1 Infection. J. Infect. Dis. 193:1557–66 314. Walsh PT, Benoit BM, Wysocka M, Dalton NM, Turka LA, Rook AH. 2006. A role for regulatory T cells in cutaneous T-Cell lymphoma; induction of a CD4+ CD25+ Foxp3+ T-cell phenotype associated with HTLV-1 infection. J. Invest. Dermatol. 126:690–92 315. Molling JW, de Gruijl TD, Glim J, Moreno M, Rozendaal L, et al. 2007. CD4+ CD25hi regulatory Tcell frequency correlates with persistence of human papillomavirus type 16 and T helper cell responses in patients with cervical intraepithelial neoplasia. Int. J. Cancer 121:1749–55 316. Estes JD, Li Q, Reynolds MR, Wietgrefe S, Duan L, et al. 2006. Premature induction of an immunosuppressive regulatory T cell response during acute simian immunodeﬁciency virus infection. J. Infect. Dis. 193:703–12

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317. Pereira LE, Villinger F, Onlamoon N, Bryan P, Cardona A, et al. 2007. Simian immunodeﬁciency virus (SIV) infection inﬂuences the level and function of regulatory T cells in SIV-infected rhesus macaques but not SIV-infected sooty mangabeys. J. Virol. 81:4445–56 318. Karlsson I, Malleret B, Brochard P, Delache B, Calvo J, et al. 2007. FoxP3+ CD25+ CD8+ T-cell induction during primary simian immunodeﬁciency virus infection in cynomolgus macaques correlates with low CD4+ T-cell activation and high viral load. J. Virol. 81:13444–55 319. Hartigan-O’Connor DJ, Abel K, McCune JM. 2007. Suppression of SIV-speciﬁc CD4+ T cells by infant but not adult macaque regulatory T cells: implications for SIV disease progression. J. Exp. Med. 204:2679–92 320. Qin S, Sui Y, Soloff AC, Junecko BA, Kirschner DE, et al. 2008. Chemokine and cytokine mediated loss of regulatory T cells in lymph nodes during pathogenic simian immunodeﬁciency virus infection. J. Immunol. 180:5530–36 321. Mexas AM, Fogle JE, Tompkins WA, Tompkins MB. 2008. CD4+ CD25+ regulatory T cells are infected and activated during acute FIV infection. Vet. Immunol. Immunopathol. 126:263–72 322. Cavassani KA, Campanelli AP, Moreira AP, Vancim JO, Vitali LH, et al. 2006. Systemic and local characterization of regulatory T cells in a chronic fungal infection in humans. J. Immunol. 177:5811–18 323. Kandulski A, Wex T, Kuester D, Peitz U, Gebert I, et al. 2008. Naturally occurring regulatory T cells (CD4+ , CD25high , FOXP3+ ) in the antrum and cardia are associated with higher H. pylori colonization and increased gene expression of TGF-β1. Helicobacter 13:295–303 324. Lundgren A, Suri-Payer E, Enarsson K, Svennerholm AM, Lundin BS. 2003. Helicobacter pylori-speciﬁc CD4+ CD25high regulatory T cells suppress memory T-cell responses to H. pylori in infected individuals. Infect. Immun. 71:1755–62 325. Raghavan S, Suri-Payer E, Holmgren J. 2004. Antigen-speciﬁc in vitro suppression of murine Helicobacter pylori-reactive immunopathological T cells by CD4CD25 regulatory T cells. Scand J. Immunol. 60:82–88 326. Harris PR, Wright SW, Serrano C, Riera F, Duarte I, et al. 2008. Helicobacter pylori gastritis in children is associated with a regulatory T-cell response. Gastroenterology 134:491–99 327. Lundgren A, Stromberg E, Sjoling A, Lindholm C, Enarsson K, et al. 2005. Mucosal FOXP3-expressing CD4+ CD25high regulatory T cells in Helicobacter pylori-infected patients. Infect. Immun. 73:523–31 328. Kaparakis M, Laurie KL, Wijburg O, Pedersen J, Pearse M, et al. 2006. CD4+ CD25+ regulatory T cells modulate the T-cell and antibody responses in helicobacter-infected BALB/c mice. Infect. Immun. 74:3519–29 329. Quinn KM, McHugh RS, Rich FJ, Goldsack LM, de Lisle GW, et al. 2006. Inactivation of CD4+ CD25+ regulatory T cells during early mycobacterial infection increases cytokine production but does not affect pathogen load. Immunol. Cell Biol. 84:467–74 330. Roberts T, Beyers N, Aguirre A, Walzl G. 2007. Immunosuppression during active tuberculosis is characterized by decreased interferon- γ production and CD25 expression with elevated forkhead box P3, transforming growth factor-β, and interleukin-4 mRNA levels. J. Infect. Dis. 195:870–78 331. Hougardy JM, Place S, Hildebrand M, Drowart A, Debrie AS, et al. 2007. Regulatory T cells depress immune responses to protective antigens in active tuberculosis. Am. J. Respir. Crit. Care Med. 176:409–16 332. Chen X, Zhou B, Li M, Deng Q, Wu X, et al. 2007. CD4+ CD25+ FoxP3+ regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease. Clin. Immunol. 123:50–59 333. Faal N, Bailey RL, Jeffries D, Joof H, Sarr I, et al. 2006. Conjunctival FOXP3 expression in trachoma: Do regulatory T cells have a role in human ocular Chlamydia trachomatis infection? PLoS Med. 3:e266

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T Cell Activation Jennifer E. Smith-Garvin,1 Gary A. Koretzky,1,2,3 and Martha S. Jordan1,2 1

Abramson Family Cancer Research Institute, 2 Department of Pathology and Laboratory Medicine, 3 Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; email: [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2009. 27:591–619

Key Words

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

signal transduction, immunoreceptor, integrin

This article’s doi: 10.1146/annurev.immunol.021908.132706

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0591$20.00

This year marks the 25th anniversary of the ﬁrst Annual Review of Immunology article to describe features of the T cell antigen receptor (TCR). In celebration of this anniversary, we begin with a brief introduction outlining the chronology of the earliest studies that established the basic paradigm for how the engaged TCR transduces its signals. This review continues with a description of the current state of our understanding of TCR signaling, as well as a summary of recent ﬁndings examining other key aspects of T cell activation, including cross talk between the TCR and integrins, the role of costimulatory molecules, and how signals may negatively regulate T cell function.

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INTRODUCTION TCR: T cell antigen receptor

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Signal transduction: biochemical events linking surface receptor engagement to cellular responses

Twenty-ﬁve years ago, Annual Review of Immunology published its ﬁrst review describing features of the structure we now know as the T cell antigen receptor (TCR) (1). In recognition of this anniversary, this article begins by highlighting a sampling of seminal observations made in the decade following the initial description of the TCR. The discoveries made during this period established the basic paradigm for how TCR engagement initiates the earliest biochemical events leading to cellular activation, described nicely in an Annual Review of Immunology article in 1996 (2) (Figure 1a). This historical perspective sets the stage for a discussion of our current state of understanding of the molecular and biochemical events critical for T cell activation that have emerged from the work of multiple laboratories.

DESCRIBING THE TCR COMPLEX In the early 1980s, several groups began experiments to identify and characterize the antigen receptor on T cells. One approach used newly developed molecular techniques, ultimately leading to the identiﬁcation of the genes responsible for the antigen-binding proteins (3–5). An even earlier approach relied on im-

munization of mice with T cell clones of a deﬁned speciﬁcity, hybridomas, or clonal T cell tumors in the hope that antibodies would be generated that would react with the receptor responsible for binding to antigen (6–8). Such antibodies were produced and were then used ﬁrst to demonstrate interference with antigenic responses and later to perform the initial biochemical characterization of the receptor itself. These studies revealed a complicated cell surface structure that included proteins reactive with antibodies against the nonpolymorphic CD3 proteins (initially thought of as three polypeptides, γ, δ, and ε) (9), as well as variable proteins designated α and β. The antigen-binding function of αβ was obvious early on, by inference owing to their highly polymorphic nature and similarity to immunoglobulin and experimentally owing to early gene transfer experiments that demonstrated that antigen/major histocompatibility complex (MHC) reactivity tracked with expression of these receptor components (10, 11). However, it was less obvious what role the CD3 proteins played in TCR function. Several lines of evidence suggested that the CD3 molecules were critical for signal transduction: Unlike αβ, the CD3 molecules had long cytoplasmic tails, and anti-CD3 antibodies resulted in T cell activation, although it was difﬁcult to demonstrate signaling function conclusively. One early

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 TCR proximal signaling then and now: ﬁlling in the gaps. Then (a): TCR signaling mid-1990s (adapted from Reference 2). Ligation of the TCR/CD3 results in activation of Src and Syk family PTKs associated with the intracellular CD3 domains that then activate PLCγ1 and Ras-dependent pathways. PLCγ1 hydrolizes PIP2 to form IP3 and DAG. Now (b–d ): Current understanding of how the TCR couples to downstream pathways (b), the molecular basis for Ca2+ inﬂux (c), and the positive feedback loop responsible for Ras activation (d ). (b) The link between PTKs and downstream pathways is a multimolecular signaling complex nucleated by the adapter proteins LAT, Gads, and SLP-76. Lck activates ZAP-70 to phosphorylate (p) tyrosine residues on LAT, which then recruits Gads and its constitutive binding partner SLP-76. ZAP-70-mediated phosphorylation of SLP-76 results in the recruitment of multiple SH2 domain–containing effector molecules (circles) and adapter proteins (octogons). SH3 domains (shaded overlapping areas) also link effectors to adapters and contribute to stabilization of the complex. (c) The link between depletion of ER Ca2+ stores and CRAC channel activation is STIM. TCR-induced IP3 production (see b) results in the activation of IP3 receptors (IP3 R), which release Ca2+ from the ER into the cytoplasm. STIM contains paired EF hands within the ER lumen, which bind a single Ca2+ . Upon depletion of ER stores, Ca2+ -free STIM molecules oligomerize and move to areas of ER/plasma membrane proximity, where they colocalize with and induce dimerization of Orai1 dimers, resulting in a functional CRAC channel and subsequent inﬂux of Ca2+ . (d ) Activation of Ras involves two Ras GEFs (SOS and RasGRP) in a positive feedback loop. TCR-induced production of DAG (see b) results in the membrane recruitment of RasGRP, where it is phosphorylated and activated by PKCθ. RasGRP then facilitates the removal of GDP from Ras, which can then bind GTP and become activated. GTP-bound Ras then binds SOS, which is bound constitutively to GRB2 and is inducibly recruited to LAT, increasing its GEF activity, resulting in a positive feedback loop and robust Ras activity. 592

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NOW: Current understanding of TCR signaling TCR/CD3 LCK

ZAP-70 NCK p85 P

LAT

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SLP-76

ITK

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b THEN: Mid-1990s understanding of TCR signaling

PIP2

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IP3

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Ca2+

PIP2 RAF-1 Ras MKK GRB2 SOS

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IP3 + DAG

Ca2+

IP3

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Ca2+

2+

ERK1, 2

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Ca2+ Ca2+ Ca2+

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ORAI1 ORAI1

IP3R

Ca2+

Calcineurin

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STIM STIM STIM

Ca2+

AI1 OR AI1 OR

Ca2+

Ca2+ Ca2+ STIM

ER

Calcium

TCR/CD3

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P GTP

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PKC

Ras GTP

d Ras activation

Ras GDP

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PKC: protein kinase C PLC: phospholipase C

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PTK: protein tyrosine kinase Immunoreceptor tyrosine-based activation motif (ITAM): a short peptide sequence in the cytoplasmic tails of key surface receptors on hematopoietic cells that is characterized by tyrosine residues that are phosphorylated by src family PTKs, enabling the ITAM to recruit activated Syk family kinases

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effort involved an attempt to create cell lines expressing αβ without CD3 or CD3 without αβ, thereby creating reagents to investigate the independent role of the receptor components. These experiments were not successful, as it became clear that there is an obligatory coexpression of αβ with CD3 (12). These studies did result, however, in the creation of the ﬁrst in a long line of genetically altered Jurkat T cells that have been instrumental in our understanding of how the TCR complex couples to its signaling machinery (13).

EARLY SIGNALING STUDIES Initial investigations into how the TCR transduces its signals began with the observation that TCR-deﬁcient Jurkat T cells could be stimulated pharmacologically with the combination of phorbol esters and Ca2+ ionophores (14). This observation led to speculation that engagement of the TCR might stimulate the same signals that these reagents induced. This notion was tested directly in experiments demonstrating increases in intracellular free Ca2+ following CD3 or TCR stimulation in both Jurkat cells and primary T cells. The source of the Ca2+ increase was shown to be a combination of Ca2+ released from an intracellular pool in response to inositol trisphosphate (IP3 ) production and inﬂux of Ca2+ from outside of the cell (15). It was presumed that phorbol esters were important because of their ability to activate protein kinase C (PKC, now known to be a family of enzymes), whose activity is regulated physiologically by diacylglycerol (DAG). Concurrent studies in other systems had demonstrated that a single enzymatic reaction, hydrolysis of the membrane PI(4,5)P2 (phospholipid phosphatidylinositol 4,5 bisphosphate) by phospholipase C (PLC), generates both IP3 and DAG, suggesting that the TCR may function to regulate PLC activity. Testing this notion proved somewhat difﬁcult at ﬁrst, as PLC regulation was initially described downstream of heterotrimeric guanosine triphosphate (GTP)-binding proteins associated with seven-transmembrane Smith-Garvin

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domain receptors. An exhaustive but unsuccessful search ensued for the GTP-binding protein critical for coupling the TCR to PLC activation. Insight into how the TCR may initiate PLC activation emerged from a conﬂuence of discoveries in immune cells and other lineages. For example, stimulation of the TCR results in changes in protein phosphorylation, including inducible phosphorylation of the newly described ζ chain of the CD3 complex (16). Furthermore, growth factor receptors with intrinsic protein tyrosine kinase (PTK) activity also stimulate PLC function. In contrast to seven-transmembrane domain receptors, however, receptor PTKs stimulate another PLC isoform, PLCγ. Although none of the TCR components possessed enzymatic activity themselves, cytosolic PTKs of the src family (in particular Lck and Fyn) were being described in T cells. These PTKs were associated either with the TCR (17) or with the CD4 and CD8 coreceptors (18, 19), both important for TCR signaling. These observations, coupled with the new knowledge that T cell activation requires PTK function (20), led to a new TCR signaling paradigm in which the TCR recruits cytosolic PTKs to activate key second messengers.

HOW CD3 TRANSDUCES ITS SIGNALS Testing this model led to a search for additional substrates of the TCR-stimulated PTKs. Among the most attractive candidates were tyrosines within the CD3 molecules themselves that fell within a motif present once within CD3 ε, γ, and δ and in triplicate within each ζ chain and also in key immunoreceptors on other immune cell lineages. The signature of these motifs (21), eventually designated immunoreceptor tyrosine-based activation motifs (ITAMs), is two tyrosines ﬂanking a series of amino acids, including key leucine/isoleucines with stereotypic spacing. Numerous laboratories demonstrated that the ITAM tyrosines are in fact phosphorylated upon TCR ligation, but deﬁning the importance of this

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posttranslational modiﬁcation for TCR function was not trivial. Because the CD3 molecules could not be expressed without the αβ chains, several groups created chimeric molecules fusing the cytoplasmic domains of individual CD3 chains to extracellular and transmembrane domains from other proteins (22–25). These cDNAs in chimeric proteins were then transfected into T cell lines selected for TCR loss. Antibody crosslinking of the extracellular domain of the chimeras recapitulated all the known TCRmediated signaling events leading to cellular activation. Mutation of the key ITAM tyrosines (or altering their spacing) abrogated the ability of the chimeras to activate the cells, thus demonstrating that tyrosine phosphorylation of the CD3 ITAMs is an early and requisite step for TCR-mediated T cell activation. Studies of the CD3 chimeras led naturally to the question of the purpose for ITAM phosphorylation. Unlike growth factor receptors whose intrinsic enzymatic activity is enhanced by tyrosine phosphorylation, the CD3 molecules have no such effector function on their own. Investigators speculated, therefore, that tyrosine phosphorylation of the ITAMs might serve as docking sites for interactions with other proteins. Indeed, it was soon shown that phosphorylated CD3ζ (and later other ITAM-containing proteins) is a recruitment site of a 70-kDa phosphoprotein, the Syk kinase family member ZAP-70 (ζ-associated protein of 70 kDa) PTK (26). A model therefore emerged that engagement of the TCR leads to Src family PTK activity resulting in ITAM phosphorylation and recruitment of ZAP-70. This converted the TCR with no intrinsic enzymatic function to an active PTK able to phosphorylate a spectrum of substrates leading to a myriad of downstream signals that, when integrated appropriately (along with signals from other coreceptors), lead to T cell activation (27). The basic tenets of this model have stood the test of intensive investigation. In the 15 years since ZAP-70 was cloned, investigators have ﬁlled in many of the gaps between the TCR and initiation of effector functions. Much has been learned about substrates of the PTKs (in-

ARGUING BY ANALOGY: COMPARING AND CONTRASTING SIGNALING PATHWAYS BY MULTIPLE RECEPTORS AND LINEAGES Key insights important for understanding T cell activation have come from studies in nonimmune mammalian cells and cells of lower organisms, including the observation that PTKs could link to the phosphatidylinositol pathway and the paradigm describing adapter proteins as integrators of signal transduction. T cell biologists have also provided unique insights to their nonimmunologist colleagues. Examples include a mechanism for recruitment of nonreceptor PTKs to enzymatically inactive surface receptors and the notion that protein tyrosine phosphatases may be positive as well as negative regulators of PTK pathways. Within the immune system, biologists studying TCR signaling have been the donors and recipients of information that has been useful for investigators examining the signaling pathways of other cell surface receptors (e.g., integrins) and other hematopoietic lineages. It is clear that although basic paradigms may be similar, each cell type and receptor utilizes unique ways to regulate signal transduction cascades. Additionally, recent studies have demonstrated that pathways thought to be distinct (e.g., integrins and immunoreceptors) instead intersect at multiple levels. Future insights into how diverse signaling pathways are integrated to result in the appropriate biologic response will undoubtedly continue to beneﬁt from comparing and contrasting activation events downstream of multiple receptors in different immune and nonimmune cell lineages.

cluding Src, Syk, and more recently Tec family members) activated by the TCR and how these molecules participate in T cell activation, about how signaling complexes are organized by adapter proteins to bring effector proteins together, and about the unexpected intersection of particular signaling pathways (see also the side bar, Arguing by Analogy: Comparing and Contrasting Signaling Pathways by Multiple Receptors and Lineages). With the accumulation of data, it has also become clear that signaling via the TCR complex is not a linear event starting at the receptor and ending in the nucleus. Instead, there appears to be complex feedback and feedforward regulation at each step. Ironically, one of the most central signaling questions that remains is how receptor www.annualreviews.org • T Cell Activation

Adapter protein: cellular protein that functions to bridge molecular interactions via characteristic domains able to mediate protein/protein or protein/lipid interactions

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binding translates most proximally into an activating signal. Many models have been proposed, but none has yet withstood the rigor of subsequent investigation. This review summarizes our current understanding of many of these issues and poses some of the intriguing questions that remain.

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Since identiﬁcation of the TCR as a complex consisting of the variable αβ chains noncovalently associated with the nonpolymorphic CD3 proteins, considerable work has gone into deﬁning the stoichiometry of these interacting molecules. We now know that the CD3 proteins exist as a series of dimers including γε, δε, and ζζ associated with a single αβ heterodimer. Although it has been clear for more than 15 years that CD3 transduces signals from the engaged receptor via its ITAMs, exactly how ligation of the TCR is translated into the ﬁrst signal remains controversial. Current models suggest that both TCR aggregation and conformational changes may play roles in signal initiation. Two separate but not mutually exclusive conformational changes within the CD3 cytoplasmic tails have been proposed as mechanisms for TCR-inducible ITAM phosphorylation. The ﬁrst mechanism is based on the observation that in the presence of acidic lipid designed to mimic the inner leaﬂet of the plasma membrane, the cytoplasmic tails of CD3ε, and to a lesser extent of CD3ζ, fold or interact with the lipid in such a way as to prevent phosphorylation. Conversely, in aqueous solution or in the presence of zwitterionic lipid, the CD3 tails are more readily able to be phosphorylated by Lck (28, 29). These ﬁndings have led to the hypothesis that in resting T cells the CD3ζ and/or CD3ε tails are tightly associated with the lipid-rich inner leaﬂet of the plasma membrane, rendering them inaccessible to Lck phosphorylation, but following TCR ligation, they are released from the membrane and phosphorylated. Investigations have begun to test this hypothesis in T cells, and recently

pMHC: peptide major histocompatibility complex (MHC) complex

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the interaction of the CD3ε cytoplasmic domain with the plasma membrane has been conﬁrmed in resting Jurkat T cells by ﬂuorescence energy transfer (FRET) (29). A second possible conformational change in CD3ε has garnered much attention following the observation that upon TCR ligation a proline-rich region (PRR) in CD3ε is exposed and available to recruit an SH3 domain of the adapter protein noncatalytic tyrosine kinase (Nck). Importantly, this occurs prior to CD3ζ and CD3ε ITAM phosphorylation (30). Investigators speculated that Nck could then recruit and activate effector molecules required for subsequent ITAM phosphorylation. However, recent data using mice with retrogenic or knockin mutations of the CD3ε PRR have failed to show a requirement for the CD3ε/Nck interaction in the activation of peripheral T cells (31, 32). This observation must be considered in light of the T cell developmental abnormalities in the knockin mice. Resolving the importance of the PRR for mature T cell function requires temporal control of expression of the mutant. Although the seminal ﬁnding that there is a non-ITAM region in CD3 that is critical for TCR signaling appears ﬁrm, several controversies remain regarding the CD3ε PRR. There is disagreement regarding whether the interaction between Nck and CD3ε is constitutive rather than inducible (31, 33–35). The role of the CD3ε/Nck interaction in mature T cells is also controversial, as it has been suggested that the interaction (a) is not required for mature T cell activation (32), (b) is required for signal ampliﬁcation and ITAM phosphorylation following weak TCR ligation (34), or (c) is required for regulation of TCR activity by inhibiting ITAM phosphorylation and promoting TCR degradation (35). Ongoing work will provide insights into these controversies. Although CD3 conformational changes may explain the initiation of the intracellular kinase cascade, we do not know how peptide/MHC (pMHC)/TCR interactions result in conformational changes in the intracellular regions of the CD3 complex. Models based on ligand-induced conformational changes in

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Table 1

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Recent models for initial TCR triggering

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Model

Description

Piston-like movement

An external force from the APC/T cell interaction pushes the TCR/CD3 complex in a piston-like fashion perpendicular to the plasma membrane, exposing activating motifs on the intracellular regions of CD3 chains

Receptor deformation

The force of the motility of the T cell as it moves over an APC induces TCR triggering only when pMHC/TCR interactions are of a high enough afﬁnity to withstand the force long enough to undergo a conformational change

Permissive geometry

pMHC dimers bind TCR/CD3 dimers inducing a rotational scissor-like conformational change in CD3 chain(s) that reveal previously hidden intracellular activation motif(s)

Kinetic segregation

Signal initiation occurs because of exclusion of inhibitory molecules in the tight contact zone between the T cell and the APC, thereby shifting the enzymatic steady state toward an activating state

Diffusion trapping

Expanded kinetic segregation model in which the afﬁnity/diffusion coefﬁcient of the pMHC/TCR dictates the valency of TCRs required to trap the complex in the tight contact zone and therefore initiate a productive signal

Pseudodimer

Agonist pMHC/TCR recruits a second TCR via interaction with its associated CD4, the second TCR then binds a coagonist endogenous pMHC forming a stable pseudodimer that triggers signaling via proximity of ITAMs to CD4-associated Lck and/or by conformational change

the TCR and/or TCR aggregation have been proposed (Table 1). The crystal structures of several pMHC/TCR complexes have revealed little conformational changes in the TCR heterodimer outside of the binding domain (reviewed in 36). However, owing to the current technical limitations of crystallography and protein nuclear magnetic resonance spectroscopy, only pMHC/TCR structures in the absence of transmembrane domains and CD3 components have been elucidated. Therefore, changes between the components of the TCR/CD3 complex in relation to one another or to the plasma membrane cannot be ruled out. The rigid, rod-like transmembrane domain of the CD3εγ could mediate a piston-like movement of the TCR complex perpendicular to the plasma membrane after ligand binding, in a manner similar to that reported for the aspartate receptor (37). Such a model would require external forces that could be provided by cell-to-cell contact, but this possibility is difﬁcult to reconcile with the ability of soluble ligands to activate the TCR. Similarly, an external force is required for the recently proposed receptor deformation model, in which the movement of the T cell as it travels across the antigen-presenting cell (APC) exerts a force

on transient pMHC/TCR interactions. Only when the force required to induce conformational changes in the TCR is less than the force required to break the pMHC/TCR interaction will TCR triggering occur (38). Alternatively, the permissive geometry model for TCR triggering predicts that dimeric pMHCs bind two TCR/CD3 complexes (39). This interaction results in a rotation of the TCR heterodimers around one another, displacing the extracellular domains of the associated CD3 molecules. The transmembrane interactions between the CD3 and TCR dimers serve as pivot points, and the CD3 movement is scissor-like, exposing previously shielded ITAMs and/or other functionally important domains. This model is consistent with studies that suggest that TCR aggregation is required for TCR triggering. TCR aggregation has long been implicated as a mechanism for TCR triggering. In the classic aggregation model, crosslinking of TCR/CD3 complexes with multimeric pMHC enables close contact and transphosphorylation between the CD3 tails and associated PTKs. Clustering of several TCR complexes also may result in competition for membrane lipid between the CD3 chains, resulting in dissociation of the cytoplasmic domains from the www.annualreviews.org • T Cell Activation

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membrane and subsequent ITAM phosphorylation (28). Aggregation is supported strongly by observations that soluble multimeric but not monomeric pMHC can trigger TCR activation. However, recent biochemical and microscopic studies suggest that preformed TCR aggregates are present on nonactivated T cells (reviewed in 40). In the kinetic segregation model, adhesion and other accessory molecules with short extracellular domains such as CD2 initiate close contact zones between an APC and a T cell (41). Inhibitory phosphatases with long extracellular domains such as CD45 are excluded because of their size. TCR complexes that engage pMHC on the APC surface remain in the close contact zone, where they are segregated from phosphatases and are able to initiate signaling. TCR complexes that do not engage pMHC are free to diffuse outside of the close contact zone. Shortening the extracellular domains of inhibitory molecules or lengthening the extracellular domains of adhesion or accessory molecules can abrogate TCR signaling, suggesting that kinetic segregation is an important aspect of TCR signal initiation (reviewed in 41). This notion was expanded with the diffusion trapping model in which immobilization or trapping of pMHC/TCRs in the close contact zones is responsible for TCR signal initiation (42). The valency, or degree of aggregation, of the pMHC/TCR required for trapping and therefore triggering depends on the afﬁnity and diffusion coefﬁcient of the pMHC/TCR interaction. Another model has been proposed that suggests that endogenous pMHCs amplify signals produced by the rare agonist pMHCs by promoting TCR aggregation and a subsequent phosphorylation cascade (40). Similarly, the pseudodimer model proposes that an agonist pMHC/TCR can recruit a second TCR in a CD4-dependent manner, and this second TCR binds a coagonist endogenous pMHC, forming a stable pseudodimer that could trigger signaling through ITAM/Lck proximity or through CD3 conformational changes (43). Many of these models are not mutually exclusive, and it is likely that TCR aggregation, conformational changes within the TCR complex, and exclu-

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PI3K: phosphoinositide 3-kinase

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sion of inhibitory molecules are all required for TCR triggering, perhaps in a stepwise fashion.

PROXIMAL SIGNALING COMPLEX The earliest step in intracellular signaling following TCR ligation is the activation of src (Lck and Fyn) PTKs, leading to phosphorylation of the CD3 ITAMs. Recruitment of ZAP-70 follows, leading to a cascade of phosphorylation events. The past decade has seen the description of a subcellular assembly and activation of an adapter protein nucleated multimolecular signaling complex (Figure 1b). This complex is responsible for propagating the TCR/PTK signal into multiple and diverse distal signaling pathways. Among the most important of the ZAP-70 targets are the transmembrane adapter protein linker for the activation of T cells (LAT) and the cytosolic adapter protein src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kDa (SLP-76) (44, 45). These two adapters form the backbone of the complex that organizes effector molecules in the correct spatiotemporal manner to allow for the activation of multiple signaling pathways. The importance of these adapters is underscored by studies showing that the loss of either LAT or SLP-76 results in a near complete loss of TCR signal transduction reminiscent of Syk/ZAP-70 or Lck/Fyn double-deﬁcient T cells (46–48). LAT contains nine tyrosines that are phosphorylated upon TCR engagement, which bind the C-terminal SH2 domain of PLCγ1, the p85 subunit of phosphoinositide 3-kinase (PI3K), and the adapters growth factor receptor-bound protein 2 (GRB2) and GRB2-related adapter downstream of Shc (Gads) (reviewed in 47). SLP-76 is then recruited to phosphorylated LAT via their mutual binding partner Gads (49). SLP-76 itself contains three modular domains: an N-terminal acidic domain with three phosphorylatable tyrosines that interact with the SH2 domains of Vav1, Nck, and IL2-induced tyrosine kinase (Itk); a PRR that binds constitutively Gads and PLCγ1; and a

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C-terminal SH2 region that can bind adhesion and degranulation–promoting adapter protein (ADAP) and hematopoietic progenitor kinase 1 (HPK1) (reviewed in 46). Although LAT and SLP-76 serve to nucleate this large signaling complex, the effector molecules themselves also are important for stabilizing the complex. For example, the Tec family kinase Itk is required in a kinase-independent manner for the recruitment of the guanine nucleotide exchange factor (GEF) Vav1 to the APC contact site, whereas Vav1 is required for optimal SLP-76 phosphorylation and recruitment to LAT as well as for Itk activation (50–52). These and other data suggest that the formation of the complex is more complicated than the linear model most often invoked for simplicity. For example, PLCγ1 directly binds to SLP-76, LAT, and Vav1 as well as to its activating kinase Itk (reviewed in 53). It is thought that these interactions collectively are required to stabilize PLCγ1 in the correct conformation within the complex to allow for its optimal activity (54). Advances in biochemical and structural techniques are needed to elucidate the precise allosteric and perhaps stoichiometric changes within the multimolecular complex that allow for signal transduction. To investigate more precisely the importance of these complex interactions in primary T cells, several laboratories have generated mice expressing transgenic or knockin mutations in speciﬁc binding regions in various molecules involved in proximal signaling (55– 57). Tyrosine to phenylalanine mutations in SLP-76 at residues 112 and 128 or 145 in primary thymocytes and T cells do not result in a loss of SLP-76/Vav1/Nck/Itk interactions, as would be expected from earlier phosphopeptide mapping studies and studies in cell lines (57). However, these tyrosine mutations still result in severe defects in downstream signaling pathways consistent with defective Vav1 or Itk activity. Similarly, mutation of tyrosines of Vav1 does not result in a loss of interaction with their proposed binding partners, although it does result in dysregulated Vav1-dependent signaling (55). Although the continued interactions of

these proteins seen by immunoprecipitation experiments is likely due to tertiary interactions with other domains or other molecules, these studies suggest that SH2/phosphotyrosine interactions may play important regulatory roles for the activation of effector molecules. Indeed, structural studies have suggested that the interaction between the SH2 domain of Itk and a phosphotyrosine results in a conformational switch allowing kinase activity (58). Consistent with these data, investigators recently showed in Jurkat T cell lines that an Itk/SLP-76 interaction is required for Itk kinase activity, although we do not yet know if it is speciﬁcally the SH2/phosphotyrosine interaction that mediates this kinase activity (59). Further studies are required to determine how the activities of molecules beyond Itk are affected by speciﬁc domain/domain interactions within the complex. The proximal signaling complex results in the activation of PLCγ1-dependent pathways including Ca2+ - and DAG-induced responses, cytoskeletal rearrangements, and integrin activation pathways. Ligation of costimulatory receptors such as CD28 augments these pathways. Below, we discuss the mechanisms by which these pathways are activated and regulated.

PLCγ1 ACTIVATION AND SIGNAL TRANSDUCTION Following TCR ligation, PLCγ1 is found in the proximal signaling complex bound to SLP76, Vav1, and LAT, where it is phosphorylated and activated by Itk. Activated PLCγ1 then hydrolyzes the membrane lipid PI(4,5)P2 , producing the second messengers IP3 and DAG. These two messengers are essential for T cell function, and therefore the regulation of PLCγ1 activation has been the subject of intensive studies. Localization of PLCγ1 to the proximal signaling complex is dependent on LAT and the Gads-binding region of SLP-76 (54). Activation of PLCγ1 is dependent on Itk kinase activity that, in turn, is dependent on Vav1, Lck, ZAP-70, LAT, and SLP-76 (52, 60, 61). www.annualreviews.org • T Cell Activation

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Following TCR ligation, Itk is recruited to the membrane through PH domain interactions with PIP3 , which has been locally generated by Lck-induced PI3K activity (reviewed in 61). At the membrane, Lck phosphorylates Itk, and the SH2 and SH3 domains of Itk interact with phosphorylated tyrosine 145 and the PRR of SLP-76, respectively (62–64). The role of Vav1 and ZAP-70 in Itk activation is not understood but may relate to their involvement in the phosphorylation of SLP-76 or PI3K activation (45, 51, 52). A second Tec family kinase, Rlk, can also phosphorylate PLCγ1, resulting in a relatively mild defect in Itk-deﬁcient mice and requiring the study of Rlk/Itk double-deﬁcient mice to better understand the role of Tec kinases in T cell activation (reviewed in 61).

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DAG-MEDIATED SIGNALING PATHWAYS TCR-induced production of DAG results in the activation of two major pathways involving Ras and PKCθ. Ras is a guanine nucleotide– binding protein and is required for the activation of the serine-threonine kinase Raf-1, which initiates a mitogen-associated protein kinase (MAPK) phosphorylation and activation cascade. Raf-1 is a MAPK kinase kinase (MAPKKK) that phosphorylates and activates MAPK kinases (MAPKKs), which in turn phosphorylate and activate the MAPK’s extracellular signal-regulated kinase 1 (Erk1) and Erk2. Erk kinase activity results in the activation of the transcription factor Elk1, which contributes to the activation of the activator protein-1 (AP-1) ( Jun/Fos) transcription complex via regulation of Fos expression. Additionally, Erk activity can result in the transcriptional activation of signal transducer and activator of transcription 3 (STAT3) and in the serine phosphorylation of Lck (reviewed in 65). Ras is only active in the GTP-bound state, and its activation is facilitated by GEFs and is suppressed by GTPase-activating proteins (GAPs). Two Ras GEFs are present in T cells, son of sevenless (SOS) and Ras guanyl nucleotide-releasing protein (RasGRP). 600

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RasGRP appears more dominant for early activation of Ras, as SOS cannot compensate for RasGRP deﬁciency (66–68). RasGRP is inducibly recruited to the membrane through a DAG-binding domain (69), where it is phosphorylated by PKCθ (70). SOS is constitutively bound to the adapter protein GRB2, and upon TCR stimulation, the GRB2 SH2 domain is recruited to and binds phosphorylated tyrosines on LAT, thereby bringing SOS into the proximal signaling complex, where it can facilitate the localized activation of Ras (71). The signiﬁcance of these two modes of Ras activation was unclear until recently, when it was shown that RasGRPdependent RasGTP production catalyzes SOS activity, resulting in a positive feedback loop and robust TCR-induced Ras activation (72) (Figure 1c). This uniﬁed model explains the RasGRP dominance, as RasGRP would be required to initiate the production of RasGTP, which upon reaching threshold levels would catalyze SOS activity and further amplify the signal. The second major signaling pathway regulated by DAG is mediated by PKCθ, a PKC family member that contains a lipid-binding domain speciﬁc for DAG, which is important for recruiting PKCθ to the plasma membrane following T cell activation. However, phosphorylation by Lck (reviewed in 73) may be required to induce a conformational change that enables binding to the lipid phosphatidyl serine (PS), which in turn enhances binding to DAG, resulting in PKCθ activation (74). Other proximal signaling molecules, including Vav1, PI3K, and 3-phosphoinositide-dependent kinase 1 (PDK1), also play roles in PKCθ localization, but details of their contributions are not completely deﬁned (73). One critical pathway that PKCθ regulates is NF-κB activation. Because both the canonical and noncanonical activation of this pathway in T cells is described in detail in this volume of Annual Review of Immunology [see review by Karin and colleagues (75)], we highlight only the major steps in the classical activation of NF-κB downstream of the TCR. The NF-κB

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family of transcription factors consists of ﬁve members. In resting cells, NF-κB is found in the cytosol associated with inhibitor of NF-κB (IκB) family members that keep NF-κB from moving into the nucleus. Upon T cell activation, IκB is phosphorylated by the IκB kinase (IKK) complex, ubiquitinylated, and degraded, allowing NF-κB to translocate into the nucleus, where it activates genes involved in the function, survival, and homeostasis of T cells (reviewed in 76). Although we have known the general pathway of NF-κB activation for some time, the speciﬁcs of how PKCθ activation leads to nuclear import of NF-κB in T cells are still being elucidated. Over the past several years, the identiﬁcation and characterization of a lymphocytespeciﬁc activation complex have provided some insight into this question. Following TCR stimulation, a trimolecular complex forms between CARMA1 [caspase recruitment domain (CARD) and membrane-associated guanylate kinase (MAGUK)-containing scaffold protein], the CARD-containing adapter protein Bcl10, and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) (73, 76). The assembly of this complex is regulated by PKCθ through its phosphorylation of CARMA1, which is required for CARMA1 oligomerization and association with Bcl10 (77, 78). MALT1 binds to Bcl10 and contributes to the degradation of the regulatory subunit of the IKK complex (IKKγ) by facilitating its polyubiquitination, possibly via activation of the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) (79, 80). Degradation of this regulatory subunit allows for phosphorylation of IκB by the IKK catalytic subunits, subsequent IκB degradation, and release of NF-κB, resulting in NF-κB nuclear localization and gene activation. Recently, MALT1 was shown to enhance NF-κB signaling through its ability to degrade the deubiquitinating enzyme A20, a negative regulator of NF-κB activation (81, 82). One additional component of the complex that was recently identiﬁed as a CARMA1associating protein is ADAP (83). ADAP,

originally deﬁned as a SLP-76-binding partner, associates with the MAGUK domain of CARMA1 following T cell stimulation. Investigators proposed that this interaction might alter the conformation of CARMA1 and enhance its association with Bcl10 and MALT1 and/or stabilize the CARMA1/Bcl-10/MALT1 complex.

Ca2+ -MEDIATED SIGNALING PATHWAYS Ca2+ ions are universal second messengers in eukaryotic cells. The IP3 generated by TCRstimulated PLCγ1 activity stimulates Ca2+ permeable ion channel receptors (IP3 R) on the endoplasmic reticulum (ER) membrane, leading to the release of ER Ca2+ stores into the cytoplasm. Depletion of ER Ca2+ triggers a sustained inﬂux of extracellular Ca2+ through the activation of plasma membrane Ca2+ release-activated Ca2+ (CRAC) channels in a process known as store-operated Ca2+ entry (SOCE) (reviewed in 84) (Figure 1d ). For decades the CRAC channels had only been identiﬁed by their biophysical properties, and it has just been in the past few years that the pore-forming subunit of the channels was identiﬁed as the four-transmembrane domain– containing molecule Orai1 (85–87). Additionally, studies in the past few years have revealed the sensor for depleted ER Ca2+ stores and the activator of CRAC channels as stromal interaction molecule (STIM) (88, 89). STIM is an ER-resident transmembrane protein with a C-terminal cytoplasmic coiled-coil motif and, within the ER lumen, an N-terminal sterile α motif (SAM) and paired EF hands, where one hand binds a single Ca2+ ion with low afﬁnity (88, 89). Two STIM proteins, STIM1 and STIM2, are found in mammals, and recent work has shown that STIM1 is important for the initial robust phase of SOCE, whereas STIM2 is important for maintaining basal Ca2+ levels and sustaining the late phase of SOCE (90, 91). Following ER Ca2+ depletion, STIM1 molecules aggregate in clusters that preferentially localize to sites of ER plasma membrane apposition, where they colocalize with www.annualreviews.org • T Cell Activation

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NFAT: nuclear factor of activated T cells

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clusters of Orai1, forming punctae (92–94). The biochemical mechanism by which STIM1 couples Ca2+ depletion to CRAC activation is not yet fully understood and is an area of intense investigation. Recent work has shown that STIM1 oligomerization is sufﬁcient to induce punctae formation and CRAC channel activation independent of Ca2+ store depletion (95). Further studies showed that the C-terminal coiled-coil domain of STIM1 alone can induce dimerization of Orai1 dimers, which is sufﬁcient for CRAC channel activation independent of punctae formation and store depletion (96). How STIM1 oligomers translocate to areas of ER plasma membrane apposition and how, once there, they induce the Orai1 tetramerization remain unknown. Interestingly, the WASp family verprolin homologous protein (WAVE2) complex is also required for SOCE activity, through a mechanism that remains to be fully elucidated, although independent of its function in actin remodeling (97, 98). CRAC channels appear to be the dominant mode of Ca2+ entry in T cells, but other Ca2+ channels exist. Their relevance remains unclear (reviewed in 84). TCR-induced increases in intracellular Ca2+ levels result in the activation of Ca2+ and calmodulin-dependent transcription factors, including myocyte-enhancing factor 2 (MEF2) and downstream regulatory element antagonist modulator (DREAM), as well as signaling proteins, including the phosphatase calcineurin and the Ca2+ -calmodulin-dependent kinase (CaMK), that in turn activate a variety of transcription programs (reviewed in 99). Activated calcineurin dephosphorylates members of the nuclear factor of activated T cells (NFAT) family, leading to their translocation to the nucleus. In the nucleus, NFAT isoforms can form cooperative complexes with a variety of other transcription factors, thereby integrating signaling pathways, resulting in differential gene expression patterns and functional outcomes, depending on the context of the TCR signal. The most well-studied interaction is NFAT/AP-1, which integrates Ca2+ and Ras signals and results in the expression of genes important for T cell activation includSmith-Garvin

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ing IL-2. In contrast, NFAT activity in the absence of AP-1 activation induces a pattern of gene expression that ultimately results in T cell anergy and a characteristic lack of IL-2 production (100). It is still unclear whether NFAT isoforms are cooperating with other transcription factors or are functioning as dimers to induce the anergic transcriptional pattern (reviewed in 101). The regulatory T cell lineage– speciﬁc transcription factor forkhead box protein 3 (FOXP3) also cooperates with NFAT and antagonizes NFAT/AP-1 gene transcription, resulting in Treg functional gene expression and a lack of IL-2 production (102). Finally, NFAT family members can also cooperate with STAT proteins to induce either Th1 or Th2 differentiation through expression of T-bet or GATA3, respectively (99).

ACTIN AND CYTOSKELETAL RESPONSES When a T cell is presented with cognate antigen by an APC, signals from the TCR initiate a program of actin cytoskeletal rearrangements that results in polarization and activation of the T cell (reviewed in 104). Actin reorganization is essential for T cell function, as actin polymerization inhibitors impede T cell/APC interactions (105) and abolish proximal TCR signals (106). T cell/APC conjugation results in morphological changes, as the stimulated T cell rounds up and accumulates ﬁlamentous actin (F-actin) at the stimulatory interface. These changes are thought to depend on a TCR-induced increase in plasma membrane ﬂuidity and a decrease in cellular motility. Cessation of motility is associated with TCR-induced Ca2+ -dependent phosphorylation and deactivation of the myosin motor protein MyH9; however, the signaling pathway linking the TCR to this event has not yet been deﬁned fully (107). Plasma membrane ﬂuidity is increased, in part, by the TCR- and Vav1-dependent transient dephosphorylation of ERM (ezrin, radixin, and moesin) proteins, resulting in the loss of their ability to link the plasma membrane to the actin cytoskeleton

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(108). Ca2+ signaling and integrin activation downstream of the TCR result in additional modiﬁcations of actin-associated proteins that may play roles in altering plasma membrane rigidity (104). Accumulation of F-actin at the T cell/APC interface is the result of TCR-induced localized activation of multiple actin regulatory and polymerizing pathways, the best studied of which involves the actin-related proteins 2/3 (Arp2/3) complex, although Arp2/3independent pathways also contribute to this process (109). Activation of Arp2/3 requires its interaction with nucleation-promoting factors (NPF) including Wiskott-Aldrich syndrome protein (WASp), WAVE2, and hematopoietic cell lineage–speciﬁc protein 1 (HS1). WASp is recruited to the site of TCR activation through its interaction with the SLP-76-associated adapter protein Nck, where it is activated via Vav1-dependent stimulation of the Rho family GTPase Cdc42 (110). Vav1-mediated activation of a second Rho family GTPase, Rac1, results in the activation of WAVE2 (104). Given the proposed reliance of WASp and WAVE2 on Vav1-mediated GTPase activation, it is interesting that actin-dependent processes that are defective in Vav1-deﬁcient T cells can be rescued with the expression of a GEFinactive Vav1 mutant, suggesting that other Rac and Cdc42 GEFs may be able to support TCR-induced actin changes (55). This result also suggests that other Vav1 functions are important for TCR-induced actin changes. Consistent with this is the observation that, through its protein interaction domains, Vav1 may contribute to WAVE2 and WASp activation through the recruitment of Dynamin2, a GTPase that is important for TCR-induced actin dynamics (111). Activation of the T cell in response to an APC also results in the polarization of the T cell, whereby the microtubular organizing center (MTOC) moves toward the T cell/APC contact site (112). Although polarization of the MTOC has long been observed as a hallmark of productive T cell/APC conjugation, the signaling mechanisms responsible for this movement

remain undeﬁned. Recent data suggest, however, that the adapter protein ADAP (a component of the SLP-76-nucleated complex) may play a role through its interaction with the microtubule motor protein dynein (113). Movement of the MTOC appears essential for the formation of the immunological synapse (IS). The IS is an organized structure that develops at the contact site between the T cell and the APC. It is composed of two concentric regions based on molecular composition: the TCRrich central supramolecular activation cluster (cSMAC), surrounded by the integrin-rich peripheral SMAC (pSMAC) (114, 115). Although the IS was described approximately 10 years ago, its precise role in T cell activation remains unclear. Initially, the concentration of receptors in the cSMAC led to the proposal that the cSMAC is the site of enhanced receptor engagement and prolonged signaling (115). However, later studies showed that TCR signals peak prior to cSMAC formation and suggested that the cSMAC is primarily the site of TCR degradation (116, 121, 122). More recently, it has been proposed that the cSMAC is the site of both TCR signal enhancement and TCR degradation, and the balance between these two processes is determined by the quality of the antigen such that the cSMAC can serve to amplify weak agonist signals (117). Most studies of the cSMAC have focused on the TCR and its associated molecules. However, a newly deﬁned subregion of the cSMAC that is rich in CD28, a costimulatory molecule (see below), and PKCθ but relatively devoid of the TCR points to the potential importance of the cSMAC for costimulatory molecule signal transduction (118). Although the precise roles of the cSMAC remain controversial, it is now well accepted that the initiation of TCR signals occurs in peripheral microclusters that begin to form prior to IS formation. pMHC/TCR ligation results in the formation of intracellular microclusters that contain the TCR complex and associated signaling molecules, including LAT and SLP76 (120). These clusters initiate and can sustain Ca2+ signals (121). The clusters persist for a short time, after which they converge www.annualreviews.org • T Cell Activation

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toward the cSMAC (121, 122). Formation and translocation of the clusters are dependent on F-actin dynamics, and new clusters continue to form even after the mature IS is established (121–123). Integrins play a key role in sustaining microclusters, emphasizing their importance for T cell activation (124). Opposite the MTOC and IS, another ordered structure forms, known as the distal pole complex (DPC). Although the role of the DPC is not known for certain, investigators speculate that the DPC is critical for sequestering negative regulators away from the TCR activation complex (104). Additionally, the DPC may contribute to the polarization of key signaling molecules that may be required for distinguishing memory versus effector fate decisions in recently divided cells (125). Formation of this complex is dependent on F-actin and the rephosphorylation of ERM proteins that migrate to the distal pole linking signaling molecules to the cytoskeleton (126). TCR signaling cascades and pathways downstream of actin reorganization are intertwined and difﬁcult to tease apart, as many of the effector molecules involved have multiple enzymatic and adapter functions. WASp, WAVE2, and Vav1 signals play roles in TCRinduced signaling that appear to be independent of their roles in actin responses (52, 97, 127). Therefore, loss of different actin regulators may result in complex TCR signaling defects (128). Future studies are required to understand fully the feedforward and feedback mechanisms that deﬁne the interdependence between cytoskeletal dynamics and T cell activation.

TCR INSIDE-OUT SIGNALING TO INTEGRINS Integrins are αβ heterodimeric receptors responsible for mediating cell/cell or cell/matrix adhesions. Key T cell integrins include leukocyte function–associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4), which bind their respective ligands intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) and ﬁbronectin on 604

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other immune cells, endothelial cells, ﬁbroblasts, and extracellular matrix proteins. Activation of integrins (increasing their afﬁnity and avidity for ligand) is critically dependent on biochemical events initiated by the TCR, a process designated inside-out signaling (129). Although the pathway from the TCR to integrin activation has not been completely elucidated, TCR-mediated activation of several key signaling molecules as well as TCR-induced actin/cytoskeletal changes have been implicated in this process. A central regulator of inside-out signaling is the small GTPase Ras-proximity-1 (Rap1). Rap1 enhances T cell activation by mediating TCR-induced adhesion to ICAM-1. This conclusion is based on studies utilizing overexpression of dominant-negative and constitutively active forms of Rap1 (130, 131) and more recently by analysis of mice deﬁcient in Rap1A, in which TCR-induced adhesion to ICAM-1 is markedly reduced (132). The importance of understanding the role of Rap1 is clear, as mutations in Rap1-mediated integrin activation have been linked to leukocyte adhesion–deﬁciency syndrome, a disease that can lead to severe bacterial infections (reviewed in 129). Many proximal signaling molecules that comprise the early TCR signalosome, including LAT, SLP-76, and PLCγ1, are required for integrin and Rap1 activation (reviewed in 129) (Figure 2). However, there are downstream effectors that have a more selective role in integrin activation, including the adapter protein ADAP. T cells from ADAP-deﬁcient mice are defective in TCR-induced LFA-1 clustering and adhesion to ICAM (133). The association of ADAP with SLP-76 appears to be required for integrin activation, as overexpression of ADAP but not a mutant of ADAP that cannot bind to SLP-76 enhances integrin function following TCR ligation (134, 135). Although mice expressing the reciprocal mutation in the SH2 domain of SLP-76 have been generated and share several characteristics with ADAP-deﬁcient mice, TCR-induced integrin activation in these mice has not yet been reported.

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Inside-out signaling TCR/CD3

PTK

Integrins

PKD1 RAPL

LAT

PLCγ1

GADS

Pathway C

SLP-76

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VAV1

ADAP

ITK

DAG

Pathway B

PKCθ

RAP1

TALIN RIAM SKAP55 ADAP

RIAM SKAP55 ADAP

WAVE2 Pathway A

ARP 2/3 Actin reorganization

Figure 2 A model of integrin activation. Upon TCR ligation, the LAT/SLP-76-nucleated signalosome assembles. This complex allows for the activation of three pathways necessary for inside-out activation of integrins. Vav1 and Itk contribute to the actin reorganization required for integrin mobility ( pathway A). Mobilization of the ADAP/SKAP55/RIAM complex is necessary for activated Rap1 plasma membrane localization ( pathway B). An active Rap1/RIAM complex induces the association of talin with integrin β tails, perhaps resulting in altered integrin afﬁnity. Thirdly, PLCγ1-mediated generation of DAG leads to PKD1 activation and association with Rap1 ( pathway C). This interaction is required for Rap1 activation and contributes to Rap1 recruitment. Rap1 in turn recruits RAPL, and subsequent RAPL binding to αL subunits results in integrin clustering and afﬁnity changes.

In addition to its inducible interaction with SLP-76, ADAP constitutively associates with Src kinase–associated phosphoprotein of 55 kDa (SKAP55). The ADAP/SKAP55 complex is important for proper localization of activated Rap1 (134). How ADAP and SKAP55 recruit Rap1 to the membrane was unclear until recently when investigators showed that a third adapter, Rap1-GTP-interacting adapter molecule (RIAM), which is also constitutively bound to SKAP55, associates with activated Rap1 upon TCR ligation, resulting in Rap1 movement to the membrane (136). How RIAM itself relocalizes to the plasma membrane following T cell stimulation is currently unknown, although it is hypothesized that recruitment may be through the inducible interaction of PI(3,4)P2 (a product of T cell activation) and the pleckstrin homology (PH) domain of RIAM (137).

In addition to mobilization of the ADAP/SKAP55/RIAM complex that is downstream of early TCR signals and is likely SLP-76 dependent, Rap1-mediated integrin activation is also dependent on other TCRtriggered signals, including those leading to activation of PKC. One PKC target important for Rap1 activation is the serine-threonine kinase protein kinase D1 (PKD1) (also known as PKCμ). Following TCR stimulation, Rap1 associates with the PH domain of PKD1 (138). Interestingly, it is this interaction and not the kinase activity of PKD1 that is required for Rap1 membrane recruitment and activation. Because PKD1 also inducibly recruits the Rap1 GEF C3G (Crk SH3 domain GEF) to the membrane in a TCR-dependent fashion, it is tempting to speculate that this event contributes to the dependence of Rap1 on PKD1 for its activation (138).

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Costimulation: signals delivered to T cells by cell surface receptors other than the TCR itself that potentiate T cell activation

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Activated Rap1 can also associate with the effector regulator of cell adhesion and polarization enriched in lymphoid tissues (RAPL). This interaction is coincident with RAPL membrane localization and necessary for binding of RAPL to the αL subunit of LFA-1. In cell line models, this association is important for LFA-1 clustering as well as afﬁnity modulation (139). In addition to regulating the activation of Rap1, signals from the TCR also regulate cytoskeletal attachments to integrins. One cytoskeletal binding protein important for integrin activation is talin. Recent studies in platelets have demonstrated that Rap1 activity enhances the association of talin with β-integrin subunits through association with RIAM (140). It is possible that formation of a talin/RIAM/Rap1 complex may enable talin to bind integrins, which may induce the highafﬁnity ligand-binding state. Talin is not the only actin-binding protein implicated in TCRinduced integrin activation. Vinculin, WAVE2 (98), and the Arp2/3 complex also play roles in this process, and deﬁning the precise steps that link early TCR signals to activation of these molecules is an area of active investigation.

COSTIMULATION One central tenet of T cell activation is that signaling solely through the TCR results in a nonresponsive state (anergy) in which T cells are refractory to restimulation. Coligation of other cell surface receptors provides additional signals required for anergy avoidance and productive T cell activation. Although many cell surface receptors can enhance signaling through the TCR, CD28 does so more robustly than other costimulatory molecules. Numerous studies have shown that CD28 promotes T cell proliferation, cytokine production (via gene transcription and mRNA stability), cell survival, and cellular metabolism (reviewed in 141). One key effector downstream of CD28 is PI3K. Following binding of CD28 to its ligands CD80 or CD86 on APCs, the p85 regulatory subunit of PI3K associates with a pYMNM motif located in the cytoplasmic 606

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tail of CD28 (142). This regulatory subunit recruits the p110 catalytic subunit of PI3K, which converts PIP2 to phosphatidylinositol (3,4,5) trisphosphate (PIP3 ) at the membrane. Localized PIP3 generation serves as a docking site for the PH domains of PDK1 (3-phosphoinositidedependent protein kinase 1) and its target Akt. Akt phosphorylates multiple proteins, enabling it to affect numerous cellular responses. Activation of Akt enhances the nuclear translocation of NF-κB, which has positive effects on the expression of prosurvival genes including Bcl-xl. Emerging data suggest that Akt accomplishes this function by associating with CARMA1 and facilitating the assembly of the CBM complex (143, 144), a step critical for NF-κB activation (see above). The ability of Akt to promote prosurvival gene expression, coupled with the ability of Akt to inhibit transcription factors that promote cell cycle arrest, results in Akt-driven cell survival and proliferation (141). Akt also affects optimal transcription of NFAT-regulated genes, such as IL-2. One well-known target of Akt is GSK-3 (glycogensynthase kinase 3), a serine-threonine kinase that promotes nuclear export of NFAT (145). Thus, inactivation of GSK-3 by Akt might be one pathway responsible for prolonged NFAT nuclear localization and thus IL-2 transcription following CD28 costimulation. Recently, a GSK-3-independent mechanism by which Akt may regulate NFAT activity was suggested. This model posits that phosphorylated NFAT is bound by the scaffolding protein Homer, thus inhibiting access of calcineurin to NFAT (146). Investigators proposed that CD28 ligation induces Akt-mediated phosphorylation of Homer, resulting in its dissociation from pNFAT. Unbound pNFAT would then be susceptible to calcineurin phosphatase activity and nuclear entry. Whether this pathway is Akt dependent remains to be rigorously tested. Lastly, TCR/CD28 coligation increases the cell surface expression of the insulin transporter Glut1, leading to increased glucose uptake and glycolysis (147, 148), which is also mediated by Akt. Together, these data provide a framework for

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how Akt mediates T cell growth and survival downstream of CD28. The CD28-mediated generation of PIP3 also serves as a docking site for the PH domain of Itk. Although Itk inducibly associates with the LAT/SLP-76/Gads/PLCγ1 complex that forms following TCR ligation, its localization and activation also depends on PI3Kgenerated PIP3 (61). Itk can also associate directly with CD28 via the CD28 proximal PxxP motif (61). It is possible that this interaction keeps Itk close to Lck (which binds to the distal PxxP motif of CD28), allowing for Lckmediated phosphorylation and activation of Itk (149) and enhanced Ca2+ ﬂux, another characteristic of CD28-mediated signaling. Despite the fact that Itk and CD28 associate with one another, studies in Itk-deﬁcient T cells demonstrate that some CD28 signaling is still intact in the absence of Itk, suggesting that this interaction may not be that critical to CD28 signaling (150). Although the proline motifs in the tail of CD28 are required for CD28-mediated proliferation and IL-2 production, these motifs are dispensable for Bcl-xl upregulation (151). This function appears to be more reliant on the proximal YMNM p85 binding site (151). Thus, CD28 can differentially regulate proliferation and survival in activated T cells. Another more recently described function of CD28 is induction of arginine methylation. Following CD28 ligation, protein arginine methyltransferase activity increases, and arginine methylation of multiple proteins, including Vav1, is induced. Vav1 arginine methylation appears to occur in the nucleus and correlates with IL-2 production (152). Although the precise biologic signiﬁcance of this posttranslational modiﬁcation is unknown, this pathway may provide yet another mechanism by which CD28 regulates TCR signaling. Many of the pathways described above are activated by TCR ligation alone; however, the magnitude of the response is considerably augmented with CD28 coligation. This observation has led to speculation that CD28 engagement results primarily in a quantitative rather than a qualitative change in T cell activation

parameters (141). Although this appears to be true, it is also true that quantitative differences in signaling can result in qualitatively distinct functional outcomes. CD28-deﬁcient mice exhibit dampened immune responses to a variety of infectious agents (reviewed in 141). Although these studies demonstrate the importance of costimulation by CD28, not all immune responses are severely impacted by its loss (153). Such observations indicate that molecules other than CD28 can provide costimulation for T cells. Indeed, multiple surface receptors have been described as having costimulatory functions. Included among these are CD2, CD5, CD30, 4-1BB, OX40, inducible costimulator (ICOS), and LFA-1. For this review, we focus on the CD28-related protein ICOS and two members of the tumor necrosis factor receptor (TNFR) family to provide examples of how costimulatory molecules can link to downstream effectors, either directly or through adapter proteins. Unlike CD28, which is expressed at constant levels on both resting and activated T cells, ICOS is inducibly expressed on activated T cells (154). ICOS deﬁciency results in impaired immune responses, similar to yet not as severe as those observed in CD28 knockout models, suggesting that these two molecules may function in similar pathways (155). Indeed, ICOS shares several structural features with CD28, including a YMXM motif in its cytoplasmic tail that associates with p85 (155). This site is presumed to be responsible for PI3K-driven Akt and/or Itk activation observed downstream of CD3/ICOS stimulation, which likely contributes to the similarities seen in CD3/CD28 and CD3/ICOS-stimulated cells (156). ICOS does not induce IL-2 gene transcription as CD28 does. Failure to induce IL-2 has been attributed, at least in part, to the inability of the YxxM motif of ICOS to associate with Grb2, an association that is present via this motif in CD28 (157). Therefore, although ICOS activates genes similar to those induced by CD28, there are differences in the degree to which particular genes are expressed. These differences have in vivo relevance, as mice doubly deﬁcient www.annualreviews.org • T Cell Activation

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in CD28 and ICOS are severely defective in generating immune responses (158). Outside of the CD28 family of costimulatory molecules, the OX40 (CD134) and 41BB (CD137) members of the TNFR family provide costimulation upon engagement with their ligands OX40L and 4-1BBL. Like CD28, OX40 or 4-1BB ligation induces activation of PI3K/Akt, NF-κB, JNK, and p38 MAPK (159). However, unlike CD28 and ICOS, OX40 and 4-1BB do not directly associate with protein kinases but rather link to downstream signaling through the TRAF (TNFR-associated factor) family of adapter proteins. The proteins involved in linking TRAF signaling to NF-κB and the JNK/p38 pathways have not been completely elucidated in primary T cells. However, studies in other systems implicate TRAFs themselves in the direct recruitment of the IKK complex as well as of serine/threonine kinases that initiate signaling to JNK and p38 (reviewed in 160). Many of the same genes are regulated downstream of CD28 and TNFR family members. One reason for such overlap may be due to the timing of receptor expression. CD28 is expressed early and is critical for induction of an immune response. It promotes expression of several other costimulatory molecules including ICOS, OX40, and 4-1BB. Once expressed, these receptors prolong or sustain an immune response and, in the case of OX40 and 4-1BB, are important for memory T cell formation (159). The use of alternative means by which to activate these similar pathways, e.g., direct binding to kinases versus use of adapter proteins, may also allow for differential negative regulation of these pathways. In fact, some TRAF proteins negatively regulate NFκB; thus, association of TNFRs with different TRAF family members can modulate the immune response (160). Lastly, a role for many costimulatory molecules, including ICOS and 4-1BB, in the development and/or function of regulatory T cells is becoming increasingly apparent (161). This layer of complexity will have to be taken into account when deciphering the roles of costimulatory molecules in vivo.

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NEGATIVE REGULATION OF TCR SIGNALING As outlined above, signaling through the TCR triggers an array of signals that activate multiple effector pathways. Activation of these pathways is regulated to ensure that T cells respond to appropriate ligands and for the proper duration. As with positive regulation of T cell signaling, negative regulation is mediated through both TCR-generated signals and those emanating from other cell surface receptors (Table 2). Even the most proximal TCR signaling events are actively regulated. For example, multiple proteins contribute to the regulation of Lck activity. C-terminal src kinase (Csk) is responsible for phosphorylating Lck on its inhibitory tyrosine residue (Y505) and maintaining Lck in an inactive state (162, reviewed in 163). Countering this is the phosphatase CD45 that dephosphorylates the inhibitory site allowing for Lck autophosphorylation and activation. Interestingly, CD45 can also limit Lck activity by dephosphorylating its active site (163). Whether CD45 negatively or positively impacts TCR signaling is likely to be controlled by its proximity to TCR-stimulated effector molecules during TCR engagement and whether CD45 itself is in an enzymatically favorable conformation. An additional layer of Lck regulation initiated by TCR signals has been proposed. As a means to explain how the TCR can distinguish between strong and weak ligands, investigators showed that weak or antagonistic TCR ligation results in rapid Lck-mediated phosphorylation of SHP1 (SH2 domain–containing proteintyrosine phosphatase) (164). SHP1 then dephosphorylates the active site of Lck, resulting in cessation of the TCR signal. Conversely, in the presence of strong or agonistic TCR ligation, Erk is rapidly activated and phosphorylates Lck on Ser59. This activity is thought to prevent SHP1 binding, thus keeping Lck active to sustain TCR signals and further amplify Erk activity. The extent to which this regulatory loop operates in vivo in the context of agonist stimulation awaits the generation and analysis

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Sampling of inhibitors of TCR signalinga

Molecule

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Interactions and/or function

Deficiency phenotype

Csk (162, 163) (kinase)

Phosphorylates inhibitory Y505 of Lck

Csk knockdown in primary T cells: increased proliferation and IL-2 production

HPK1 (169, 170) (kinase)

Phosphorylates SLP-76 providing 14-3-3 binding site

Increased SLP-76, PLCγ1, LAT, ZAP-70, Vav1 phosphorylation Enhanced T cell proliferation Enhanced disease in EAE model

SHP1 (164, 165) (phosphatase)

Dephosphorylates Lck and ZAP-70 Association with Lck inhibited by Erk activation

Defective positive selection Splenomegaly, lymphadonopathy Alopecia, inﬂamed tissues

Sts-1 (171, 172, 174) (phosphatase)

Dephosphorylates ZAP-70 Binds c-Cbl and ubiquitinylated proteins

Sts-1−/− Sts-2−/− : hyperproliferative T cells and increased susceptibility to EAE

Dok-1, Dok-2 (167, 168) (adapters)

Binds Csk, RasGAP, SHIP-1 PTB domain can associate with CD3ζ ITAMs

Dok-1−/− Dok-2−/− : increased T cell cytokine production, enhanced pZAP-70, pLAT, and pErk Lupus-like renal disease Production of antidouble stranded DNA antibodies

c-Cbl, Cbl-b (175–177) (E3 ligase)

Catalyzes attachment of ubiquitin to proteins Promotes TCR downregulation

c-Cbl−/− : splenomegaly, increased TCR expression and ZAP-70 phosphorylation Cbl-b−/− : multiorgan lymphocytic inﬁltration, increased IL-2 production, autoantibody production

CTLA-4 (179, 182) (inhibitory receptor)

Binds ligands CD80 and CD86 Recruits phosphatases SHP1 and PPA2

Multiorgan lymphocytic inﬁltrates Splenomegaly Elevated serum immunoglobulin

PD-1 (180, 181) (inhibitory receptor)

Binds ligands PD-L1 and PD-L2 Recruits SHP1

Lupus-like disease Increased serum immunoglobulin

a

Abbreviations: Csk, C-terminal src kinase; CTLA-4, cytotoxic T lymphocyte antigen-4; Dok, downstream of kinase; HPK1, hematopoietic progenitor kinase 1; ITAM, immunoreceptor tyrosine-based activation motif; LAT, linker for the activation of T cells; PD-1, programmed death-1; PD-L1/PD-L2, PD-1 ligands; PLC, phospholipase C; PPA2, protein phosphatase A2; PTB, phosphotyrosine-binding domain; RasGAP, Ras GTPase-activating protein; SHIP-1, SH2 domain–containing inositol phosphatase 1; SHP1, SH2 domain–containing tyrosine phosphatase 1; SLP-76, SH2 domain–containing leukocyte phosphoprotein of 76 kDa; Sts-1, suppressor of T cell receptor signaling; ZAP-70, ζ-associated protein of 70 kDa.

of mice harboring a mutation at the indicated Lck serine residue. The importance of SHP1 function is evident, as mice deﬁcient in SHP1 develop severe autoimmunity (165). SHP1 can be recruited quickly to the TCR complex via its association with Lck. However, SH2 domain–containing phosphatases are typically recruited to phosphorylated ITIMs (immunoreceptor tyrosinebased inhibitory motifs) present in the cytoplasmic tails of cell surface receptors. Recently, the ITIM-bearing inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule

1 (CEACAM1) was identiﬁed as a potential candidate for SHP1 recruitment in human T cells (166). CEACAM1 upregulation occurs hours after stimulation; thus, SHP1 likely regulates initial TCR signaling events as well as late signaling termination events. Given the impact SHP1 activity has on immune regulation, it will be important to understand exactly how SHP1 is recruited into the TCR-ligated signaling complex. Adapter proteins also play a critical role in negatively regulating TCR signals. The downstream of kinase (Dok) adapter proteins,

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Dok-1 and -2, are expressed in T cells. Coordinated deletion or knockdown of these proteins results in increased TCR cytokine production and proliferation; prolonged phosphorylation of ZAP-70, LAT, SLP-76, and Akt; and the development of a lupus-like renal disease with high antidouble-stranded DNA antibody titers (167, 168). Dok can associate with several negative regulators including SHIP-1 (SH2 domain–containing inositol phosphatase), Csk, and Ras GTPase-activating protein (RasGAP); however, how these associations mediate the negative regulatory role of Dok proteins remains to fully elucidated. Recent structure/function studies in T cell lines showed that the phosphotyrosine-binding domain (PTB) of Dok-1 and -2 can bind the ITAM motif in CD3ζ, making it tempting to speculate that Dok may compete with ZAP-70 for ITAM binding (167). Whether this interaction is relevant in vivo remains to be shown. Dok proteins can also be recruited inducibly to a LAT-, Grb2-, and SHIP-1-containing molecular complex (168). This ﬁnding is intriguing and begs the question as to how LAT participates in both positive and negative signaling pathways. Determining the domains necessary for the assembly of this negative regulatory complex and whether it is separate from or a part of the stimulatory LAT-containing complex may provide insight into how early phosphorylation events are regulated. It has been suggested that SLP-76 can also be a target of negative regulation. The serine/ threonine kinase HPK1 inducibly binds to the SH2 domain of SLP-76. This kinase has both positive and negative effects on TCR signal transduction; however, its role as a prominent negative regulator was conﬁrmed recently by the description of HPK1-deﬁcient mice. HPK1-deﬁcient T cells exhibit enhanced phosphorylation of several early signaling molecules, and HPK1-deﬁcient mice are more susceptible to experimental autoimmune encephalomyelitis (EAE) than are wildtype mice (169). As a possible explanation for these phenotypes, two groups have shown that

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HPK1 can phosphorylate a serine residue in SLP-76 that mediates recruitment of 14-3-3 family members (169, 170). 14-3-3 proteins have been implicated in the regulation of several signaling pathways. Thus, although the precise mechanism by which HPK1 disrupts TCR signal transduction remains unclear, identiﬁcation of a SLP-76/14-3-3 interaction may indicate that this mechanism involves a conserved method of signal transduction regulation. The role of a novel family of proteins in regulating TCR signaling has become appreciated more recently. The suppressor of T cell receptor signaling (Sts) family of proteins contains two members, Sts-1 (TULA2) and -2 (TULA). Combined deﬁciency in these proteins leads to hyperproliferative T cells and an increased susceptibility to autoimmunity (171). When the phenotype of Sts-1 and -2 knockout mice was described, the mechanism of negative regulation was unclear. Although still not understood fully, the C-terminal phosphoglycerate mutase domain of Sts-1 acts as a phosphatase with speciﬁcity for Syk and, to a lesser degree, Src family members, thus providing a model for how Sts-1 may negatively regulate TCR signal transduction (172). Interestingly, Sts-2 has very little phosphatase activity and, in cell line models, can enhance ZAP-70 activation (173). Recent studies demonstrating that Sts-2 can bind to and induce the degradation of c-Cbl (a negative regulator of TCR signaling; see below) have provided a mechanism by which Sts-2 may act as a positive regulator of T cell activation (174). Whether net ZAP-70 activity is ultimately increased or decreased in response to an in vivo challenge likely depends on the relative expression of these two family members. Cbl proteins play an important role as negative regulators of T cell signaling. Two family members, c-Cbl and Cbl-b, are immune modulators, as deletion of each gene in vivo leads to hypercellularity and, in the case of Cbl-b, spontaneous mulitorgan inﬁltration (175, 176). Both c-Cbl and Cbl-b facilitate the ubiquitination of proteins, targeting them for degradation. They also mediate the downregulation of the TCR

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following stimulation with antigenic peptides and have been recently shown to play a role in the dissipation of the early signaling complex (176–178). Thus, Cbl proteins, by way of their ability to regulate protein degradation, provide an example of yet another means by which TCR signaling is terminated. Similar to the necessity of signals provided by the TCR to be complemented by costimulatory molecules, TCR-generated regulatory signals are also aided by coreceptor signals. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) are two examples of such receptors that limit the expansion and activation of TCR-triggered T cells. These molecules are found on activated T cells with peak expression 24–48 h after stimulation. Genetic studies have documented the importance of both for maintaining selftolerance. By 5–6 days after birth, CTLA-4deﬁcient mice exhibit activated peripheral T cells, splenomegaly, and lymphocytic inﬁltrates into nonlymphoid organs (179). Although less striking, PD-1-deﬁcient mice also develop autoimmune features, including the development of a lupus-like disease by 6 months of age (180). Like costimulatory receptors, inhibitory receptors utilize similar motifs and molecular pathways to those used by the TCR when propagating a negative signal; they may even provide docking sites for these shared molecules (181, 182). Another mode by which inhibitory receptors have been proposed to function is to utilize the same ligand and/or signaling molecules as costimulatory receptors, thus setting up the potential for competition or sequestration of ligands or key substrates. This means is best illustrated by CTLA-4 and CD28, which share the ligands CD80 and CD86 and counter each other in the regulation of cell cycle proteins, cytokine expression, and Cbl-b expression.

CONCLUSIONS As in the ﬁrst 10 years after identiﬁcation of the TCR, the past 15 years have seen a dramatic expansion in our understanding of the biochemical pathways triggered downstream of the TCR. We now appreciate more fully how PTKs couple to effectors and second messengers via the utilization of adapter proteins, how Ras and NF-κB pathways are activated, how Ca2+ ions are sensed, that TCR signaling can be required for the activation of other cell surface receptors, and that the actin cytoskeleton does more than just dictate cell shape. Studies investigating signal transduction by costimulatory molecules have highlighted the importance of both signal ampliﬁcation and the induction of additional signaling pathways, and discoveries of spontaneous or induced models of autoimmunity have brought negative regulation of these signaling pathways to the forefront. However, as often noted in this review, links between many signaling molecules and cellular outcomes remain poorly deﬁned. Future studies will be required to ﬁll gaps in our knowledge and likely will reveal new and unexpected interactions between currently known and unknown molecules. This review primarily focused on the signal transduction pathways in naive T cells. Over the next 15 years, it will be important to apply current and new knowledge to other T cell lineages, including T regulatory cells, natural killer T cells, and memory T cells, to establish whether these paradigms are universal. In the coming years, the population-based analyses that established the ﬁeld of signal transduction will be driven toward single cell analyses. We can anticipate that this technological shift will allow for better analysis of human T cells and the application of basic science to human disease.

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

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ACKNOWLEDGMENTS We thank Christopher P. Garvin for ﬁgure design, Dr. Art Weiss for helpful comments, and Justina Stadanlick for editorial assistance. LITERATURE CITED

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22–25. Three papers describing chimeric proteins that demonstrate the signaling capability of CD3ζ, supporting the notion that CD3 is the signaling component of the TCR complex.

26. Describes the cloning and initial characterization of ZAP-70, the Syk family PTK essential for coupling the TCR to its downstream signaling machinery.

30. The first ITAMindependent function for a CD3 molecule is identified in the PRR of CD3ε as a binding site for Nck.

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48. LAT−/− mice reveal a complete block in T cell development that, along with studies describing SLP-76−/− mice, exemplifies in vivo the essential role of adapter proteins for signal transduction.

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159. Watts TH. 2005. TNF/TNFR family members in costimulation of T cell responses. Annu. Rev. Immunol. 23:23–68 160. Chung JY, Park YC, Ye H, Wu H. 2002. All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction. J. Cell Sci. 115:679–88 161. So T, Lee SW, Croft M. 2008. Immune regulation and control of regulatory T cells by OX40 and 4-1BB. Cytokine Growth Factor Rev. 19:253–62 162. Vang T, Abrahamsen H, Myklebust S, Enserink J, Prydz H, et al. 2004. Knockdown of C-terminal Src kinase by siRNA-mediated RNA interference augments T cell receptor signaling in mature T cells. Eur. J. Immunol. 34:2191–99 163. Hermiston ML, Xu Z, Weiss A. 2003. CD45: a critical regulator of signaling thresholds in immune cells. Annu. Rev. Immunol. 21:107–37 164. Stefanova I, Hemmer B, Vergelli M, Martin R, Biddison WE, Germain RN. 2003. TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways. Nat. Immunol. 4:248–54 165. Tsui HW, Siminovitch KA, de Souza L, Tsui FW. 1993. Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene. Nat. Genet. 4:124–29 166. Nagaishi T, Pao L, Lin SH, Iijima H, Kaser A, et al. 2006. SHP1 phosphatase-dependent T cell inhibition by CEACAM1 adhesion molecule isoforms. Immunity 25:769–81 167. Yasuda T, Bundo K, Hino A, Honda K, Inoue A, et al. 2007. Dok-1 and Dok-2 are negative regulators of T cell receptor signaling. Int. Immunol. 19:487–95 168. Dong S, Corre B, Foulon E, Dufour E, Veillette A, et al. 2006. T cell receptor for antigen induces linker for activation of T cell-dependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2. J. Exp. Med. 203:2509–18 169. Shui JW, Boomer JS, Han J, Xu J, Dement GA, et al. 2007. Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses. Nat. Immunol. 8:84–91 170. Di Bartolo V, Montagne B, Salek M, Jungwirth B, Carrette F, et al. 2007. A novel pathway downmodulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76. J. Exp. Med. 204:681–91 171. Carpino N, Turner S, Mekala D, Takahashi Y, Zang H, et al. 2004. Regulation of ZAP-70 activation and TCR signaling by two related proteins, Sts-1 and Sts-2. Immunity 20:37–46 172. Mikhailik A, Ford B, Keller J, Chen Y, Nassar N, Carpino N. 2007. A phosphatase activity of Sts-1 contributes to the suppression of TCR signaling. Mol. Cell 27:486–97 173. Agrawal R, Carpino N, Tsygankov A. 2008. TULA proteins regulate activity of the protein tyrosine kinase Syk. J. Cell. Biochem. 104:953–64 174. Feshchenko EA, Smirnova EV, Swaminathan G, Teckchandani AM, Agrawal R, et al. 2004. TULA: an SH3- and UBA-containing protein that binds to c-Cbl and ubiquitin. Oncogene 23:4690–706 175. Bachmaier K, Krawczyk C, Kozieradzki I, Kong YY, Sasaki T, et al. 2000. Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b. Nature 403:211–16 176. Naramura M, Jang IK, Kole H, Huang F, Haines D, Gu H. 2002. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nat. Immunol. 3:1192–99 177. Murphy MA, Schnall RG, Venter DJ, Barnett L, Bertoncello I, et al. 1998. Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice. Mol. Cell. Biol. 18:4872–82 178. Balagopalan L, Barr VA, Sommers CL, Barda-Saad M, Goyal A, et al. 2007. c-Cbl-mediated regulation of LAT-nucleated signaling complexes. Mol. Cell. Biol. 27:8622–36 179. 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 270:985–88 180. Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, et al. 2001. Autoimmune dilated cardiomyopathy in PD-1 receptor-deﬁcient mice. Science 291:319–22 181. Chemnitz JM, Parry RV, Nichols KE, June CH, Riley JL. 2004. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J. Immunol. 173:945–54 182. Teft WA, Kirchhof MG, Madrenas J. 2006. A molecular perspective of CTLA-4 function. Annu. Rev. Immunol. 24:65–97

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175–177. Three papers demonstrating autoimmunity in Cbl−/− mice and identifying Cbl family members as critical negative regulators of T cell activation.

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Horror Autoinﬂammaticus: The Molecular Pathophysiology of Autoinﬂammatory Disease∗ Seth L. Masters,1 Anna Simon,2 Ivona Aksentijevich,1 and Daniel L. Kastner1 1

The National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892 and 2 Department of General Internal Medicine, Radboud University Nijmegen Medical Center, The Netherlands; email: [email protected], [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2009. 27:621–68

Key Words

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

innate immunity, IL-1β, inﬂammasome, type 2 diabetes mellitus, pulmonary ﬁbrosis, Crohn’s disease, ankylosing spondylitis, atherosclerosis

This article’s doi: 10.1146/annurev.immunol.25.022106.141627 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0621$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 The autoinﬂammatory diseases are characterized by seemingly unprovoked episodes of inﬂammation, without high-titer autoantibodies or antigen-speciﬁc T cells. The concept was proposed ten years ago with the identiﬁcation of the genes underlying hereditary periodic fever syndromes. This nosology has taken root because of the dramatic advances in our knowledge of the genetic basis of both mendelian and complex autoinﬂammatory diseases, and with the recognition that these illnesses derive from genetic variants of the innate immune system. Herein we propose an updated classiﬁcation scheme based on the molecular insights garnered over the past decade, supplanting a clinical classiﬁcation that has served well but is opaque to the genetic, immunologic, and therapeutic interrelationships now before us. We deﬁne six categories of autoinﬂammatory disease: IL-1β activation disorders (inﬂammasomopathies), NF-κB activation syndromes, protein misfolding disorders, complement regulatory diseases, disturbances in cytokine signaling, and macrophage activation syndromes. A system based on molecular pathophysiology will bring greater clarity to our discourse while catalyzing new hypotheses both at the bench and at the bedside.

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INTRODUCTION autoinflammatory: innate immune activation by endogenous pathways TRAPS: TNF receptor–associated periodic syndrome

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FMF: familial Mediterranean fever autoimmune: adaptive immune activation by endogenous pathways HIDS: hyperimmunoglobulinemia D with periodic fever syndrome crystalline arthropathies: diseases caused by uric acid or calcium pyrophosphate dihydrate crystal deposition fibrosing disorder: inﬂammatory disease that leads to ﬁbrosis

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The possibility of maladies in which the immune system turns against its host has long held a special fascination, dating back to the time of Paul Ehrlich and “Horror Autotoxicus” (1). Inherited illnesses, so-called “experiments of nature,” extend our understanding of genes and proteins we may have thought we understood while opening windows to the heretofore unimaginable. The publication of this review marks the tenth anniversary of the concept of systemic autoinﬂammatory diseases. The idea was initially proposed with the identiﬁcation of ectodomain mutations in the p55 tumor necrosis factor (TNF) receptor in patients with a dominantly inherited syndrome of fever and widespread inﬂammation (the TNF receptor– associated periodic syndrome, TRAPS) (2). This discovery followed close on the heels of the positional cloning of the gene for a similar recessively inherited illness, familial Mediterranean fever (FMF) (3, 4), and thereby raised the possibility that these disorders might be prototypes for an emerging family of inﬂammatory diseases. Both FMF and TRAPS are characterized by seemingly unprovoked, recurrent episodes of fever, serositis, arthritis, and cutaneous inﬂammation, but the usual hallmarks of autoimmunity, namely high-titer autoantibodies and antigen-speciﬁc T cells, are usually absent. The term autoinﬂammatory was coined to draw the distinction between this category of illnesses and the more classically recognized autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, in which the hallmarks of adaptive immunity are more evident. The following year witnessed the identiﬁcation of the gene underlying yet another hereditary fever, the hyperimmunoglobulinemia D with periodic fever syndrome (HIDS) (5, 6), and the positional cloning of a dominantly inherited gene that causes a curious syndrome of fever, malaise, and hives-like rash upon generalized cold exposure (7). Neither of these latter mendelian disorders ﬁts under the rubric of classical autoimmunity, thus further kin-

Masters et al.

dling the notion that autoinﬂammation might serve as the basis for a new taxonomy of human diseases that are in a sense complementary to the established autoimmune diseases. Based more on their clinical presentation than on a detailed understanding of their molecular basis, several other categories were added to the autoinﬂammatory universe, including metabolic disorders such as gout and other crystalline arthropathies, complement diatheses such as hereditary angioedema, granulomatous diseases such as Blau syndrome (chronic granulomatous synovitis with uveitis and cranial neuropathy), storage diseases such as Gaucher’s disease and Hermansky-Pudlak syndrome, ﬁbrosing disorders such as idiopathic pulmonary ﬁbrosis, and vasculitic syndromes such as Behc¸et’s disease (8). This formulation not only widened the phenotypic scope associated with autoinﬂammation but also extended the concept into the realm of genetically complex disease. Even more recently, several other classes of diseases have been placed under the autoinﬂammatory banner, including idiopathic febrile syndromes [systemic-onset juvenile idiopathic arthritis (SoJIA); adult Still’s disease; the syndrome of periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA)] (9, 10); pyogenic disorders such as the syndrome of pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) (11); and the autoinﬂammatory bone diseases (12). The notion that the inﬂammatory manifestations of these diseases are truly unprovoked is, of course, a relative matter, since we now know that a number of factors, including psychological stress, trauma, immunizations, cold exposure, and dietary indiscretion, may trigger some of these illnesses, but in all cases the autoinﬂammatory appellation implicitly posits a signiﬁcant host predilection. A truly useful disease nosology reﬂects not only clinical phenotype but also underlying biology, thereby suggesting previously unexpected relationships between/among illnesses, spawning new pathogenic hypotheses, and directing the clinician to novel therapeutic

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targets. At ﬁrst in parallel and relatively independently, the biologic basis for autoinﬂammation was established with the recognition of innate immunity as a phylogenetically ancient, hardwired, rapid-response system distinct from but, in mammals, intertwined with adaptive immunity (13). Over time, it became clear that the myeloid effector cells and germline molecules of innate immunity play a major role in the pathogenesis of many of the illnesses clinically classiﬁed as autoinﬂammatory, while the lymphoid cells and somatically plastic molecules of the adaptive immune system play a more signiﬁcant role in the classic autoimmune diseases. A watershed in the convergence of the clinical concept of autoinﬂammatory disease with the biology of innate immunity came with the discovery that three well-established autoinﬂammatory diseases are all caused by activating, gain-of-function mutations in NLRP3 (originally denoted CIAS1 for cold-induced autoinﬂammatory syndrome 1; also known as NALP3, PYPAF1, and CLR1.1), encoding what was then a newly recognized molecular linchpin in the innate immune system (7, 14, 15). The discovery of disease-associated mutations in NLRP3 solidiﬁed the nexus between autoinﬂammatory disease and innate immunity for several reasons. First, the NLRP3 protein product, originally called cryopyrin, now ofﬁcially denoted NLRP3, is a component of a macromolecular complex, the inﬂammasome, that senses various microbial products and endogenous “danger signals” (damageassociated molecular patterns, DAMPs) to activate caspase-1, thereby initiating IL-1β and IL-18 processing, a key step in the innate immune response (16). Second, the NLRP3 inﬂammasome may directly or indirectly interact with proteins mutated in other putative autoinﬂammatory diseases, including pyrin (in FMF) (17) and PSTPIP1 (in PAPA syndrome) (11). Third, the inﬂammasome has now been implicated in the pathogenesis of a number of diseases, such as gout (18) and pulmonary ﬁbrosis (19–21), included in the expanded deﬁnition of autoinﬂammation, thus providing

molecular vindication for the clinical classiﬁcation. Finally, NLRP3 is a prototype for a family of proteins, now known as the NLR family (22–24), that is intimately involved in the innate immune system, and recent evidence implicates other members of this protein family in human disease. With the recognition that these inﬂammatory diseases without hallmarks of adaptive immunity are in fact disorders of the innate immune system, it is possible to propose a new schema based upon underlying molecular mechanisms (Table 1). It should be stated at the outset that, as noted by McGonagle & McDermott (25), the spectrum of self-reactive immunological disease represents a continuum between autoimmune disorders based primarily on lesions of the adaptive immune system and autoinﬂammatory conditions rooted primarily in the innate immune system. Particularly for genetically complex disorders, multiple lesions of both branches of the immune system, with potentially self-amplifying loops, are quite possible. In keeping with the foregoing discussion, the ﬁrst types of autoinﬂammatory disease enumerated in the Table are IL-1β inﬂammasomopathies, deﬁned as disorders of macromolecular IL-1β-activating complexes, the prototypes of which are nucleated by NLRP3, but which may also include complexes of a number of related proteins. Intrinsic inﬂammasomopathies represent molecular lesions in the constituent proteins of the complex, while extrinsic inﬂammasomopathies denote disorders of various upstream or downstream regulatory elements. Although the IL-1β inﬂammasome represents a major conceptual advance in our understanding of innate immunity and related human disease, it is by no means the only molecular engine of innate immunity. In the Table, the proposed Type 2 autoinﬂammatory diseases, such as Crohn’s disease (CD) and Blau syndrome (BS), are caused in part by sequence variants in NOD2/CARD15 (26–28). While IL-1β undeniably plays a role in both illnesses, the NOD2/CARD15 protein plays a central role in NF-κB activation in response to intracellular www.annualreviews.org • Horror Autoinﬂammaticus

DAMP: dangerassociated molecular patterns NLR: nucleotidebinding domain and leucine-rich repeat containing

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Table 1

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Provisional molecular/functional classification of autoinflammatory disease

Disease

Gene (chromosome)

Protein (synonyms) or pathogenic stimulus

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Type 1: IL-1β activation disorders (inflammasomopathies) Intrinsic FCASa , MWSb , NOMIDc /CINCAd Extrinsic FMFf PAPAg CRMOj /SAPHOk Majeed syndrome HIDSl Recurrent hydatidiform mole DIRAm Complex/acquired Gout, pseudogout Fibrosing disorders Type 2 diabetes mellitus Schnitzler syndrome

NLRP3/CIAS1 (1q44)

NLRP3e (cryopyrin, NALP3, PYPAF1)

MEFV (16p13.3) PSTPIP1 (15q24–25.1) Complex LPIN2 (18p11.31) MVK (12q24) NLRP7 (19q13) IL1RN

Pyrin (marenostrin) PSTPIP1h (CD2BP1i )

Complex Complex Complex Sporadic

Uric acid/CPPD Asbestos/silica Hyperglycemia

Complex NOD2 (16p12) ATG16L1 (2q37.1) IRGM (5q33.1) NOD2 (16p12) NLRP12 (19q13.4)

Muramyl dipeptide NOD2n (CARD15) ATG16L1◦ IRGMp NOD2 (CARD15) NLRP12 (NALP12)

Lipin-2 Mevalonate kinase NLRP7 (NALP7, PYPAF3, NOD12) IL-1Ra

Type 2: NF-κB activation disorders Crohn’s disease

Blau syndrome FCAS2 (Guadaloupe periodic fever)

Type 3: Protein folding disorders of the innate immune system TRAPSq Spondyloarthropathies

TNFRSF1A (12p13) Complex HLA-B (6p21.3) ERAP1 (5q15)

TNFRSF1Ar (TNFR1, p55, CD120a)

CFH (1q32) MCP (1q32) CFI (4q25) CFB (6p21.3) Complex Complex CFH (1q32)

Complement factor H MCPv (CD46) Complement factor I Complement factor B Autoantibodies

SH3BP2 (4p16.3)

SH3-binding protein 2

UNC13D (17q21.1) PRF1 (10q22) STX11 (6q24.2) Complex LYST (1q42.3)

Munc13-4 Perforin 1 Syntaxin 11 Virus LYSTy (CHS1)

HLA-B27s ERAP1t (ARTS1)

Type 4: Complement disorders aHUSu

AMDw

Complement factor H

Type 5: Cytokine signaling disorders Cherubism Type 6: Macrophage activation Familial HLHx

Chediak-Higashi syndrome

(Continued )

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(Continued )

Disease

Gene (chromosome)

Protein (synonyms) or pathogenic stimulus

Griscelli syndrome X-linked lymphoproliferative syndrome Hermansky-Pudlak syndrome Secondary HLH Atherosclerosis

RAB27A (15q21.3) SH2D1A (Xq25) HPS1-8 Complex Complex

RAB27A SAPz HPS1-8aa Cholesterol

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a

Familial cold autoinﬂammatory syndrome; b Muckle-Wells syndrome; c neonatal-onset multisystem inﬂammatory disease; d chronic neurologic cutaneous and articular syndrome; e nucleotide-binding domain, leucine-rich repeat, and pyrin domain containing protein 3; f familial Mediterranean fever; g pyogenic arthritis, pyoderma gangrenosum, and acne; h proline serine threonine phosphatase-interacting protein; i CD2-binding protein 1; j chronic recurrent multifocal osteomyelitis; k synovitis acne pustulosis hyperostosis osteitis; l hyperimmunoglobinemia D with periodic fever syndrome; m deﬁciency of the interleukin-1 receptor antagonist; n nucleotide-binding oligomerization domain-containing; ◦ autophagy-related 16-like 1; p immunity-related GTPase family M; q TNF receptor–associated periodic syndrome; r TNF receptor superfamily 1A; s human leukocyte antigen B27; t endoplasmic reticulum aminopeptidase 1; u atypical hemolytic uremic syndrome; v membrane cofactor protein; w age-related macular degeneration; x hemophagocytic lymphohistiocytosis; y lysosomal trafﬁcking regulator; z SLAM-associated protein; aa Hermansky-Pudlak syndrome 1–8.

microbial products. Emphasizing the interplay between innate and adaptive immunity, granulomas feature prominently in both disorders. In a third member of the proposed NF-κB activation disorders, Guadaloupe variant periodic fever syndrome, mutations occur in NLRP12, encoding a regulator of NF-κB activation (29). A third group of autoinﬂammatory diseases is due to the biologic consequences of protein misfolding in cells of the innate immune system. The mendelian prototype of this process is TRAPS, in which missense substitutions in the p55 TNF receptor lead to misfolding (30) and ligand-independent activation of kinases and aberrant cytokine production. Similarly, in the spondyloarthropathies, misfolding of HLA-B27 appears to trigger the unfolded protein response (UPR) in macrophages and consequently inappropriate cytokine secretion (31–33). Disorders of the complement system, long recognized as a key component of innate immunity, can lead to a host of immunologic disorders. In some cases, such as the deﬁciency of the fourth component of complement, abnormal clearance of immune complexes leads to a classical autoimmune lupus-like picture. Here we focus on those instances in which the deﬁciency of complement regulatory factors produces an autoinﬂammatory phenotype, such as age-related macular degeneration and atypical

hemolytic uremic syndrome (Type 4 disease in the Table). Cherubism, a relatively newly recognized autoinﬂammatory disorder of the bone, is caused by mutations in an SH3-binding protein (34), which in animal models leads to heightened responsiveness to the cytokines M-CSF and RANKL, and increased osteoclastogenesis (35). Given the importance of cytokine signaling in the innate immune response, additional examples of aberrant cytokine responses will likely be identiﬁed in other autoinﬂammatory diseases, and hence the proposal of Type 5 disorders. Finally, macrophage activation is a common theme among a host of inﬂammatory diseases, and the genetics of familial hemophagocytic lymphohistiocytosis implicates loss-offunction lesions in the adaptive immune system as one cause of this (36, 37). Other mechanisms leading to activation of effector cells in the innate immune system and elaboration of a proinﬂammatory cytokine milieu also characterize Type 6 autoinﬂammatory disease, which possibly includes the leading cause of death in the Western world, atherosclerosis (38). There remain a number of human illnesses that are clearly autoinﬂammatory but do not fall neatly into any of the proposed categories. For some disorders, such as PFAPA, the molecular details are still too few to permit classiﬁcation,

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whereas for other syndromes, such as SoJIA, multiple molecular mechanisms are emerging. In yet other instances, such as Behc¸et’s disease, lack of detail and heterogeneity appear paradoxically to go hand in hand. Undoubtedly, the six types of autoinﬂammatory disease enumerated in Table 1 represent only a beginning but should serve not only to organize our thinking about existing data, but also to stimulate hypotheses about less well-understood phenotypes.

IL-1β ACTIVATION DISORDERS (INFLAMMASOMOPATHIES) Intrinsic Inflammasomopathies Cryopyrin-associated periodic syndromes. As disorders of an essential inﬂammasome protein, the cryopyrin-associated periodic syndromes (CAPS, also known as cryopyrinopathies) encompass a spectrum of disease states. In order of severity, from mild to severe, these include familial cold autoinﬂammatory syndrome (FCAS), which presents with coldinduced fevers, urticaria-like rash, and constitutional symptoms; Muckle-Wells syndrome (MWS), with fevers, hives, sensorineural hearing loss, and arthritis unrelated to cold exposure; and neonatal-onset multisystem inﬂammatory disease (NOMID) [or chronic infantile neurologic cutaneous articular (CINCA) syndrome], which presents with fever, urticaria, epiphyseal overgrowth of the long bones, and chronic aseptic meningitis. The cryopyrinopathies are due to autosomal dominant or de novo mutations of NLRP3 (7, 14, 15). The protein product of this gene (Figure 1) was ﬁrst denoted cryopyrin, although the current convention is NLRP3 (24). To avoid confusion, we have retained the cryopyrinopathy/CAPS disease nomenclature in this review, although we use the new terminology to denote the gene and protein. The original protein name was derived from the aforementioned association of symptoms with cold exposure in some patients (cryo), and its invariant ∼90 amino acid N-terminal death-fold 626

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motif, the pyrin domain (PYD) (39–41), that mediates cognate interactions with other proteins, and that it shares with the FMF protein, pyrin. Through this domain both NLRP3 and pyrin interact with an adaptor protein denoted ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain). NLRP3 belongs to the family of NLR (nucleotidebinding domain and leucine-rich repeat containing) proteins (24) implicated in inﬂammation and apoptosis, providing an additional clue as to its function. C-terminal leucine-rich repeats (LRRs) had already been implicated in sensing bacterial components, suggesting a role in the innate immune response. The case was strengthened with the discovery that, through pyrin domain–ASC interaction, the related protein NLRP1 nucleates the ﬁrst recognized IL-1β activating inﬂammasome (42), and the subsequent ﬁnding that NLRP3 itself participates in a somewhat different inﬂammasome that also exhibits pro-IL-1β processing activity (16). Although the inﬂammasome is also required for processing of IL-18, IL-33, and several other nontraditionally secreted proteins (43), most research has focused on how NLRP3 mutations affect IL-1β production, and certainly IL-1β is upregulated in this disease (15). Another domain of the NLRP3 protein is the ATP-binding cassette (nucleotide binding domain, NBD, or NACHT domain; Figure 1), which has now been conﬁrmed as such both in a cell-free system (44) and in a number of other in vitro settings (45), and is the major locus of CAPS-associated mutations. This leads to the intriguing possibility that these sequence variants may confer activation of the protein without the usual ATP costimulus, and certainly many of the mutations are structurally proximate to a hypothetical ATP-binding site (46). Indeed, Gattorno et al. in a recent publication, showed that mutation-positive NOMID and MWS patient peripheral blood mononuclear cells (PBMCs) secrete higher levels of IL-1β in response to LPS alone than healthy controls, but that with the addition of ATP, secreted IL-1β levels are comparable (47). This suggests that the mutations in NLRP3 have

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bypassed a requirement for ATP in IL-1β production. However, inactivating mutations at the ATP-binding site ablate the constitutive IL-1β production associated with MWS mutations in an overexpression system (45). This emphasizes the importance of ATP binding to NLRP3 activation and indicates that the ATP independence of MWS mutations may not be related to the requirement of ATP binding to the NACHT domain. Instead, NLRP3 mutations may function independently of exogenous ATP due to heightened downstream sensitivity to the effect of ATP on the P2X7 receptor at the cell surface, which is a well-documented stimulus for the processing and secretion of IL-1β (48, 49). It would be interesting to test these hypotheses in a cell-free system and to know the exact mechanism behind the gain of function of NLRP3 mutations. In 2006, four laboratories published data describing the role of NLRP3 in vivo from analysis of knockout mice. These ﬁndings are presented in greater depth in another review appearing in this issue of the Annual Review of Immunology (50). The results that are most relevant to the current discussion include the requirement of NLRP3 for macrophage IL1β production in response to Toll-like receptor (TLR) agonists plus ATP (51, 52), to Grampositive bacteria such as Staphylococcus aureus or Listeria monocytogenes (51), to bacterial RNA (53), to dsRNA and viral RNA (54), and to uric acid crystals (18). While NLRP3 is required to produce IL-1β in response to these many and varied insults, the disease-causing mutations in this protein do not seem to render CAPS patients clinically overresponsive when faced with these challenges in natura, although some patients anecdotally report increased resistance to common viral infections. Sutterwala and colleagues also observed that, like IL-1β, NLRP3 was required to generate contact hypersensitivity to the hapten trinitrophenylchloride (TNPCL) (52). This observation agrees with a recent body of work highlighting the critical role of the innate immune system in contact hypersensitivity, which is predominantly a T cell–mediated disease (55). Again, we have not observed that

CAPS patients manifest an increased delayedtype hypersensitivity reaction. One further aspect of the function of NLRP3 was indicated by the reduced level of macrophage cell death observed for cells of knockout mice in response to Gram-positive bacteria (Staphylococcus) (51). Willingham et al. showed that this was also true for infection with Shigella ﬂexneri, and independent of the inﬂammasome component ASC and of IL-1β (56). Monocyte/macrophage cell death has also been examined in overexpression systems (57), and with cells from patients directly (56, 58); in both cases, LPS-induced necrosis appears to be associated with the mutations in NLRP3 that cause CAPS. This comports with previous work that reported on a CAPS patient with low-level somatic mosaicism who did not inherit a mutated NLRP3 allele from either parent (59). Because the mutated cells are only a fraction of the total, being able to identify and sort them could facilitate better detection of this cause of disease. Indeed, by looking at just those cells undergoing necrotic cell death, Saito et al. found somatic mosaicism in three of four previously mutation-negative patients (58). However, this approach would seem to rely heavily on the genetic ﬁdelity of a dead or dying population of cells, which has not been established. Indeed, the very nature of a mutation undergoing selection in a small population of cells that proceed to rapid cell death seems somewhat contradictory, without some driving force for their continued production. Further lines of investigation that are sure to attract interest include the elucidation of possible genotype-phenotype correlations in the cryopyrinopathies and investigation of the structural basis for disease-causing mutations. While such studies would provide important mechanistic insight, it is very fortunate that therapy for these diseases based on current understanding is remarkably effective. For example, the IL-1 receptor antagonist, anakinra, has a very signiﬁcant impact in even the most severe cryopyrinopathy, NOMID/CINCA, with dramatic effects on not only the rash and acute-phase proteins, but also on the aseptic meningitis www.annualreviews.org • Horror Autoinﬂammaticus

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ery eight weeks). Unlike anakinra, which blocks IL-1β signaling at the receptor, rilonacept and canakinumab target IL-1β directly, and in the studies completed to date, these agents show great promise (61–63). It is interesting that patients with all of the clinical features of CAPS

and cochlear inﬂammation, which often lead to severe disability (60). Improved therapy for CAPS patients now focuses on new biologics with more favorable treatment schedules, such as rilonacept (administered once per week) and canakinumab (administered once ev-

Most common mutations Functional polymorphisms

G196W S208AfsX39 E225D P180R S242R S179I T267I T177I A268V P283L E167D R151S A289V E148Q E299G R143P

P780T M680I I772V M680L R761H T681I S749C G678E Y688X M692del A744S M694del F743L D661N M582L M694V V726A M694L R717S L649P M694I L709R A595V K695R P646L V704I I591T

G514E I506V F479L H478Y V469L

R354W P369S R408Q

T309M

Pyrin

PYD 1

b

bZIP

A89T

92

266

B-box

280

370

Coiledcoil

412 420

B30.2/SPRY

440

597

776

NOMID/CINCA MWS FCU/FCAS Mutations found in more than one disease

H358R A374D G307V F309S Q306L E354D M406I F523L(c>g) L305P L353P T405P F573S E304K E311K T436A F523L(c>a) A352V T587I I572F T436I,P,N F523C(t>g) R260L D303G H312P L571F V351M,L T436del R260P D303H Y570C,F E627G P315L R260W D303N A439V T348M G569A L632F G326E G301D A439T A495V G569R M659K C259W V262A A439P S331R E567K M662T G755A V262G V198M Y563N E688K R488K L264H,R,F I172T E690K G755R F443L Y859C L264V C148Y, R168Q E525K N477K S710C I480F

PYD 6

NACHT/NBD

W157R A140T R138Q

CARD 28

CARD 124 127

Figure 1 (Continued ) 628

T189M

T605P R587C E383K 558delLG D382E M513T R373C H496L R334Q C495Y R334W W490L R311W L469F A301V

ATP

Crohn’s disease Blau syndrome

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LRR

536

c

NOD2/CARD15

NAD

220

89

220

273

NACHT/NBD

573

742

990

N670K V793M E778K R703C R702W

M863V N853S

NAD

Cryopyrin

ATP

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A918D

1007fs

R1019X

G908R

LRR 744

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Mevalonic aciduria HIDS Mutations found in both diseases

G326R S329N,R T322S G25fs L234P V293MV310M A334T H44fs R388X H20P L39P T237S P288L G309S G211A A141fsX46 W62X W188X D386N H20N,Q G336S T243I L35S Q190fs G211E R277C A141fsX47 A147T N301T L246P G202R L29fs K13X E296Gfsx14 H380R G140fs A148T R277H V250I V203fs S378P S135L S272F Y149X L6fs V203A delEXON3 V377I I119M V132I I268T R215Q delEXON2 P167L G376V N205D L265R Y114fs delEXON5 C367S L265F L168fs E93fs T209A D366fs G171R delEXON8 L264F

Mevalonate kinase

GHMP-kinase

GHMP-kinase

130

TNF receptor type 1

F60L (C>G) F60L (A>G) T37I F60S C33Y Y38C C33G L39F C55R C30Y D42del C55S C55Y C30S C43R C30R C43Y C30F C43S C29Y C29F P46L H22D T50M T50K H22Y C52F Y20H C52Y Y20D C52R

-29

15

53

54

287

366

T61N N65I L67P H69fs C70G C70R C70S C70Y C73R C73W S74C S86P

Mutations aﬀecting cysteines Functional polymorphisms H105P F112I C129delins I170N V173D

C88R C88Y R92Q V95M C96Y CRD2

CRD1

212

CRD3 97

98

CRD4 138

139

Cleaveage

e

Signal pep

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Death domain

TM 183

205

316

412

Figure 1 Schematic representation of mutations in ﬁve proteins that cause autoinﬂammatory diseases. (a) For pyrin, mutations that are most frequently found to cause FMF are presented in red, while those in black are less common disease-causing variants. Residues in purple are found at approximately 1% allele frequency in the general population and may therefore represent functional polymorphisms. (b) Cryopyrin mutations cause a spectrum of disease states that range in severity from severe (NOMID/CINCA, red ), to intermediate (MWS, blue), to mild (FCAS, purple). In some instances, it is difﬁcult to distinguish between NOMID/CINCA and MWS. Residues for which disease presentation overlaps are depicted in black. (c) NOD2/CARD15 mutations can cause Blau syndrome/early-onset sarcoidosis (blue), which cluster within the NACHT/NBD and NAD domains. Other variants that are spread throughout this protein are associated with Crohn’s disease (red ). (d ) Mevalonate kinase mutations can cause the severe metabolic disease mevalonic aciduria (red ), or the less severe autoinﬂammatory disease HIDS (blue). HIDS mutations are recessively inherited and often include one mild mutation coupled with a severe mutation; thus, the severe mutations can be found in both HIDS and mevalonic aciduria (black). (e) Dominantly inherited missense mutations in TNFR1 that cause TRAPS are now known to affect almost every cysteine residue within the ﬁrst two cysteine-rich domains of the extracellular region of the protein (red ). These mutations appear to affect the protein folding, whereas at least two mutations, I170N and V173D, can affect ectodomain cleavage that generates the soluble form of the receptor. P46L and R92Q ( purple) are probably functional polymorphisms also present in unaffected individuals. The numbering system for TNFR1 used here begins at residue 30, which is at the N terminus after removal of the 29-residue signal peptide, as per common convention. Abbreviations used: PYD, pyrin domain; NBD, nucleotide-binding domain; NAD, NACHT-associated domain; LRR, leucine-rich repeats; CRD, cysteine-rich domain; TM, trans-membrane domain.

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but no demonstrable NLRP3 mutations also respond to therapy targeting IL-1β. This suggests an excellent opportunity for discovering additional disease-causing genes on the IL-1β axis.

Extrinsic Inflammasomopathies

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Familial Mediterranean fever (FMF). FMF is characterized by 1- to 3-day episodes of fever with sterile peritonitis, pleural inﬂammation, arthritis, and/or rash, sometimes complicated by systemic amyloidosis. While FMF is the prototypic autoinﬂammatory disease, the effect of the underlying disease-causing mutations on IL-1β production may be primarily extrinsic to the inﬂammasome (Figure 2). Linkage analysis (64) and positional cloning based on an autosomal recessive model of inheritance allowed two independent consortia to identify mutations in a single gene that cause this disease (3, 4). The gene, denoted MEFV for MEditerranean FeVer, encodes a protein product (Figure 1) alternatively termed pyrin (after the Greek for fever) or marenostrin (after the Latin for the Mediterranean Sea). The mutations causing FMF are present at a very high frequency in several populations, and for this reason the disease is more prevalent in the Mediterranean basin and Middle East (65). In the years following the discovery of mutations in MEFV, much has changed in our understanding of this disease. Perhaps most strikingly, as the sequencing of this gene has become widespread in patients and families outside those studied in the initial linkage analysis, we have discovered many patients with compatible symptoms, sometimes even classic manifestations, but only a single MEFV mutation. It is also true that many of the obligate carriers for single MEFV mutations may actually have subclinical evidence of inﬂammation (66), or even be periodically symptomatic for FMF (67) but not to the extent that would prompt medical evaluation or genetic diagnosis. Although most FMF patients inherit two pyrin mutations recessively, it appears that in some instances a more broadly deﬁned FMF is inherited in a 630

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dominant fashion, and in fact there are select mutations for which this does seem to be the case (68). However, for the remaining people with only one pyrin mutation, what determines if disease will present or not? An attractive hypothesis is that there is digenic or even multigenic inheritance of FMF. In such a scenario, a single pyrin mutation may require permissive alleles at one or more additional loci before FMF is manifest. Mutations are found throughout the MEFV gene (http://fmf.igh.cnrs.fr/infevers), but the most severe are clustered in exon 10 (69, 70), which encodes a motif known as the B30.2/SPRY domain (PRYSPRY), at the C terminus of the protein (Figure 1). Pyrin is the exemplar for the N-terminal domain that bears its name, and also contains B-box, bZIP basic, and coiled-coil domains. Pyrin is also known as TRIM20, as it is a part of a larger family termed the TRIpartite Motif (TRIM) proteins that typically have a RING domain (not present in pyrin), B-box, and coiled-coil domain, and frequently also the B30.2/SPRY domain (71). Initial speculation concerning the function of pyrin hypothesized that it was a transcription factor (3). Although DNA binding activity has not been ascribed to pyrin, the protein does contain two nuclear localization motifs (3, 72), and the endogenous protein does localize to the nucleus in granulocytes and dendritic cells (73). We have also observed the translocation of a speciﬁc N-terminal fragment of pyrin to the nucleus after cleavage by caspase-1 (74). N-terminal pyrin appears to activate NF-κB through increased calpainmediated degradation of IκB-α, and was also observed in patient leukocytes. Pyrin can also be demonstrated in the cytoplasm of monocytes, and much more has now been published regarding the role of the protein in this locale. One interesting ﬁnding was that pyrin interacts with tubulin and colocalizes with microtubules (75), suggesting a rationale for current highly efﬁcacious treatment of the disease using colchicine, a microtubule-destabilizing agent (76). More recently, pyrin was found to interact with ASC, the apoptosis-associated speck-like

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NF-κ κB activation disorders

IL–1β activation disorders

Activation MDP

Uric acid, CPPD, asbestos, silica, Aβ, cholesterol

Inhibition Translocation

Phagosomal destabilization

Production Association

ROS CathepsinB

MK*

ASC

RIP2

RAC1

NOD2

NLRP3*

NOD2*

pyrin*

NLRP12* NF-κB

caspase 1

5 p6

PI3K

NLRP7*

0

p5

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PSTPIP1*

Inflammasome

PKB

Nucleus RI -1 IL

proIL-1β

IL-1Ra*

Cytoplasm

IL-1β

Extracellular

Proinflammatory cytokines

Figure 2 Mechanisms of autoinﬂammatory disease regulated by IL-1β (inﬂammasomopathies) and NF-κB. The NLRP3 protein interacts with ASC and caspase-1 to form a complex termed the inﬂammasome, which is a macromolecular complex that processes IL-1β into its active form. Mutations (denoted by asterisks) in proteins that affect the function of this complex such as pyrin, PSTPIP1, mevalonate kinase (MK), and NLRP7 thus represent extrinsic inﬂammasomopathies. Mutations in NLRP3 clearly activate the molecule; however, the precise mechanism by which pyrin and PSTPIP1 mutations regulate inﬂammasome activity has not been determined. As NLRP7 is a negative regulator of IL-1β production, these mutations are likely to inactivate the protein, and it has been shown that MK mutations are also loss of function, although the pathway that leads from these mutations to Rac1/PI3K/PKB activation is not yet formally described. Preliminary data from our laboratory suggest that mutations in the IL-1 receptor antagonist (IL-1Ra) cause another extrinsic inﬂammasomopathy. For NF-κB activation disorders, NOD2 could become activated by mutations that may relieve autorepression of the molecule; however, there are also persuasive data showing that mutations inactivate the protein from tolerizing the host to the bacterial cell wall component muramyl dipeptide (MDP). NLRP12 is a negative regulator of NF-κB, and thus a proinﬂammatory disease would be caused by what appear to be loss-of-function mutations. Abbreviations used: CPPD, calcium pyrophosphate dihydrate; Aβ, amyloid-β; ROS, reactive oxygen species; PKB, protein kinase B. www.annualreviews.org • Horror Autoinﬂammaticus

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protein with a caspase-recruitment domain (CARD), through cognate pyrin domain association. When this interaction was ﬁrst described, it was the apoptotic effect of ASC that was addressed, and indeed wild-type pyrin, when overexpressed in HeLa cells, appeared to increase ASC speck formation and, paradoxically, increase the survival of these cells (77). Macrophages from mice harboring a truncated form of pyrin have subsequently been shown to manifest a defect in apoptosis (17). In addition to its role in apoptosis, ASC also nucleates inﬂammasome complexes through the homotypic interactions of its pyrin domain and CARD with NLRP proteins and inﬂammatory caspases, respectively (78), thus activating IL1β (Figure 2). The direct interaction of pyrin with ASC suggests potential molecular mechanisms for the inﬂammatory hallmarks of FMF, either if pyrin inhibits IL-1β activation by competing with caspase-1 for ASC (17) or if pyrin itself forms an inﬂammasome complex (79). Depending on experimental conditions, there are data to support both of these formulations and the corresponding antiinﬂammatory and proinﬂammatory effects for wildtype pyrin. The initial observation to support an antiinﬂammatory role for pyrin concerned mice deﬁcient for MEFV that exhibit increased cytokine production and subsequent lethality induced by LPS (17). Because murine MEFV lacks a signiﬁcant portion of exon 10, including the region where FMF mutations reside, many experiments using human constructs have subsequently been performed. We have shown that the B30.2 domain of pyrin interacts with caspase-1 and that this inhibits the production of mature IL-1β (80). Papin et al. have veriﬁed this interaction and the consequent inhibition of IL-1β processing (81), but the effect of FMFassociated mutations in the B30.2 domain remains controversial. On the other hand, Yu et al. reported that overexpression of pyrin in 293T cells that stably express ASC and procaspase-1 appears to activate an inﬂammasome complex, although overexpressed mutant forms of pyrin did not result in higher levels of caspase-1 activation (79). A different approach to address this

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issue is to study the effect of a reduction in the level of pyrin. Using siRNA to decrease the level of pyrin in THP-1 cells resulted in increased IL-1β production in vitro (80, 81). Nevertheless, under different experimental conditions ectopic silencing of pyrin resulted in decreased IL-1β production (82), suggesting that the function of pyrin may be context dependent, with dual roles that are physiologically relevant. Additional studies of peripheral blood leukocytes from FMF patients and healthy controls have been performed to identify a predominant role for pyrin in vivo, but again the results are contradictory. In favor of a proinﬂammatory effect for wild-type pyrin, the ability of macrophages from healthy donors to process IL-1β compared to the monocytes from which they are derived is signiﬁcantly reduced, corresponding to the reduced amount of pyrin that these cells express (82). In contrast, the same laboratory showed an association of reduced mRNA levels of pyrin, IL-10, and IL1 receptor–associated kinase (IRAK)-M with survival in pediatric multiple organ dysfunction syndrome, consistent with an antiinﬂammatory role for pyrin in cytokine production (83). In FMF patients, Ustek et al. observed reduced levels of MEFV message at baseline, with further reductions during disease ﬂares (84), whereas our preliminary analysis of pyrin protein expression in peripheral blood neutrophils shows an increase in FMF patients. Further complicating the picture, it is not clear whether changes in mRNA or protein expression reﬂect the underlying pathophysiology of FMF or a compensatory mechanism. Given the autoinﬂammatory phenotype in FMF, one might associate an antiinﬂammatory effect of the wild-type protein and lossof-function mutations with recessively inherited disease, or a proinﬂammatory role of the wild-type protein and gain-of-function mutations with dominantly inherited disease. At present, neither the effect of the pyrin protein in experimental models, nor the effect of mutations on gene expression, nor the mode of inheritance of the disease is entirely unambiguous. It has therefore been difﬁcult to construct

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a detailed molecular account of FMF pathogenesis, and we have provisionally opted to denote FMF as an extrinsic inﬂammasomopathy, recognizing that further reﬁnements may be necessary. These points notwithstanding, pyrin does play an important role in the regulation of IL-1β in experimental systems, suggesting that parenteral administration of the recombinant IL-1 receptor antagonist, anakinra, would ameliorate the inﬂammatory attacks of FMF. Indeed, there are now several anecdotal reports vindicating this concept (80, 85–89), although anakinra appears not to have as uniformly dramatic an effect as that seen in the cryopyrinopathies. The microtubule-inhibitor colchicine, which is given orally and is markedly less expensive than anakinra, remains the drug of choice in FMF. IL-1β inhibitors may play an adjunctive role in the treatment of the 5–10% of FMF patients for whom colchicine is ineffective or not tolerated. The proposed mechanisms by which colchicine prevents the attacks of FMF relate both to the mutant protein, pyrin (which binds to microtubules), and to the granulocytes that are a major locus of pyrin expression. A more complete understanding of how colchicine works in FMF and why it sometimes does not work would likely shed additional light on the function of pyrin, reinforcing the possibility of as yet undiscovered additional functions for this protein. Perhaps the most engaging concept pertinent to any discussion of FMF is the evolution of disease-causing mutations. Several aspects in particular are noteworthy. First, carriers of FMF mutations are far more frequent in certain populations than would be expected. Under the simplifying assumption of autosomal recessive inheritance, even if homozygotes for disease mutations all die before reproductive age, their frequency varies with the square of the mutant allele frequency, which is much less than the frequency of heterozygous carriers, and thus natural selection would have increased the frequency of mutations only if they conferred a heterozygote advantage. Second, different mutations predominate in different populations

(Figure 3a). This reinforces the possibility of a selective advantage, as genetic drift would be unlikely to account for the increased carrier rate of several different FMF mutations in these various populations. Third, the most common and potentially most severe mutations cluster in one domain of the protein, the B30.2/SPRY domain. This domain is not conserved among lower species (90), which highlights the relatively recent evolution of pyrin. This is also true for the majority of the TRIM protein family that has rapidly expanded in higher organisms. More than 20 TRIM family proteins exhibit the ability to affect the retroviral life cycle (to date, pyrin has not been found to have such an effect) and some of these TRIM proteins confer innate immune resistance to retroviruses (91). Finally, in nonhuman primates the wildtype pyrin protein includes several residues that are associated with disease in humans (92). For humans, FMF mutations often represent a reversion to ancestral amino acids, and modeling suggests episodic positive selection for the current human wild-type sequence. This argues that nonhuman primates may live with endemic pathogens against which pyrin mutations confer resistance, and that FMF mutations may also confer resistance against a similar, perhaps geographically restricted, pathogen for humans. Recent NMR and crystallographic data suggest a possible structural basis for the evolutionary data. The ﬁrst structure for the B30.2/SPRY domain was an NMR model derived from a murine protein SSB-2 (93). Even in this ﬁrst model, it was apparent that FMF mutations predominantly affected one face of the molecule, and mutational analysis of the SSB-2 protein showed that a protein-binding site was present at this region. Further information has come from crystal structures of other B30.2/SPRY domains in complex with their binding partners (94, 95). This more deﬁnitively described the protein interaction surface of the domain, and models of the pyrin B30.2/SPRY domain show where FMF-associated residues map close to the binding pocket (Figure 3b). Although several FMF mutations do not map near this region, the initial observation that the least severe www.annualreviews.org • Horror Autoinﬂammaticus

inflammasomopathy: diseases that result from increased levels of secreted IL-1β, either directly or indirectly

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a Ashkenazi

V726A M694V

V726A 3.0%

E148Q Central Europe 4.0%

E148Q Silk Road

M694V 0.2% Armenia

Turkey

Japan Iraq

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North Africa

China

Japanese

Sephardi

M694V 7%

E148Q 23%

India

E148Q 5% V726A 0.5%

b

Predicted structure of the pyrin B30.2/SPRY domaIn M694V B30.2/SPRY binding pocket

V726A

90°

M680I

Figure 3 The evolution and structural ramiﬁcations of ancestral mutations in pyrin. (a) This ﬁgure highlights both that pyrin mutations are very common in several Mediterranean populations and that it is not the same mutation that is expanded in each population. Blue and red lines indicate the migration of populations carrying the M649V and V726A mutations, respectively, while the E148Q mutation ( purple) is common to countries along the medieval trade route (the Silk Road) that extended from the Middle East through to Japan. Pie diagrams indicate the carrier frequencies of selected mutations in Ashkenazi Jewish, Sephardi Jewish, and Japanese populations. (b) A model for the structure of the B30.2/SPRY domain of pyrin (based on that of TRIM21) indicates the preponderance of FMF mutations (cyan) affecting one side of the molecule that is thought to present a protein-binding pocket. A phenotype-genotype correlation is proposed that links the more severe disease-causing mutations (M694V blue, M680I green) to the putative binding site, whereas a less severe mutation (V726A red ) is located further away from this region.

FMF mutations map further from the binding interface gives a structural understanding to the potential genotype-phenotype correlation in FMF (93). Together, these evolutionary and structural data point to the possibility that pyrin is capable of directly binding to a pathogen, or to another moiety that acts as a surrogate marker of infection. Identiﬁcation of 634

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such a mechanism would represent a major advance in our understanding of the innate immune host defense as it relates to the mechanisms of autoinﬂammatory disease. Pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) syndrome. PAPA syndrome is a dramatic hereditary

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autoinﬂammatory disorder of the skin and joints for which there is clear evidence of an important, although not necessarily exclusive, pathogenic role for IL-1β. PAPA syndrome was initially described by two different groups, several years apart (96, 97). Both groups observed families with recurrent arthritis, sterile but purulent synovial ﬂuid, and cutaneous manifestations such as pyoderma gangrenosum (large, open purulent lesions) and severe cystic acne. The genetic basis for this dominantly inherited condition was found to be mutations of CD2-binding protein 1 (CD2BP1) (98). This protein is now usually denoted by the name of its murine ortholog, proline serine threonine phosphatase-interacting protein 1 (PSTPIP1), which interacts with a PEST [rich in proline (P), glutamic acid (E), serine (S), and threonine (T)] type protein tyrosine phosphatase (PTPPEST). Initially, it was shown that the mutations that cause PAPA syndrome diminish the interaction of PSTPIP1 with PTP-PEST, but the relevance of this ﬁnding for the promotion of an autoinﬂammatory disease was unclear. Subsequent data from our laboratory established that PSTPIP1 interacts with the prototypic autoinﬂammatory protein, pyrin (11), thus suggesting potential molecular mechanisms for the proinﬂammatory aspects of PAPA syndrome (Figure 2). Furthermore, the PAPA mutations of PSTPIP1 lead to a stronger interaction with pyrin (FMF mutations in pyrin, however, do not have a similar effect). The increased interaction with pyrin is most likely because the PSTPIP1 variants bind PTP-PEST less avidly and are therefore hyperphosphorylated, and because the avidity of PSTPIP1 for pyrin varies with PSTPIP1 phosphorylation status. Both in vitro and ex vivo, PAPAassociated variants were associated with increased IL-1β production. If one takes the view that pyrin inhibits inﬂammation, PSTPIP1 mutations could be seen to function by increased sequestration and impairment of pyrin function (11). On the other hand, Yu and colleagues (99) have shown that the domain of pyrin to which PSTPIP1 binds (the B-box) is an autoinhibitory domain that constrains ASC binding

and thereby prevents the formation of the pyroptosome, a large molecular assembly of ASC and pyrin molecules of which there is only one per cell (100). Pyroptosome formation rapidly proceeds to cell death and the release of proinﬂammatory cytokines such as IL-1β. By this formulation, the stronger interaction of the PAPA mutants with the B-box of pyrin would relieve the autoinhibitory constraint and promote inﬂammation via pyrin engagement in pyroptosomes. While the mechanisms proposed by Shoham et al. (11) and Yu et al. (99) differ respectively as to whether wild-type pyrin is an intermolecular inhibitor of IL-1β production or is itself prevented from activating IL-1β by intramolecular interactions, both agree that increased binding by PSTPIP1 mutants would work against this constraint. Despite anecdotal evidence for the efﬁcacy of the IL-1 receptor antagonist anakinra in the articular attacks of PAPA syndrome (101), broader experience suggests that IL-1 inhibition is probably not as effective a strategy in PAPA syndrome as for CAPS, and thus raises the possibility that PSTPIP1 mutations may have additional pathophysiologic effects. That PSTPIP1 is also very highly expressed in T cells lends itself to the hypothesis that PAPA syndrome may at some level arise due to improper activation of the adaptive immune response. PSTPIP1 interacts with a number of T cell proteins, including the WiskottAldrich syndrome protein (WASp), CD2, and Fas ligand (FasL) (102–104). PSTPIP1/PTPPEST binding inhibits WASp phosphorylation, which is required for activated T cell transcriptional activity, actin polymerization, and immunologic synapse formation. Although PAPAassociated PSTPIP1 mutations did not affect WASp binding in yeast, in mammalian cells such mutations may decrease binding as a result of PSTPIP1 hyperphosphorylation and could thereby potentiate T cell activation (98). PTP-PEST/PSTPIP1 interaction with CD2 inhibits T cell activation (103), and, if PAPA mutations inhibit this interaction, T cell activation could ensue. PSTPIP1 also interacts with FasL in T cells, and when PSTPIP1 is www.annualreviews.org • Horror Autoinﬂammaticus

Pyroptosome: macromolecular complex of ASC and pyrin molecules, of which there is only one per cell

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overexpressed, FasL is retained in the cytoplasm of the T cell, thus preventing its cytotoxic activity (104). Although the net effect of PAPA-associated PSTPIP1 mutants on T cell function in vivo is unknown, the adaptive immune response could provide at least a trigger for PAPA ﬂares, notwithstanding the fact that the neutrophil is by far the predominant effector cell in PAPA lesions. Among a number of unanswered questions is the relative contribution of innate and adaptive immunity in the pathogenesis of PAPA. The dominant mode of inheritance suggests a gain of function for mutant PSTPIP1, consistent with its documented increased binding to pyrin, but which may extend to its interactions with binding partners in T cells or other lymphocyte subpopulations. Careful examination of protein interactions in patient cells, and the development of knockout and knockin animal models, may help address basic mechanistic issues.

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Chronic recurrent multifocal osteomyelitis (CRMO) and synovitis acne pustulosis hyperostosis osteitis (SAPHO) syndrome. In the cmo mouse model, spontaneous recessive mutations in Pstpip2 (a murine gene encoding a protein homologous to the PAPA syndrome protein PSTPIP1) cause autoinﬂammatory bone disease (105, 106). This most closely resembles CRMO, a human disorder presenting as bone pain, lytic bone lesions on radiographs, and culture-negative inﬂammatory inﬁltrates of the bone, with or without fever (12, 107, 108). CRMO usually presents early in life (∼10 years of age), and in most patients treatment with NSAIDs can ameliorate symptoms. CRMO is often associated with other inﬂammatory syndromes, such as palmar-plantar pustulosis, psoriasis vulgaris, and inﬂammatory bowel disease. SAPHO syndrome is characterized by the conditions abbreviated in its acronym (synovitis, acne, pustulosis, hyperostosis, and osteitis), and most of these are coincident with CRMO, although skin involvement plays a larger role in SAPHO. Therefore, CRMO and SAPHO syndrome probably form a spectrum of disease with one difference being that CRMO is usu636

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ally a pediatric disease, whereas for the most part SAPHO syndrome is not. A genetic inﬂuence is clear in some families, with affected siblings, concordant monozygotic twins, and indeed an autosomal recessive form of CRMO with congenital dyserythropoietic anemia, known as Majeed syndrome, that is caused by mutations in the gene LPIN2. Although little is known about the function of LPIN2, it has been suggested that its upregulation in macrophages with oxidative stress could relate to a possible role in regulating the innate immune response (107). By this hypothesis, disease-associated LPIN2 mutations might disturb its protective function against oxidative stress, thus causing tissue damage and polymorphonuclear (PMN) cell inﬂux. This is also how pathology in the spontaneous cmo murine disease proceeds, with neutrophilic osteomyelitis and ensuing bone resorption that closely mirrors both Majeed syndrome and CRMO as a whole (105, 106). A second mouse model, termed Lupo, exhibiting a similar phenotype primarily in distal appendages, was subsequently generated by random mutagenesis and found to have a different missense mutation in Pstpip2 (106, 109). Lupo is intrinsic to the hematopoietic system as documented by adoptive transfer of affected bone marrow, and the phenotype is unchanged when examined in mice lacking both T and B cells. This characteristic agrees well with the expression of human PSTPIP2 predominantly in monocytes and macrophages and, because expression of mutant Pstpip2 is much lower in Lupo mice relative to controls, suggests that the function of the gene is antiinﬂammatory. The functional relationship between PSTPIP2 and PSTPIP1 remains unclear, particularly because PSTPIP2 lacks the C-terminal SH3 domain that mediates interactions with many different binding partners for PSTPIP1 including pyrin. The human PSTPIP2 gene is encoded within a genomic interval found to be associated with sporadic CRMO by transmission disequilibrium testing (110), although to date no PSTPIP2 mutations have been identiﬁed in sporadic cases.

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The classiﬁcation of CRMO and SAPHO as disorders of IL-1β inﬂammasome regulation is conjectural at the present juncture, but it does immediately suggest additional lines of investigation both in mice and humans. In the animal model, breeding of intercrosses of Pstpip2 mutant mice with mice deﬁcient in various IL-1β inﬂammasome components will be of great interest. In humans, the potential efﬁcacy of IL-1 inhibition in patients with the CRMO/SAPHO disease spectrum may provide additional data to support or refute the importance of this cytokine in disease pathogenesis. Hyperimmunoglobulinemia D with periodic fever syndrome (HIDS)/mevalonate kinase deficiency (MKD). Initially, mevalonate kinase (MVK), the gene responsible for HIDS (5, 6), was enigmatic as a gene causing autoinﬂammatory disease without clear links to the immune system. The mevalonate kinase (MK) enzyme is ubiquitously expressed, and by converting mevalonic acid to 5-phosphomevalonic acid it catalyzes an early step in the biosynthetic pathway for cholesterol and nonsterol isoprenes. The most severe loss-of-function mutations in MVK, when present on both chromosomes, result in mevalonic aciduria, with pathology including recurrent fever, mental retardation, developmental abnormalities, and often early death (111). In HIDS there may be approximately 5% residual enzymatic activity (often due to a mild mutation in trans with a more severe mutation), and these patients have the recurrent fever, lymphadenopathy, abdominal pain, and rash associated with HIDS, but not the more severe manifestations of mevalonic aciduria. Whereas the mutations associated with mevalonic aciduria tend to be clustered in sites important to the catalytic activity of the enzyme, HIDS-associated mutations are more broadly distributed throughout the protein sequence (Figure 1). Preliminary hypotheses suggested opposing mechanisms for the disease pathogenesis in HIDS, one being that mevalonic acid is present in excess and is therefore toxic, the other that isoprenoid biosynthesis is deﬁcient, leading to perturba-

tions in signaling pathways (5, 6). Defective apoptosis of lymphocytes from HIDS patients is also suggested to be a cause of disease, although the molecular basis of this is uncertain (112). More recently, investigation into the signaling pathways that may be affected by alterations in the MK pathway has implicated the small GTPase Rac1, phosphoinositide 3kinase (PI3K), and protein kinase B (PKB) as key molecules that could give rise to activated caspase-1 (and hence IL-1β production and inﬂammation) due to isoprenoid deﬁciency (113). These data come from a model of isoprenoid deﬁciency created using simvastatin, an inhibitor of HMG-CoA reductase that should mirror the effect of loss-of-function mutations in MK by lowering mevalonate levels. Simvastatin treatment of THP-1 cells after exposure to LPS resulted in increased IL-1β secretion that was abrogated by Rac1 and PI3K inhibition, but further increased when constitutively active PKB was overexpressed (Figure 2). Replication of these ﬁndings in vivo, perhaps using the recently developed murine model of HIDS from a mouse lacking one Mvk allele (114), is necessary. Nevertheless, it is very encouraging to see that inhibition of Rac-1 lowers the levels of IL-1β produced by HIDS PBMCs ex vivo, as this suggests a new avenue for treatment of HIDS that may be directly targeted to the disease mechanism (113). In the aforementioned murine model (114), heterozygous knockout mice for the Mvk gene have a clear accumulation of mevalonic acid in several organs and signiﬁcantly increased serum concentration of IgD. These mice also have a higher incidence of hepatomegaly (25%) and splenomegaly (33%) and a higher serum concentration of TNF-α. The involvement of IgD in the pathogenesis of HIDS is unlikely (115). Symptoms can occur in children some years before the elevation of serum IgD concentration is found, and IgD concentrations do not correlate with disease activity or severity. In addition, a number of mutation-positive patients have a normal concentration of IgD, and occasional patients with other recurrent www.annualreviews.org • Horror Autoinﬂammaticus

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fever syndromes have modestly elevated serum IgD (116). There is not yet consensus in the literature about treatment of HIDS, although preliminary reports on the efﬁcacy of anakinra are consistent with the involvement of IL-1β in this disease (117, 118). Furthermore, it seems that, especially for adult HIDS patients with infrequent and/or less severe attacks, anakinra need not be used prophylactically but instead can be administered at symptom onset to lessen the severity and duration of attacks. For this reason, and because of the new biochemical data linking MK to IL-1β, we feel comfortable discussing HIDS as an extrinsic inﬂammasomopathy. An argument could also be made to classify HIDS as a protein-folding disorder because the mutations in MK appear to affect protein folding (119). Culture of patient ﬁbroblasts in circumstances that promote a more controlled protein folding increased the residual mevalonate kinase enzyme activity. Intercrossing Mvk heterozygous mice with ASC- and/or Nlrp3deﬁcient mice should conﬁrm the role of the inﬂammasome in disease or suggest an alternative mechanism of disease pathogenesis.

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Recurrent hydatidiform mole. Recurrent hydatidiform mole is a relatively rare condition that underscores the important connections between reproductive biology and innate immunity. In general, hydatidiform mole is a benign trophoblastic tumor presenting as a pregnancy with the absence of embryonic development and with cystic degeneration of chorionic villi (120). This condition may occur in as many as 1 in 1500 pregnancies, although recurrent hydatiform mole (RHM) comprises only about 2% of the total. RHM has a strong genetic contribution, and multiple cases within individual families have been described (120). Although there is biparental inheritance of RHM, DNA methylation at imprinted loci is abnormal, with either a defective maternal contribution or paternalspeciﬁc methylation patterns on maternally inherited alleles (121). Therefore it was somewhat surprising when the ﬁrst genetic cause of RHM was identiﬁed as NLRP7 (NALP7, PYPAF3) 638

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(122), an innate immune gene without a known direct role in DNA methylation. Women with RHM are homozygous or compound heterozygous for NLRP7 mutations, whereas males homozygous for the same mutation have normal reproduction. The NLRP7 protein is similar in structure to NLRP3 and NLRP1, with an Nterminal pyrin domain, a NACHT/NBD motif, and a C-terminal LRR domain. NLRP7 is not known to function as NLRP1 and NLRP3, to activate IL-1β, but instead is thought to act as a negative regulator of IL-1β, perhaps induced by inﬂammatory cytokines as part of a negative feedback loop (123). NLRP7 was also found to be expressed in target organs such as uterus, endometrium, and ovary (122), and therefore the regulation of the involvement of inﬂammatory processes during pregnancy becomes the new hypothetical mechanism of disease, and DNA methylation differences may instead only be as a result of tissue inﬂammation. The role of the innate and adaptive immune systems during pregnancy is a fascinating topic, as they control a very delicate equilibrium between fetal rejection and defense against infection. The balance appears to change during the course of pregnancy, with a predominance of pro- or antiinﬂammatory cytokines required at different stages (124). For brevity, we discuss only IL-1, although functional data implicate many other cytokines, chemokines, receptors, and complement molecules (125). Regarding IL-1, it has been shown that IL-1β induces preterm delivery in mice and that an intronic polymorphism in the IL-1 receptor antagonist (IL-1RN) may be associated with preterm birth of humans (126). The relevant IL-1RN polymorphism is also associated with increased IL-1β production in vitro (127), and thus one could hypothesize that inactivating mutations in NLRP7, a protein that negatively regulates IL-1β, could similarly shift the balance in favor of increased IL-1β activity in vivo. Perhaps this speculation is somewhat premature because the key biochemical experiments looking at IL-1β production in the presence of mutant NLRP7 molecules have not yet been reported, let alone the in vivo correlates of this in animal models.

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It is interesting that two NLRP7 mutations may preclude the possibility of normal pregnancy (128), whereas heterozygous mutations in females predispose to still birth and spontaneous abortions (129). The emerging phenotypic picture may be a complex interplay of NLRP7 genotypes with other genetic and environmental effects. Understanding how innate immunity, IL-1β, and NLRP7 are regulated to allow successful pregnancy is certain to advance over the next few years.

Complex/Acquired Inflammasomopathies Gout/pseudogout. Gout and pseudogout are common rheumatic diseases caused by the respective deposition of monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals in the joints and periarticular tissues, leading to acute and chronic inﬂammation. In the sense that metabolic perturbation provokes inﬂammatory activation, the crystal deposition diseases could be considered autoinﬂammatory (8), but the molecular details, particularly regarding the innate immune system, were at ﬁrst lacking. Martinon and colleagues (18) subsequently made a key breakthrough by showing that NLRP3 plays a pivotal role in the inﬂammatory complications of MSU and CPPD crystal deposition (Figure 2). Both MSU and CPPD crystals increased caspase-1 activation and IL-1β secretion from macrophages stimulated with LPS, but this did not occur in ASC- or Nlrp3deﬁcient macrophages (18). Furthermore, the neutrophil inﬂux generated in a mouse model of crystal-induced peritonitis was reduced in mice deﬁcient for inﬂammasome components. More evidence for a role of IL-1 in crystalline arthropathies comes from mice deﬁcient in the adaptor protein MyD88, which are resistant to MSU-initiated gouty inﬂammation (130). MyD88 transduces both TLR and IL-1 receptor signaling, and although mice deﬁcient for various TLRs still exhibit inﬂammation, those deﬁcient for the IL-1β receptor do not, suggesting that IL-1 signaling speciﬁcally con-

trols disease progression in this model. Bone marrow reconstitution experiments demonstrated that the IL-1 receptor was required in nonhematopoietic cells, but not hematopoietic cells, for acute gouty inﬂammation. Small pilot studies of anakinra suggest that this research is translatable into the clinic (131, 132). While this supports the role of the IL-1β inﬂammasome in the crystalline arthropathies, IL-1 inhibition is unlikely to have the same impact in these illnesses as in CAPS, given the availability of alternative approaches. Lifestyle change and nonbiological agents such as colchicine will likely remain the mainstay of treatment. This is not to say that IL-1 inhibition will not become increasingly useful as the drugs targeting this pathway are improved for half-life, cost, or oral absorption. Even with currently available agents, randomized controlled studies may support the efﬁcacy and cost-effectiveness of IL-1 inhibition in the crystalline arthropathies. Fibrosing disorders. The ﬁbrosing disorders are a heterogeneous group of conditions that include idiopathic pulmonary ﬁbrosis, cryptogenic cirrhosis, retroperitoneal ﬁbrosis, sclerosing cholangitis, and scleroderma. There are a few well-documented cases of ﬁbrosing disease secondary to environmental insults, as was recently demonstrated for nephrogenic systemic sclerosis induced by the magnetic resonance contrast agent gadolinium (133). Genetic factors, such as mutations at the telomerase locus, have also been associated with rare familial cases of pulmonary ﬁbrosis (134). While there are anecdotes and small series suggesting that the ﬁbrosing disorders may be manifestations of a broader susceptibility to inﬂammation (135), until recently the details have remained elusive. However, recent data point to an important role for the inﬂammasome in two environmentally induced forms of pulmonary ﬁbrosis. Asbestos and silica can cause severe inﬂammatory and ﬁbrotic disease when inhaled. Alveolar macrophages lavaged from individuals with prolonged asbestos exposure secrete enhanced basal amounts of IL-1β in vitro www.annualreviews.org • Horror Autoinﬂammaticus

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(136). Several labs have now reported that IL-1β production in response to these particles is abrogated in Nlrp3-deﬁcient mice (19– 21) (Figure 2). One paper further suggests that NLRP3-activating crystals/ﬁbers share the ability to destabilize lysosomes (21). Similarly, phagocytosis of amyloid-β in the central nervous system may also lead to lysosomal destabilization, inﬂammasome activation, and IL-1β release (137). While the role of IL-1β in the pathogenesis of Alzheimer’s disease is still not conclusive, there is now a growing rationale for clinical trials targeting IL-1β in the ﬁbrosing disorders caused by substance inhalation. By analogy, it is tempting to hypothesize that idiopathic pulmonary ﬁbrosis (IPF) is caused by a similar process, triggered perhaps by other inhalants or environmental exposures. Genetics appears to play an important role in susceptibility to IPF (138), with some studies suggesting an association with a polymorphism in the IL-1 receptor antagonist gene (139). In mice, direct administration of IL-1β by inhalation results in ﬁbrotic inﬂammation that is comparable to that induced by bleomycin (140), a murine model for IPF. Bleomycin-induced pulmonary ﬁbrosis is attenuated by genetic deletion of the IL-1 receptor and the inﬂammasome adaptor ASC genes, and by exogenously administered IL-1 receptor antagonist. Bearing in mind that IPF is likely a genetically complex disorder involving a number of inﬂammatory pathways, data from human and animal models suggest a pathogenic role for IL-1β and raise the possibility of pilot studies of IL-1 inhibitors in this life-threatening disease.

IPF: idiopathic pulmonary ﬁbrosis

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T2DM: type 2 diabetes mellitus

Type 2 diabetes mellitus. Diabetes mellitus is characterized by elevated fasting and postprandial blood sugar levels due to the relative or absolute deﬁciency of insulin. In type 1 diabetes (T1DM) there is strong evidence of pancreatic islet cell destruction, often early in life, eventually leading to marked reduction in insulin production. The large majority (type 1A) are caused by anti-islet cell autoantibodies (141). In contrast, type 2 diabetes mellitus (T2DM) tends to occur later in life and is due to the com640

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bination of peripheral insulin resistance and impaired islet cell insulin secretion, which are thought to be metabolic consequences of excessive weight (142). In addition, an emerging body of data suggests that the innate immune system plays an important role in the pathogenesis of T2DM. T2DM was ﬁrst proposed as a disease of the innate immune system over a decade ago (143). This work suggested that T2DM is associated with persistent elevation of acute-phase reactants and inﬂammatory mediators, and this has now been further reﬁned to a subset of proinﬂammatory cytokines, particularly IL-1β and IL-6 (144). IL-1β is a key cytokine that induces the apoptosis of pancreatic beta cells. Hyperglycemia creates a vicious circle by inducing the production of IL-1β by the β cells themselves (145), thus leading to further islet cell dropout and ever-increasing blood glucose levels. In a mouse model of diabetes, administration of the IL-1 receptor antagonist protected animals fed a high-fat/high-sucrose diet from hyperglycemia and β cell apoptosis (146). Moreover, in a randomized, doubleblind, placebo-controlled trial in 70 patients with T2DM, anakinra improved glycemia and β cell secretory function (147). Although to date variants in the genes encoding inﬂammasome proteins have not been associated with T2DM in whole-genome analyses, the biologic and clinical evidence places the inﬂammasome at a critical crossroads in the pathogenesis of T2DM. Particularly as longer-acting IL-1 inhibitors become available, this pathway could provide an important target in the treatment of this common but debilitating illness. Schnitzler syndrome. Our discussion of Schnitzler syndrome as a disease mediated primarily by the innate immune system may still be premature. This illness is characteristically associated with monoclonal IgM gammopathy and sometimes bone pain and radiographic ﬁndings (148), which might indicate involvement of the B cell lineage, and the mean age of onset is 51 years. However, the remaining constellation of urticaria, intermittent fever,

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arthralgia or arthritis, and elevated acute-phase reactants is reminiscent of the hereditary periodic fever syndromes, and of the cryopyrinopathy MWS in particular. The biochemical data available to date do indeed suggest a prominent inﬂuence of proinﬂammatory cytokines from the innate immune system (149), and there are now several reports indicating that inhibiting IL-1β signaling is also very beneﬁcial in the treatment of Schnitzler syndrome (149– 153). Gammopathy may persist after anakinra despite remission of fever, rash, and constitutional symptoms (149), suggesting more proximal molecular events in the pathogenesis of Schnitzler syndrome.

NF-κB ACTIVATION DISORDERS Crohn’s Disease (CD) NOD2/CARD15 was placed center stage in the ﬁeld of immunology when two simultaneous publications documented susceptibility alleles that predispose to CD (26, 27). CD and ulcerative colitis (UC) are the two most common forms of inﬂammatory bowel disease (IBD), but there are several clinical as well as genetic features that distinguish between them (163). In CD, inﬂammation is typically transmural and can be found discontinuously throughout the gut, whereas UC primarily affects the mucosal and submucosal layers of the rectum and colon in a continuous pattern. NOD2 appears to be a genetic discriminating factor as a locus that is only associated with CD (163–167). This is in contrast to single nucleotide polymorphisms (SNPs) in ECM1 (an intestinal glycoprotein that activates NF-κB) and IL-10 associated just with UC (165, 167), and to SNPs in IL-23R and IL-12B (encoding the p40 subunit of IL-12 and IL-23) that are associated with IBD in general (163–167). These genes highlight the involvement of both the innate and adaptive immune system with features of both autoinﬂammatory and autoimmune disease. Of note, the cellular distribution of NOD2 expression is almost exclusively within the myeloid lineage of innate immune cells, while the associations with

INNATE IMMUNE GENES AS DETERMINANTS OF AUTOIMMUNE DISEASE Recent data implicate genes of the NLR and TLR families in the development of a number of diseases that more appropriately ﬁt into the autoimmune (as opposed to the autoinﬂammatory) spectrum. One interesting example is the association of NLRP1 not only with vitiligo, but also with a range of other autoimmune diseases that affect the relatives of vitiligo probands (154). NLRP1 is highly expressed in T cells, but also in a range of innate immune cells such as monocytes and dendritic cells. In allogeneic stem cell transplantation, recipient variants in NLRP1, as well as donor variants at NLRP2 and NLRP3, have been shown to be important prognostic factors (155). Variants at another NLR gene, NOD2/CARD15, in both donor and recipient are associated with graft-versus-host disease and mortality (156). Although we have distinguished between the IL-1β activating role of the NLRP proteins, and the NF-κB regulatory effect of NOD2, both link pathogen-associated molecular patterns (PAMPs) with proinﬂammatory cytokine activation. NOD2 may directly sense an exogenous bacterial component like muramyl dipeptide (MDP) (157, 158), whereas NLRP1, NLRP2, and NLRP3 may respond to changes in homeostatic proteins due to danger signals from molecules such as uric acid that would be released with a variety of natural or iatrogenic insults. In contrast to this, TLR7 and TLR9 are activated directly by nucleic acids, and this activation is implicated in the pathogenesis of systemic lupus erythematosus (159–162). These receptors usually alert the innate immune system to viral RNA/DNA, but inappropriate presentation of self RNA/DNA may lead to autoimmune disease. Although our understanding of innate immune genes in autoimmune diseases is still rudimentary, these pathways are becoming increasingly attractive targets for therapeutic intervention.

IL-10, IL-12B, and IL-23R suggest an important pathogenic role for T cells. A unifying theory might posit that NOD2-associated SNPs cause inappropriate activation of innate immune myeloid cells to normal intestinal ﬂora, thus triggering a response inﬂuenced by genes of the adaptive immune system. Alternatively, NOD2 may normally function as an innate signal to tolerize the host adaptive immune system, for which loss-of-function mutations may render the host sensitive to normal enteric bacteria. www.annualreviews.org • Horror Autoinﬂammaticus

IBD: inﬂammatory bowel disease

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The NOD2 protein bears some structural similarities to the NLRP3 protein (Figure 1). Whereas NLRP3 has a single N-terminal pyrin domain, NOD2 has two N-terminal CARDs, but both pyrin domains and CARDs belong to the same family of motifs, which assume a threedimensional structure that permits cognate interactions (39–41). Through homotypic CARD interactions, NOD2 interacts with RICK/RIP2 to activate the NF-κB and mitogen-activated protein (MAP) kinase signaling pathways (168). Like NLRP3, NOD2 also has a NACHT/NBD domain with ATP-binding activity, and ten Cterminal LRRs through which it mediates, directly or indirectly, intracellular recognition of the bacterial cell wall component MDP (157, 158) (Figure 2). While CD mutations are found throughout NOD2, three mutations in the Cterminal third account for 80% of NOD2 CD variants (169). The mechanism by which CD mutations in NOD2 cause IBD remains controversial. One major problem is that the effect of these mutations on innate immunity is highly dependent on the type of cells being assayed. Thus, whereas NF-κB activation is increased in the lamina propria of individuals with CD (170), PBMCs exhibit decreased MDP-induced activation (171, 172). Bone marrow–derived macrophages from mice harboring a NOD2 CD mutation exhibit increased NF-κB activity and IL-1β in response to MDP (173), consistent with the patient data from intestinal tissue. On the other hand, CD patients exhibited an impaired inﬂammatory response to topical application of IL-8, which was normalized by the addition of MDP, unless the patient was homozygous for a NOD2 CD variant (174). Mice deﬁcient in the murine Nod2 are susceptible to Listeria monocytogenes infection via the oral route and express reduced amounts of a subgroup of intestinal antimicrobial peptides known as cryptdins (175). More recent data from CD patients suggest that this latter effect is due to inﬂammation and is not a primary pathogenic event (176). However, neither the aforementioned mice nor another line deﬁcient in Nod2 develop spontaneous IBD-like

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disease (175, 177). Data from yet another experimental model suggest that MDP activation of NOD2 downregulates multiple TLR responses and that the absence of such tolerization with CD mutations might increase susceptibility to intestinal inﬂammation (178). Alternatively, it has been suggested that the LRR of NLR proteins is autoinhibitory and that inactivating CD mutations would prevent autoinhibition and thus promote inﬂammation (Figure 2). Another important consideration is that NOD2 is not the only innate immune gene associated with CD, and thus it is possible that animal model systems should take into account more of the >30 genetic factors that have been associated with disease susceptibility in humans (164). Several autophagy-related genes, such as ATG16L1, are strongly associated with CD, which further suggests a role for the presentation of bacterial components to intracellularly expressed proteins such as NOD2. Paneth cells from mice deﬁcient in the murine Atg16L1 manifest abnormalities in granule exocytosis similar to those seen in CD patients (179) and exhibit heightened susceptibility to chemically induced colitis in an IL-1β-dependent fashion (180). Mice deﬁcient in a second CD-associated autophagy gene, Irgm, have an impaired capacity to eliminate intracellular pathogens. Such data suggest a complex interaction between NOD2, various autophagy genes, and a number of other loci controlling innate and adaptive immune function to confer susceptibility to CD.

Blau Syndrome (BS) Whereas certain variants in NOD2 are risk factors for CD, a different subset of NOD2 sequence substitutions are high-penetrance, dominantly inherited mutations that cause BS (28). BS is characterized by a triad of granulomatous uveitis, arthritis, and skin rash with camptodactyly (ﬂexion contractures of the ﬁngers). NOD2 mutations have also been found in early-onset sarcoidosis (181), an entity that some regard as the same disease as BS, and among patients with a somewhat broader

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phenotype than that typical for BS (182, 183). BS mutations almost exclusively target the NBD of the protein, whereas CD mutations predominantly affect its LRR (Figure 1). The central NBD of other NLR proteins is known to bind and hydrolyze ATP, and this is required for signaling. It has not yet been determined that NBD mutations in BS result in altered ATP processing, but it is known that basal NF-κB activation is upregulated (181). Mutations causing cryopyrinopathies also cluster around the NBD of NLRP3. Comparison of the predicted threedimensional structure of NLRP3 with that of NOD2 indicates that the R260W mutation of NLRP3 and the BS-associated R334W mutation of NOD2 encode the same amino acid substitution at a homologous, structurally conserved residue (184). It is intriguing why CD-associated NOD2 variants predominantly reside in or adjacent to the LRRs and induce gastrointestinal disease, while BS-associated NOD2 mutations occur mostly in the NBD/NACHT domain and produce a broader distribution of affected tissues. As is the case for NLRP3, NBD mutations in BS may produce a protein that is constitutively active. On the other hand, LRR mutations may alter thresholds for PAMP-induced signaling, which for NOD2 may occur mostly in the gastrointestinal tract, but would not lead to constitutive, ATP-independent activation.

Guadeloupe Variant Periodic Fever Syndrome (FCAS2) We have provisionally placed a newly reported periodic fever syndrome, ﬁrst described in two families from Guadeloupe (29), among the NF-κB activation disorders, although at ﬁrst glance the clinical picture suggests an IL-1βrelated disorder. Patients present with weeklong episodic fevers triggered by cold exposure and associated with arthralgia, myalgia, and other constitutional symptoms. Two affected members of one family had sensorineural hearing loss. Although clinically similar to FCAS or MWS, mutational screening in NLRP3 and other known periodic fever genes

was negative. A subsequent examination of NLRP12 (NALP12, PYPAF7, MONARCH-1), chosen because of its similarity to NLRP3 and expression in myelomonocytic cells, revealed dominantly inherited nonsense and splice-site mutations in the two families. These variants were not found in a panel of 104 ethnically matched control chromosomes, a relatively small number, but the functional case for their pathogenic role is fairly strong (see below). Based on the clinical similarities with FCAS, but unique genetics, this condition has also been termed FCAS2. NLRP12 was the ﬁrst example of an NLR protein capable of negatively regulating NFκB activation (185). The mutations identiﬁed in NLRP12 affect the protein signiﬁcantly, with the nonsense mutation truncating the protein within the NBD and the splice mutation deleting the C-terminal LRRs (29). This differs from the NLRP3 mutations in CAPS, which are predominantly missense mutations of the NBD. In overexpression systems, the NBD truncation substantially impacted normal NLRP12 inhibition of NF-κB activation, with the LRR mutation showing a less convincing effect (29). It is not known whether NLRP12 can participate in an inﬂammasome complex regulating IL-1β production.

PAMP: pathogenassociated molecular pattern

PROTEIN FOLDING DISORDERS OF THE INNATE IMMUNE SYSTEM TNF Receptor–Associated Periodic Syndrome (TRAPS) Dominantly inherited heterozygous mutations in TNFRSF1A, encoding the TNF receptor 1 (TNFR1, also known as TNFRSF1A, p55, and CD120a) cause TRAPS (2). Patients experience recurrent, often prolonged fevers that can be accompanied by severe abdominal pain, pleurisy, arthritis, a migratory skin rash with underlying fasciitis, and/or periorbital edema (186, 187). Some TRAPS patients eventually develop systemic AA amyloidosis. In contrast to FMF, there is no ethnic predilection, and www.annualreviews.org • Horror Autoinﬂammaticus

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corticosteroids are effective for the acute attacks, whereas colchicine is not. TRAPS-associated mutations exclusively affect the extracellular region of the receptor (Figure 1), and in many cases the cysteine residues that participate in disulﬁde bonds result in the most severe and penetrant disease phenotype with the highest risk of amyloidosis (188). In the initial description of TRAPS, we considered the possibility that interchain disulﬁde binding of unpaired cysteine residues might lead to constitutive activation but rejected this hypothesis based on studies of cytokine production from patients’ PBMCs (2). Experiments examining the binding of radiolabeled TNF to leukocytes from patients and controls showed no increased TNF binding (2). An alternate hypothesis was that TNFR1 mutations might lead to a defect in the ability of metalloproteases to cleave TNFR1 from the cell membrane, also called receptor shedding, which is the most common way of inactivating TNFR1 (189). Despite the fact that most TRAPS-associated TNFR1 mutations are remote from the known cleavage site, initial observations, including increased cell surface TNFR1 and decreased soluble TNFR1 in patients’ blood, were consistent with impaired ectodomain cleavage (2). By this hypothesis, TRAPS-associated mutations would lead to impaired receptor shedding, thereby permitting repeated stimulation through membrane-bound receptors and a decreased pool of potentially inhibitory soluble receptors. Implicit in this hypothesis was the concept that cell-bound mutant receptors would signal normally and that TNF signaling would be the central pathogenic mechanism. However, not all TRAPS-associated TNFR1 mutations have an effect on shedding (188), and even for TNFR1 mutations where an effect on shedding has been demonstrated, this is conﬁned to certain cell types (190). Our understanding of how the TRAPS phenotype develops has recently been advanced by in vitro analysis of mutant receptors. Overexpression experiments in cell lines suggest that the mutations cause TNFR1 to aggregate and inhibit its trafﬁcking to the cell membrane

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(30, 191, 192). The TNFR1 mutations also cause decreased binding of TNF (191). Lobito et al. demonstrated that the mutant receptors no longer oligomerized with wild-type receptors, instead forming disulﬁde-linked homooligomers (30). Furthermore, the mutant receptors were retained intracellularly where they colocalized with markers for the endoplasmic reticulum (ER), and their cell surface expression was also prevented in vivo in mice homozygous for a knockin of the T50M mutation (30). This ﬁnding is also in agreement with computational modeling studies that suggest improper folding of the mutated protein, leading to different conformations of the TNFR1 ectodomain (193), and with studies of neutrophils from C33Y TRAPS patients that show minimal surface staining for the mutant receptor (192). These recent ﬁndings are a signiﬁcant departure from our initial understanding of the disease. Undoubtedly, the structural changes induced by TRAPS mutations would interfere with recognition by proteases and thereby inhibit cleavage of whatever mutant receptor makes it to the cell surface. Moreover, there are rare patients with the TRAPS phenotype who do have mutations near the cleavage site at the transmembrane region whose leukocytes exhibit impaired receptor shedding (194, 195). However, the evidence for impaired trafﬁcking of a number of mutant receptors adds an important new dimension to the discourse on the underlying cause of TRAPS. From a theoretical view, there are at least two constraints on potential hypotheses regarding the mechanism of TRAPS. First, any proposal must explain how mutations that lead to a grossly misfolded protein, with impaired ligand binding, aberrant intracellular trafﬁcking, and impaired aggregation with wild-type receptors, still lead to an autoinﬂammatory rather than an immunodeﬁciency phenotype. Second, any viable theory must explain the dominant mode of inheritance of TRAPS. Initially (when it was still assumed that mutant TNFR1 would reach the cell surface normally), the inheritance of TRAPS was explained by the fact that TNFR1 molecules must trimerize to signal, and that

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7/8 possible trimers composed of mutant and wild-type receptors would contain at least one mutant receptor. However, in light of the recent data in transfection systems that mutant receptors seem not to associate with wild-type molecules (30), this explanation seems much less likely. Similarly, dominant-negative interference with apoptotic signaling through the wild-type TNFR1 would seem unlikely if the wild-type and mutant species do not interact. Instead, various gain-of-function hypotheses become more likely. Two such formulations that comport with the data include the notion that misfolded intracellular receptors might trigger ER stress and the UPR (196), or that intracellular aggregates of misfolded protein might constitutively induce intracellular signaling through aggregation of their death domains and subsequent recruitment of downstream signaling molecules. In contrast, certain other theories, such as the proposal that TNFR1 mutations might lead to shunting through the 75-kDa TNFR2, seem less likely because they do not explain the dominant inheritance of TRAPS. Our recent data suggest that the accumulation of mutant TNFR1 results in spontaneously increased activation of MAPK such as JNK and p38 (Figure 4). This activation may prime TRAPS cells to become more susceptible to low doses of inﬂammatory stimuli such as LPS (A. Simon, H. Park, R. Maddipati, A. Bulua, A. Jackson, et al., submitted). Increased MAPK activation was mediated by reactive oxygen species (ROS), and could be blocked by inhibition of ROS. Further exploration of a mouse model of TRAPS showed that homozygous mutant mice, which lack the wild-type TNFR1 cell surface receptor, do not develop the full phenotype seen in heterozygous mice. We hypothesize that the wild-type cell surface TNFR1 is necessary for the autocrine and paracrine feedback loops of TNF-α (Figure 4), which in turn result in a marked increase in cytokine production. Initially, treatment of TRAPS with the soluble p75:Fc fusion protein (etanercept) seemed to be the ideal substitute for a deﬁciency of sol-

uble p55 TNFR1 that was implicated by the shedding hypothesis (186). It did not come as a surprise therefore that this treatment ameliorated disease in a large number of cases. However, as our understanding of TRAPS has become more sophisticated, with ligandindependent intracellular signaling taking on new prominence, the limitations of etanercept have become clearer, and there are even anecdotal reports of the efﬁcacy of anakinra in this disease (197–199). This raises the possibility that cytokine inhibitors may work in TRAPS by mechanisms much less speciﬁc than originally envisaged. On the other hand, the fact that etanercept has provided at least some beneﬁt to a number of patients is consistent with the hypothesis that wild-type TNFR1 is required to develop the disease phenotype.

Ankylosing Spondylitis Ankylosing spondylitis (AS) is a systemic disease manifested by chronic arthritis of the spine and sacroiliac joints, leading eventually to the loss of spinal mobility. There are a number of extraarticular manifestations, including uveitis, aortitis, enthesitis, and dactylitis, and spinal fusion may lead to restrictive lung disease and consequent pulmonary hypertension. The association of ankylosing spondylitis susceptibility with the class I molecule HLA-B27 is one of the strongest known HLA disease associations and has been recognized for over 30 years (200). Initial theories of disease pathogenesis have revolved around the possible abnormal presentation of self or microbial peptides selectively by HLA-B27, but the identiﬁcation of so-called arthritogenic peptides has remained elusive (201). The inability to explain the association of HLA-B27 with AS on the basis of antigen presentation and models of adaptive immunity has led to consideration of alternative hypotheses. One such line of investigation is based on the tendency of HLA-B27 heavy chains to misfold in the ER and to form disulﬁde-linked heavy chain dimers (202, 203). In transgenic rats, disulﬁde-linked heavy chain complexes are www.annualreviews.org • Horror Autoinﬂammaticus

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Mechanisms of autoinﬂammatory protein folding disorders. Left: TNF-receptor associated periodic syndrome (TRAPS). TNF-receptor type 1 (TNFR1) accumulates in the endoplasmic reticulum (ER) when mutated (indicated by asterisk). This accumulation leads to increased reactive oxygen species (ROS) activation, and subsequent MAPK phosphorylation ( JNK and p38), which makes the cells more susceptible to inﬂammatory stimuli. TRAPS patients, who are heterozygous for TNFR1 mutations, still carry the wild-type TNFR1, which is thought to play a role in propagating the inﬂammatory cascade. Right: spondyloarthropathies, ankylosing spondylitis. An HLA-B27 variant is strongly associated with ankylosing spondylitis and may accumulate in the ER, leading to an unfolded protein response. Weaker gene associations include ARTS1 (ER-associated aminopeptidase 1, ERAP1), which could affect the pathogenic presentation of antigenic peptide fragments or the cleavage of membrane bound receptors, and IL-23R/IL-1α, which argue for the role of proinﬂammatory cytokines in this disease.

much more prone to form in animals harboring the AS-associated B27 allele rather than the disease-resistant HLA-B7 allele (204). Moreover, bone marrow macrophages from B27, but not B7, rats exhibit evidence of the UPR, ei646

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ther during the inﬂammatory phase of illness or with cytokine stimulation in premorbid animals (31, 205). When stimulated with TLR ligands, bone marrow macrophages from HLAB27 transgenic rats produce increased amounts

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of type I interferons (32) and possibly IL-23 (33). While these data have formed the basis for a new paradigm for understanding the pathogenesis of AS, considerable complexities remain in correlating the many subtypes of HLAB27 with misfolding and disease susceptibility (206, 207). Although the molecular mechanism may still require some reﬁnement, genetic studies are providing additional insights. Candidate gene analyses have implicated the IL-1 cluster of genes on chromosome 2q13 as an important AS susceptibility locus (208). The genetic contribution to AS has recently been further clariﬁed by whole-genome analysis that identiﬁed a variant ARTS1 allele as a risk factor for the disease (209). The ARTS1 protein (also known as ERAP1) has at least two immunologic functions, one related to adaptive immunity, the second more related to innate immunity. In the ER, ARTS trims peptides to optimal length for presentation to MHC class I molecules, while at the cell surface it cleaves receptors for the proinﬂammatory cytokines IL-1, IL-6, and TNF (Figure 4). Data from this large study also implicated the gene encoding the IL-23 receptor as an AS susceptibility locus. This is of especial note, given the aforementioned observations on IL-23 production by HLAB27 transgenic rat macrophages, the already established role for IL-23R in susceptibility to CD (164) and psoriasis (210), and the emerging role for IL-23 in the differentiation of the Th17 subset of T lymphocytes (211). IL-23 may therefore provide yet another connection between innate and adaptive immunity in the pathogenesis of autoinﬂammatory disease.

COMPLEMENT DISORDERS Hemolytic Uremic Syndrome Hemolytic uremic syndrome (HUS) is characterized by Coombs-negative hemolytic anemia, thrombocytopenia, and renal failure. The underlying pathological process is thrombotic mi-

croangiopathy, with uncontrolled small-vessel blood clots leading to erythrocyte fragmentation, platelet consumption, and glomerular hypoperfusion (212). HUS is most commonly caused by a strain of Escherichia coli expressing Shiga toxin, and is characterized by diarrhea followed by the aforementioned triad of clinical ﬁndings (213). With the exception of diarrhea, these clinical features are also common to the atypical form of the disease (aHUS), which is an inherited condition for which the genetic causes are now becoming understood. At ﬁrst deﬁciency of, and then simple mutations in, the complement factor H (CFH) gene was identiﬁed (214, 215), and it is now known that mutations in the gene encoding the membrane cofactor protein (MCP or CD46, which is in the same complement gene cluster as CFH on chromosome 1q32) (216, 217) and complement factor I (CFI) gene (218) also cause disease when mutated (Figure 5). The third component of complement is a critical molecule at the convergence of the classical, alternative, and lectin pathways, and its activation is tightly regulated (212, 219). CFH binds C3b to prevent the formation of the C3bBb convertase complex that further activates C3, it accelerates the dissociation of Bb from the convertase complex, and it acts as a cofactor for CFI in degrading C3b (215). Over 100 CFH mutations have been identiﬁed in aHUS. MCP/CD46 is a transmembrane glycoprotein that acts as a membrane-bound cofactor for C3I-mediated degradation of C3b. CFH is particularly important in protecting structures such as the glomerulus, which lack MCP/CD46, from C3 activation (220). CFI is a serine protease that inactivates C3b. Unchecked C3 activation leads to the production of the anaphylatoxins C3a and C5a, which are chemotactic factors that elicit the inﬂux of neutrophils and other inﬂammatory cells, and C3b, which acts as an opsonin (219). Neutrophil-mediated endothelial cell damage can, in turn, lead to the release of thrombin and the development of the microangiopathic cascade.

www.annualreviews.org • Horror Autoinﬂammaticus

aHUS: atypical hemolytic uremic syndrome

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Figure 5 Pathogenesis of the complement-mediated autoinﬂammatory disease atypical hemolytic uremic syndrome. Activating gain-of-function mutations in CFB increase C3bBb convertase stability and lead to permanent activation of the alternative pathway of complement. Autoantibodies against, and inactivating mutations within negative regulators of this process have also been described, such as for complement factor H (CFH) and membrane cofactor protein (MCP), which are cofactors for the inactivation of C3b by complement factor I (CFI). Asterisks denote proteins that carry mutations with known disease associations.

Age-Related Macular Degeneration

AMD: age-related macular degeneration

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Complement factor H variants confer risk of developing age-related macular degeneration, a leading cause of blindness in developed nations (221–223). Homozygosity at one speciﬁc variant, the substitution of histidine for tyrosine at residue 402 (Y402H), may increase the risk of developing this condition late in life by as much as sevenfold. The hallmark of this disease is the presence of drusen (protein and cell debris containing immune and complementassociated molecules) in and around the macula, the area of the retina that confers central vision and maximal visual acuity. Recent studies have focused on delineating the mechanism by which CFH variants lead to the deposition of drusen and consequent macular degeneration. In mice, a very close approximation of the human disease can be generated by immunization with carboxyethylpyrrole, an oxidation fragment of docosahexaenoic acid that has been found atMasters et al.

tached to protein in the drusen and in the serum of people with AMD (age-related macular degeneration) (224). Thus one possible scenario is that oxidative damage in the outer retina where docosahexaenoic acid is abundant can perturb the complement system, and that in individuals with high-risk variants of CFH, this leads to the development of pathogenic drusen.

CYTOKINE SIGNALING DISORDERS Cherubism Cherubism is a dominantly inherited disorder characterized by bony swelling of the upper and lower jaws during childhood (12). Patients often develop the characteristic cherubic round face after which the disease was named. Radiographs show multiple, symmetrical radiolucent cystic bone lesions of the mandibula and maxilla. The pathologic process is one of inﬂammatory cyst

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formation and bone replacement with ﬁbrous tissue composed of stromal cells and multinucleated osteoclast-like cells. Although jaw swelling regresses in puberty, cherubism is not only disﬁguring during the active phase but causes signiﬁcant permanent dental problems. A relatively recent addition to the family of autoinﬂammatory diseases, cherubism is caused by mutations in SH3BP2, which encodes an SH3-binding protein expressed in multinucleated osteoclasts and in stromal cells from cherubism lesions (34). Recently, the development of a knockin mouse model has provided important insight into the molecular pathophysiology of cherubism (35, 225). In this study, Ueki and colleagues developed a mouse line in which the P416R SH3BP2 cherubism mutation was knocked in to the mouse ortholog. Somewhat unexpectedly, the heterozygous mice exhibited only minimal trabecular bone loss in the long bones, even though cherubism is dominantly inherited in humans. However, mice homozygous for the knockin mutation developed an inﬂammatory phenotype affecting not only the bone, but also the skin, muscle, liver, lung, and stomach. Bone lesions included trabecular bone loss and pits in the mandible, with increased osteoclasts and areas of ﬁbrosis in the affected bone. Bone marrow transplantation experiments demonstrated that disease could be transferred from mutant to irradiated wild-type mice with hematopoietic cells. In order to further characterize the cellular and molecular basis of disease in the knockin mice, animals were bred that were doubly homozygous for cherubism knockin and Rag1 deﬁciency (which lack functional lymphocytes) or for the knockin and Opdeﬁciency (which lack functional myeloid cells). Bone loss occurred in the Rag1-deﬁcient mice, but not the Op-deﬁcient animals, thus establishing a key role for cells of the myeloid lineage. Moreover, upon stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), myeloid osteoclast progenitors from homozygous or heterozygous knockin mice formed

unusually large osteoclasts and macrophages that secreted excessive TNF-α (Figure 6). The mechanism of increased responsiveness to MCSF and RANKL appears to be increased ERK and Syk phosphorylation. In this model TNF-α plays an important role both as an effector and ampliﬁer of bone resorption. This cytokine is a potent activator of osteoclasts, and has been implicated in the bone loss associated with a number of inﬂammatory arthritides. It is therefore likely that hyperresponsiveness to M-CSF and RANKL may set up a positive feedback loop mediated by TNFα. In fact, the homozygous knockin mouse had elevated levels of TNF-α in the serum, and crossing this mouse to one deﬁcient for TNF-α rescued it from disease, although bone marrow cells from the double mutant mice did maintain an enhanced response to M-CSF and RANKL. The data from the SH3BP2 knockin mouse support the concept that the molecular pathophysiology of cherubism involves the aberrant response of myeloid progenitors to M-CSF and RANKL, which then induces a TNF-αmediated vicious circle of osteoclast stimulation. There remain a number of unanswered questions, including the explanation for the difference in inheritance between human (autosomal dominant) and murine (autosomal recessive) disease, the mechanism by which the positive feedback loop is short-circuited at puberty, and the question of whether therapy with TNF inhibitors will ameliorate disease. Nevertheless, we regard cherubism as the prototype for a new category of autoinﬂammatory disease characterized by aberrant cytokine responsiveness.

HLH: hemophagocytic lymphohistiocytosis

MACROPHAGE ACTIVATION Primary and Secondary Hemophagocytic Lymphohistiocytosis Although triggered by the adaptive immune system, the major effector cell of both primary and secondary HLH (hemophagocytic lymphohistiocytosis) is the macrophage, an agent of innate immunity. Primary (familial) HLH is a potentially life-threatening disorder caused by www.annualreviews.org • Horror Autoinﬂammaticus

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Inﬂammation and bone resorption Figure 6 Autoinﬂammatory disease caused by aberrant cytokine signaling. Osteoclast and macrophage differentiation is controlled by the cytokines M-CSF and RANKL. Dominantly inherited mutations in SH3BP2 alter the signals that normally come from these cytokines, which results in the differentiation of hyperactive osteoclasts and macrophages that can cause the autoinﬂammatory disease cherubism. Excess TNF-α produced by these hyperactive macrophages could also feed back through the stroma to increase M-CSF and RANKL levels, although this is still hypothetical.

Scavenger macrophage: activated macrophage that scavenges haptoglobinhemoglobin complexes via the CD163 receptor, phagocytoses erythrocytes, and secretes proinﬂammatory cytokines and ferritin

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mutations in several genes important to cytotoxic T cell and natural killer (NK) cell function (36, 37). Clinical manifestations include prolonged fever, splenomegaly, and cytopenias involving multiple lineages in the peripheral blood (erythrocytes, platelets, leukocytes). Although not required for the diagnosis, some patients have evidence of hemophagocytosis (macrophage-mediated phagocytosis of erythrocytes) in the bone marrow, spleen, lymph nodes, or cerebrospinal ﬂuid (226). Other laboratory ﬁndings include hypoﬁbrinogenemia (due to coagulopathy), hypertriglyceridemia, elevated serum ferritin, low or absent NK cell function, and elevated levels of soluble IL-2Rα. Because of the hypoﬁbrinogenemia, the erythrocyte sedimentation rate is usually unexpectMasters et al.

edly low relative to other inﬂammatory measures, such as the C-reactive protein. Exacerbations of primary HLH are sometimes associated with viral infection, and current concepts on pathophysiology revolve around (a) the inability of cytotoxic cells to clear virally infected cells, leading to persistent antigen-driven activation of macrophages, and (b) cytokine-mediated differentiation and ampliﬁcation of activated macrophages for which there are insufﬁcient apoptotic signals to remove (226, 227). The resultant hypercytokinemia leads to the expansion and activation of a subset of scavenger macrophages expressing high levels of CD163, a scavenger receptor that binds hemoglobin-haptoglobin complexes (228) and that attaches hematopoietic

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Figure 7 Molecular lesions that affect cytotoxic adaptive immune system cells lead to unregulated macrophage activation. Familial hemophagocytic lymphohistiocytosis (HLH) is caused by mutations in genes encoding proteins associated with vesicular transport and granule shedding, such as Munc13-4 (UNC13D), Perforin 1 (PERF1), and Syntaxin11 (STX11). The functions of NK and cytotoxic T cells are also controlled by several other genes that cause individual monogenic diseases when mutated (LYST, RAB27A, SH2D1A, AP3B1). Impairment of the normal efﬁcacy of cytotoxic T cells for virally infected targets and upregulation of IFN-γ production leads to the compensatory development of a subset of scavenger macrophages that mediate the macrophage activation syndrome (MAS). They secrete proinﬂammatory cytokines, phagocytose erythrocytes, express the CD163 receptor for haptoglobin-hemoglobin complexes, and produce ferritin. Their production of IL-18 in particular could amplify the production of IFN-γ in a positive feedback loop. Secondary HLH is most commonly observed as a sequella of systemic-onset juvenile arthritis, for which variants of IL-6 and MIF are risk factors (double asterisks). A clinical hallmark of MAS is profound hyperferritinemia.

cells to macrophages, facilitating hemophagocytosis (226). These cells, in turn, secrete ferritin, an acute-phase protein that binds free iron to prevent oxidative damage (229), and a number of proinﬂammatory cytokines (Figure 7). Several genes encoding proteins important in cytotoxic cell function have been implicated in recessively inherited familial HLH. These include PRF1, which encodes perforin, a soluble pore-forming protein that permeabilizes the target cell membrane (230); UNC13D, which encodes Munc13-4, a protein essential for cytolytic granule fusion with the cell membrane during degranulation (231); and STX11, encoding syntaxin 11, a member of the SNARE protein family that facilitates fusion in intracellular membrane trafﬁcking (232). HLH may also be seen sporadically in a number of immunodeﬁciency syndromes, usually caused by genes that impact the innate immune system through their roles in vesicular transport and granule shedding in cytotoxic

T cells and NK cells (Figure 7). In ChediakHigashi syndrome, mutations in LYST (CHS1) appear to have an effect on membrane ﬁssion or fusion events (233), while in Griscelli syndrome, RAB27A mutations lead to impaired function of a small Rab GTPase involved in exocytosis of cytotoxic vesicles (36). HermanskyPudlak syndrome type II, caused by mutations in AP3B1 (encoding a component of the AP3 complex, involved in vesicle formation), may also manifest defects in cytotoxic T cell and NK activity leading to HLH (234), while in X-linked lymphoproliferative syndrome there are hemizygous mutations in SH2D1A that cause abnormal NK responses and NKT cell deﬁciency (226). Secondary HLH is seen in childhood rheumatic diseases, most commonly SoJIA (227), and is often termed macrophage activation syndrome (MAS) under these circumstances. Clinical features of MAS are similar to those seen in primary HLH, including low www.annualreviews.org • Horror Autoinﬂammaticus

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NK cell function and reduced perforin expression in cytotoxic cells (235). Gene expression proﬁling in peripheral blood leukocytes suggests that a subset of untreated SoJIA patients may have some of the early features of MAS (228). Although gene variants associated with risk of primary HLH appear not to be susceptibility loci for SoJIA (236), in a panel of 18 patients with SoJIA/MAS, a genetic association with UNC13D was observed, ﬁrst for biallelic mutations that have been observed in familial HLH, and also for a series of 12 SNPs that are likely to be inherited as an extended haplotype (237). Cytokine proﬁling consistently shows that IFN-γ is elevated in HLH, and for primary HLH it is predominantly made by malfunctioning CD8 and NK cells. In secondary HLH, the effect of IFN-γ could also be ampliﬁed by increased levels of IL-18 that have been observed (Figure 7). This scenario does reveal two contradictions: One is that there is no IFN-γ gene expression signature despite these differences in cytokine levels, and the other is that in vitro, IFN-γ skews toward inﬂammatory macrophage differentiation rather than the scavenger macrophage typically observed in MAS. Taken together, these data suggest a fundamental relationship between primary HLH and MAS. In both, compensatory mechanisms elicited because of impaired cytolytic T cell and NK activity lead to runaway activation of macrophages, truly leading to a horror autoinﬂammaticus that, if not treated quickly and aggressively, can even lead to death.

Atherosclerosis Cardiovascular disease has become the leading cause of death in the Western world, and is predominantly caused by atherosclerosis (localized arterial inﬂammation). Preceding an atherosclerotic lesion, lipid-laden macrophages and some T cells accumulate beneath the endothelium, and this nidus can go on to form the core of the plaque where characteristic foam cells (activated, lipid-laden macrophages) are found (238). The top of the plaque is a mix of smooth muscle cells and collagen-rich matrix. 652

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Between and around this, T cells, macrophages, dendritic cells, and mast cells inﬁltrate and can destabilize the lesion, leading to rupture, which accounts for 60%–70% of coronary thromboses (38). The contribution of inﬂammatory cytokines and chemokines to these processes (for example, TNF-α, MCP-1, IL-1, IL-6) cannot be understated, both in the generation and effect after rupture. Aside from innate immune cells, genes of the innate immune system are also implicated in atherosclerosis. This has been shown using an ApoE-deﬁcient mouse that spontaneously develops atherosclerotic disease, which can be partially rescued by deletion of TLR2, TLR4, and MyD88 (239). In the same mouse model, overexpression or administration of LOX-1 or IL-18, respectively, dramatically worsens disease progression. These ﬁndings implicate TLR/C-type lectin activation and IL1/IL-18 signaling pathways in atherosclerosis. As a clear-cut inﬂammatory disease, further reﬁnement of atherosclerosis as an autoinﬂammatory or autoimmune disease is not straightforward. Findings that argue the autoimmune point of view include a report that adoptively transferred CD4-positive T cells promote atherosclerotic disease in ApoE-deﬁcient mice (240), and another report demonstrating that polymorphisms in the T cell costimulator TNFSF4 are associated with coronary atherosclerosis in man, and that mice with targeted mutations in the Tnfsf4 homolog had smaller atherosclerotic lesions than control mice when fed a high-fat diet (241). Also, although foam cells are characteristic of disease, it is unclear if they represent an innate immune response gone awry, or if they could actually be beneﬁcial in disease. Mouse studies of macrophages lacking the scavenging receptors that mediate uptake of oxidized low-density lipoprotein (LDL) have conﬂicting results, some progressing, others ameliorating ApoE mediated disease (242, 243). Some of this difference might be accounted for by the observation that foam cells are still generated in the scavenger receptor–deﬁcient mice, indicating that alternate mechanisms of LDL uptake may exist.

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Given the numerous factors that may contribute to the formation of atheromatous lesions, it is unlikely that any one cytokine or pathway will predominate, and hence the judgment to group atherosclerosis among the syndromes of macrophage activation. Nevertheless, it is intriguing to consider whether at least some component of susceptibility would be mediated through the inﬂammasome and IL-1β. By this line of reasoning, the uncontrolled accumulation of cholesterol could potentially act as a danger signal to an innate immune cell. Several years ago, studies of IL-1 receptor antagonistdeﬁcient mice demonstrated increased foam cell lesion area relative to controls when fed a high-cholesterol/cholate diet. Conversely, mice transgenic for the IL-1 receptor antagonist on an LDL receptor–deﬁcient background were moderately protected from cholesterol/cholate lesions (244). A clinical trial is currently under way to assess the efﬁcacy of anakinra, the recombinant IL-1 receptor antagonist, in controlling inﬂammatory markers in patients with coronary artery disease (245).

THE NEXT FRONTIER: AUTOINFLAMMATORY DISEASES OF UNKNOWN ETIOLOGY As noted in the introduction, there remain a number of disorders that appear clearly to be autoinﬂammatory, but for which there are insufﬁcient data to place them into one of the six categories described in this review. In this ﬁnal section, we brieﬂy discuss three of these illnesses, SoJIA, Behc¸et’s disease, and PFAPA. With the current burgeoning interest in autoinﬂammatory diseases, each of these illnesses is coming under considerable scrutiny. SoJIA is a systemic disease of children that may present with a daily fever, a characteristic salmon-colored skin rash, anemia, and hepatosplenomegaly; eventually arthritis develops, but systemic symptoms may precede the onset of arthritis by weeks or months. Autoantibodies are generally not observed in SoJIA patients. Pioneering studies by Virginia Pascual and colleagues have demonstrated an important

role for IL-1β in the pathogenesis of SoJIA (9). This group found that serum from SoJIA patients induces the transcription of innate immunity genes, including IL-1, in PBMCs from healthy donors, and that SoJIA PBMCs release large amounts of IL-1β when stimulated. They went on to show that nine patients refractory to other therapies responded to anakinra, and they have subsequently identiﬁed a gene expression proﬁle characteristic of SoJIA (246). Nevertheless, a substantial number of SoJIA patients do not respond to anakinra treatment (247), and there are also data implicating other cytokines in the pathogenesis of SoJIA. For example, a SNP in the promoter region of the IL-6 gene has been associated with SoJIA (248), as well as a promoter haplotype in the gene encoding macrophage migration inhibitory factor (249). For these reasons we have resisted the temptation to group SoJIA with the inﬂammasomopathies for the time being, though the data on IL-1β are clearly encouraging. Behc¸et’s disease (BD) is a genetically complex disorder that usually presents in adults rather than children, and is characterized by oral and genital ulcerations, uveitis, acneiform and papulopustular skin lesions, arthritis, and vasculitis involving both the arteries and veins (250). Some patients with BD also exhibit the phenomenon of pathergy, in which a pustule forms following a needle prick. Autoantibodies are generally not seen in patients with BD. BD is most common among populations spread along the historical Silk Road, which extended from the Mediterranean basin, through Turkey and central Asia, to Japan and Korea. Although BD is clearly not inherited as a mendelian trait, there is a strong familial tendency, and susceptibility has also been associated with the HLABW51 allele. The BD phenotype likely represents a complex interaction of multiple genes and the environment. Just as has been the case for AS and IBD, genome-wide association studies may yield important new insights into the important host factors and pathways that underlie this fascinating disorder. PFAPA, the syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical www.annualreviews.org • Horror Autoinﬂammaticus

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adenopathy, is the most common periodic fever syndrome seen in childhood (10). Important additional features include the remarkable clockwork periodicity of the attacks, the responsiveness of attacks to abortive doses of corticosteroids (although in some patients the episodes then recur more frequently), and the almost predictable remission that occurs in adolescence. Like BD, PFAPA is not inherited as a mendelian trait, but does exhibit some familial tendency. At the molecular level, current data highlight a role for proinﬂammatory cytokines IL1β, IL-6, and TNF, which were elevated in PFAPA patients even between attacks (251). In addition, the antiinﬂammatory cytokine IL-4 was decreased compared to controls at all the time-points measured, with a strong increase of IFN-γ during fever episodes. One unresolved question is whether PFAPA is triggered by a viral or other infectious agent; certainly children of this age are regularly encountering many foreign antigens, some of them for the ﬁrst time (252). While the exact cellular determinants and pathogenic molecular mediators remain elusive, the clinical picture and cytokine proﬁle of PFAPA outline a very complex, perhaps heterogeneous, but nevertheless autoinﬂammatory, disease.

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CONCLUDING REMARKS The autoinﬂammatory diseases are the consequence of natural variation in the innate immune system that is severe enough to cause illness, but not so severe as to be embryonic lethal. These disorders represent a selective sampling from the universe of what can go wrong, a reminder of the precarious equilibrium that deﬁnes human health, and a vindication of the nonredundant role of speciﬁc genes in human biology. Monogenic illnesses such as the cryopyrinopathies, caused by rare mutations conferring no apparent selective advantage, have profoundly informed our understanding of each of the six categories of autoinﬂammatory disease discussed in this review. Diseases such as FMF, in which disease-associated recessive 654

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variants appear to have been selected in certain geographic regions perhaps by endemic pathogens, promise a more nuanced understanding of gene-environment and gene-gene interactions. Genetically complex illnesses such as CD and the spondyloarthropathies have been associated with multiple common variants of innate immune genes and extend the reach of autoinﬂammation squarely into the realm of everyday experience. Regrettably, chance and the inherent fallibility of DNA replication provide an inexhaustible font of new subjects for this science. During the course of the preparation of the present manuscript, we have found evidence for a new autoinﬂammatory disorder with severe skin and bone manifestations, caused by recessive loss-of-function mutations in the IL1 receptor antagonist. This illness promises yet deeper insights into the role of IL-1 in human and the importance of its soluble inhibitor, the absence of which can be fatal. In other cases, rare recessive mutations and incompletely penetrant dominant mutations may underlie disorders that initially appear not to be genetic at all. In the era of genome-wide association studies and targeted biologics, it is now also possible not only to discover new etiologic pathways in the genetically complex diseases in a completely hypothesis-neutral fashion, but also to interrogate the role of one’s favorite mediator with exquisite speciﬁcity. Table 1 also underscores the prominence of the inﬂammasome and its regulation in a large number of autoinﬂammatory diseases. It remains to be seen whether IL-1β occupies some special place in the ﬁrmament of cytokines, unique in an apparent ability to trigger innate immunity without major provocation of the adaptive immune system, or whether the prominence of IL-1β among the autoinﬂammatory diseases merely reﬂects the bias of increased vigilance. The latter possibility notwithstanding, the number of molecular stimuli for the NLRP3 inﬂammasome has become quite large, raising questions as to whether the LRR of NLRP3 could actually bind such a disparate group of partners. This

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has given rise to a formulation termed the guard hypothesis, whereby NLR proteins monitor homeostatic pathways that can be perturbed by individual PAMPs or DAMPs to trigger activity of the inﬂammasome. If true, the proteins comprising these homeostatic pathways would become prime suspects in the search for new autoinﬂammatory disease loci. The recently recognized molecular consequences of protein misfolding in innate immune cells may represent yet another opportunity for gene discovery in unexplained syndromes. Another theme that emerges from our analysis of autoinﬂammatory disease is the opportunity for the innate immune system to make mischief in sites of immunologic privilege. The central nervous system, eye, and pregnant uterus are three such anatomic sites, yet each is a major site of involvement for NOMID (central nervous system, eye), age-related macular degeneration (eye), and recurrent hyda-

tidiform mole (pregnant uterus). Possibly, the restraints that place these anatomic compartments off limits for the adaptive immune system do not similarly conﬁne the cells or molecules of innate immunity. As diseases of human beings, the autoinﬂammatory disorders are not merely guideposts to the innate immunome, but also opportunities to better the human condition. The use of IL1 inhibitors in NOMID and DIRA (deﬁciency of the interleukin-1 receptor antagonist) represent triumphs of molecular medicine, but the application of similar therapies to conditions like T2DM and atherosclerosis may represent the next breakthrough, and the development of small molecule inhibitors, if effective, would be revolutionary. It is our hope that as the study of autoinﬂammatory disease moves into its second decade, we stand on the threshold of understanding and treating a broad spectrum of human afﬂictions, both rare and common.

Guard hypothesis: mechanism of innate immune protection based on the detection of alterations in homeostatic parameters as a proxy for pathogenic insults

SUMMARY POINTS 1. Intrinsic inﬂammasomopathies: NLRP3 nucleates a complex including ASC and caspase1 called the inﬂammasome that processes pro-IL-1β into a mature, active form. To date, mutations in only one component, NLRP3, have been identiﬁed, and they cause a spectrum of diseases known as the cryopyrinopathies (CAPS). 2. Extrinsic inﬂammasomopathies: These diseases are caused by mutations in proteins that regulate the production of IL-1β, or can gain the function to do so, but are not constituents of the inﬂammasome complex. 3. Complex/acquired inﬂammasomopathies: Multigenic or environmentally predisposed diseases result in increased secretion of IL-1β by the inﬂammasome. 4. NF-κB activation disorders: These diseases are mediated predominantly by improper regulation of NF-κB within the innate immune system. 5. Protein folding disorders of the innate immune system: Multiple mechanisms can lead from the buildup of misfolded proteins to the production of proinﬂammatory cytokines by innate immune cells. 6. Complement disorders: Complement was the ﬁrst described arm of the innate immune system, and it is tightly regulated to prevent autoinﬂammatory diseases. 7. Cytokine signaling disorders: Cytokines control the differentiation and activation of innate immune cells, so perturbations at the cytokine receptor or of downstream signaling pathways can cause autoinﬂammatory disease.

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8. Macrophage activation: Diseases have been identiﬁed in which the activation of macrophages occurs indirectly, perhaps as a result of defective adaptive immune cells or environmental and inﬂammatory pathways.

FUTURE ISSUES

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1. How exactly do mutations all clustered in the NACHT domain of NLRP3 activate the inﬂammasome and lead to a wide spectrum of inﬂammation as observed in CAPS patients? 2. What are the intermediate(s) and direct upstream activator(s) of NLRP3, given that NLRP3 is probably incapable of directly detecting all the known agents that stimulate its activity? 3. What is the role of pyrin in humans, pro- or antiinﬂammatory, and what is the effect of mutations that cause FMF? 4. What are other genes mutated in patients with monogenic autoinﬂammatory diseases, and what susceptibility alleles are yet to be found in patients with complex autoinﬂammatory diseases? 5. What are the DAMPs or PAMPs that activate the inﬂammasome in IPF, T2DM, atherosclerosis, and other complex inﬂammasomopathies? 6. Are IL-1 and IL-23, which participate in the differentiation of naive T cells into Th17 cells, present and perhaps pathogenic in autoinﬂammatory diseases that are caused by increased production of IL-1β or associated with polymorphisms in IL-23? 7. Does the NOD2 molecule normally tolerize or sensitize the host to bacteria and what is the effect of NOD2 mutations on this function? 8. Why does the activation of IL-1 in autoinﬂammatory disease not predispose patients to autoimmune disease, given that this pathway is thought to prime an adaptive immune response and account for the adjuvant effect of alum? 9. Is there cross talk between the different mechanisms causing autoinﬂammatory disease? We know that IL-1 plays a role beyond the bounds of the inﬂammasomopathies. Might ER stress likewise play a role in the other categories of disease such as T2DM and UC/IBD?

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS This work was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases. 656

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LITERATURE CITED 1. Ehrlich P. 1910. Studies in Immunity. London: Wiley 2. McDermott MF, Aksentijevich I, Galon J, McDermott EM, Ogunkolade BW, et al. 1999. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 97:133–44 3. International FMF Consortium. 1997. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. Int. FMF Consort. Cell 90:797–807 4. French FMF Consortium. 1997. A candidate gene for familial Mediterranean fever. The French FMF Consort. Nat. Genet. 17:25–31 5. Houten SM, Kuis W, Duran M, de Koning TJ, van Royen-Kerkhof A, et al. 1999. Mutations in MVK, encoding mevalonate kinase, cause hyperimmunoglobulinaemia D and periodic fever syndrome. Nat. Genet. 22:175–77 6. Drenth JP, Cuisset L, Grateau G, Vasseur C, van de Velde-Visser SD, et al. 1999. Mutations in the gene encoding mevalonate kinase cause hyper-IgD and periodic fever syndrome. International Hyper-IgD Study Group. Nat. Genet. 22:178–81 7. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD. 2001. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinﬂammatory syndrome and MuckleWells syndrome. Nat. Genet. 29:301–5 8. Galon J, Aksentijevich I, McDermott MF, O’Shea JJ, Kastner DL. 2000. TNFRSF1A mutations and autoinﬂammatory syndromes. Curr. Opin. Immunol. 12:479–86 9. Pascual V, Allantaz F, Arce E, Punaro M, Banchereau J. 2005. Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade. J. Exp. Med. 201:1479–86 10. Thomas KT, Feder HM Jr, Lawton AR, Edwards KM. 1999. Periodic fever syndrome in children. J. Pediatr. 135:15–21 11. Shoham NG, Centola M, Mansﬁeld E, Hull KM, Wood G, et al. 2003. Pyrin binds the PSTPIP1/ CD2BP1 protein, deﬁning familial Mediterranean fever and PAPA syndrome as disorders in the same pathway. Proc. Natl. Acad. Sci. USA 100:13501–6 12. Ferguson PJ, El-Shanti HI. 2007. Autoinﬂammatory bone disorders. Curr. Opin. Rheumatol. 19:492–98 13. Medzhitov R, Janeway CA Jr. 1997. Innate immunity: the virtues of a nonclonal system of recognition. Cell 91:295–98 14. Feldmann J, Prieur AM, Quartier P, Berquin P, Certain S, et al. 2002. Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes. Am. J. Hum. Genet. 71:198–203 15. Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT, et al. 2002. De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inﬂammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinﬂammatory diseases. Arthritis Rheum. 46:3340–48 16. Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, Tschopp J. 2004. NALP3 forms an IL-1β-processing inﬂammasome with increased activity in Muckle-Wells autoinﬂammatory disorder. Immunity 20:319–25 17. Chae JJ, Komarow HD, Cheng J, Wood G, Raben N, et al. 2003. Targeted disruption of pyrin, the FMF protein, causes heightened sensitivity to endotoxin and a defect in macrophage apoptosis. Mol. Cell 11:591–604 18. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. 2006. Gout-associated uric acid crystals activate the NALP3 inﬂammasome. Nature 440:237–41 19. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. 2008. Innate immune activation through Nalp3 inﬂammasome sensing of asbestos and silica. Science 320:674–77 20. Cassel SL, Eisenbarth SC, Iyer SS, Sadler JJ, Colegio OR, et al. 2008. The Nalp3 inﬂammasome is essential for the development of silicosis. Proc. Natl. Acad. Sci. USA 105:9035–40 21. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, et al. 2008. Silica crystals and aluminum salts activate the NALP3 inﬂammasome through phagosomal destabilization. Nat. Immunol. 9:847–56 www.annualreviews.org • Horror Autoinﬂammaticus

2. Demonstrated that mutations in TNFR1 cause TRAPS and coined the phrase autoinflammatory.

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26–28. Discovery that variants of NOD2 are associated with CD and dominantly inherited in BS.

30. Suggested that an alternative mechanism for the pathogenesis of TRAPS mutations in TNFR1 would exist.

35. Mice with cherubism mutations in SH3BP2 were made and found to differentiate hyperactive macrophages and osteoclasts in response to normal RankL and M-CSF stimulation.

42. First description of the macromolecular inflammasome complex.

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26 December 2008

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Blood Monocytes: Development, Heterogeneity, and Relationship with Dendritic Cells Cedric Auffray,1 Michael H. Sieweke,2 and Frederic Geissmann1,3 1

INSERM U838, Universit´e Paris-Descartes, 75015 Paris, France

2

Centre d’Immunologie de Marseille-Luminy, Campus de Luminy, 13288 Marseille, France

3

Division of Immunology, Infection, and Inﬂammatory Diseases, King’s College London School of Medicine at Guy’s Hospital, London, UK, SE1 9RT; email: [email protected]

Annu. Rev. Immunol. 2009. 27:669–92

Key Words

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

macrophages, subsets, inﬂammation

This article’s doi: 10.1146/annurev.immunol.021908.132557

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0669$20.00

Monocytes are circulating blood leukocytes that play important roles in the inﬂammatory response, which is essential for the innate response to pathogens. But inﬂammation and monocytes are also involved in the pathogenesis of inﬂammatory diseases, including atherosclerosis. In adult mice, monocytes originate in the bone marrow in a Csf-1R (MCSF-R, CD115)-dependent manner from a hematopoietic precursor common for monocytes and several subsets of macrophages and dendritic cells (DCs). Monocyte heterogeneity has long been recognized but in recent years investigators have identified three functional subsets of human monocytes and two subsets of mouse monocytes that exert specific roles in homeostatis and inflammation in vivo, reminiscent of those previously described classically and alternatively activated macrophages. Functional characterization of monocytes is in progress in humans and rodents and will provide a better understanding of the pathophysiology of inﬂammation.

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INTRODUCTION

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DCs: dendritic cells

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The innate immune system has been shaped by evolution to allow multicellular organisms to live together with microorganisms and parasites. In most species, including invertebrates such as Drosophila and vertebrates such as zebraﬁsh, mice, and humans, the innate immune system is composed of a humoral arm, which consists of antimicrobial peptides and opsonins, and a cellular arm, which mainly involves specialized cells known as phagocytes. Phagocytes are cells able to internalize and digest bacteria and other cells; to scavenge toxic compounds produced by the metabolism; and to produce inﬂammatory mediators that can kill bacteria, parasites, and viruses and contribute to the activation of other cell types and to the walling off of parasites. The cellular innate immune system thus contributes to keeping the growth of microbes under more or less tight control. However, its activation has side effects—collectively known as inﬂammation—mainly owing to tissue damage to the host. In the long term, inﬂammation contributes to the development of inﬂammatory diseases, including atherosclerosis. Inﬂammatory diseases, which are leading causes of morbidity and mortality in developed countries, could thus be seen as long-term natural side effects of innate immunity in the context of individual genetic susceptibility. This implies that tuning down but not turning off innate surveillance may delay the aging of tissues and prevent or attenuate inﬂammatory disorders. In this context, understanding the cellular basis and the molecular mechanisms of the innate surveillance of tissues is a worthy goal. Monocytes represent 10% of leukocytes in human blood and 4% of leukocytes in mouse blood. They are distinct from polymorphonuclear (PMNs) and natural killer (NK) cells, which also belong to the innate arm of the immune system, as well as from lymphoid T and B cells, which represent the adaptive arm of the immune system. Monocytes are present in mammals, birds, amphibians, and ﬁsh (1–3), and a related population of hemocytes (called plasmatocytes) is present in the ﬂy (4, 5), which does

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not have lymphocytes. Monocytes play an important role in development and homeostasy, in part via the removal of apoptotic cells and scavenging of toxic compounds (4, 6). Strikingly, monocyte/macrophage specialization can already be observed among unicellular eukaryotic organisms, as phagocytes able to scavenge toxic compounds and kill bacteria differentiate inside colonies of social amoeba (Dictyostelium discoideum) (7). In mammals, monocytes also represent accessory cells, which can link inﬂammation and the innate defense against microorganisms to adaptive immune responses. Indeed, the best known function of monocytes is as a considerable systemic reservoir of myeloid precursors for the renewal of some tissue macrophages and antigen-presenting dendritic cells (DCs) (8–11). However, differentiation of monocytes into DCs is mostly observed in inﬂammatory conditions, e.g., during an active infection, and evidence indicates that the renewal of tissue macrophages and DCs does not rely solely on blood monocytes (12–14). As discussed above, blood monocytes also represent a large pool of scavenger and potential effector cells inside blood vessels in homeostasis as well as during inﬂammatory processes (15). Monocytes are equipped with a large array of scavenger receptors that recognize microorganisms but also of lipids and dying cells, and stimulated monocytes can produce large quantities of effector molecules involved in the defense against pathogen, as reviewed recently (16–18), and in the pathogenesis of several inﬂammatory diseases, including arthritis and atherosclerosis (19). The role of monocytes in the control of microorganisms is likely to be under evolutionary pressure, although their detrimental effects may not be under such pressure given that, in most cases, these detrimental effects are only apparent after several decades of life. Studying the biology of monocytes is therefore useful for the understanding of susceptibility to infection, but it may be even more important for providing ideas and tools to control, delay, or alleviate the long-term detrimental side effects of the inﬂammatory response.

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Studying the functions of human monocytes in biological processes is still a difﬁcult task, however, because monocytes permanently survey their environment and rapidly react to its modiﬁcation or alteration. Isolating them, purifying them on gradients, and culturing them in vitro notably affect their phenotype and behavior. A better understanding of the functions of human monocytes comes from whole-genome array analysis on sorted prospective subsets and from a careful comparison with the results obtained from studies in mice and maybe other model systems in which an in vivo analysis of the effector functions of prospective subsets is possible by combining genetic engineering and intravital studies (15).

MOLECULAR CONTROL OF MONOCYTE DEVELOPMENT FROM HEMATOPOIETIC PRECURSORS CSF1-R Controls Monocyte Development The development of blood monocytes is dependent on the growth factor Csf-1 (also known as M-CSF and CD115). In mice deﬁcient in Csf-1R (c-fms, M-CSFR, CD115) and its ligand Csf-1, the number of blood monocytes is dramatically reduced (20–22), and expression of an M-CSF transgene rescues the differentiation of monocytes (22a). Csf-1R is a hematopoietic growth factor receptor expressed in monocytes, macrophages, and DCs and their precursors (23, 24), a population of cells sometimes referred to as the mononuclear phagocyte system (MPS). The two known ligands of Csf-1R, Csf-1/M-CSF (25) and the more recently described IL-34 (26), are both important for the development of this lineage, as M-CSF-deﬁcient mice (op/op and csf1−/− ) have a milder phenotype than do Csf-1Rdeﬁcient mice (20). Other cytokines, such as GM-CSF, Flt3, and lymphotoxin α1β2 (12, 27, 28),

control the development and homeostasis of the macrophage and DC networks but appear to be dispensable for monocyte development.

MDP: macrophage and DC precursor

Transcription Factors that Control Monocyte Development from Hematopoietic Stem Cells Monocytes develop from hematopoietic stem cells in the bone marrow via several commitment steps and intermediate progenitor stages that, in the prevalent model, pass through the common myeloid progenitor (CMP), the granulocyte/macrophage progenitor (GMP), and the macrophage/DC progenitor (MDP) stages (14, 29) (see below). Each of these differentiation steps involves cell fate decisions that successively restrict developmental potential. In several of these steps, the Ets family transcription factor PU.1 plays an important role. PU.1 can induce myeloid commitment in immature multipotent progenitor cells (30) and is required for the generation of CMP in early myelopoiesis (31, 32). Besides PU.1’s role in early commitment, we also know from gain-of-function and retroviral reconstitution experiments of PU.1-deﬁcient cells that PU.1 controls several cell fate decisions along the myelo-monocytic pathway by engaging in antagonistic interactions with different transcription factors. Initially, inhibitory interactions with GATA-1 shut down the megakaryocytic/erythroid pathway, and repression of GATA-2 blocks mast cell development (33). At the later bipotent GMP stage, PU.1 is critical for driving monocytic differentiation, at the expense of granulocytic differentiation (31), by antagonizing C/EBPα (34), a transcription factor required for granulocytic development (35). During myelopoiesis, PU.1 thus appears to successively close development options by overruling key regulators of other pathways. The antagonism with these factors, however, is not absolute but exquisitely dependent on relative expression levels and the balance of both factors. Whereas PU.1 expression over a certain threshold of antagonist can block the associated cell fate, low or equal levels may actually result in cooperative readouts www.annualreviews.org • Blood Monocytes

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(33, 34, 36). For example, the combination of C/EBPα and PU.1 is thus required ectopically to induce macrophage fate in B cells (37), T cells (38), and ﬁbroblasts (39), underscoring the importance of partner molecules in deﬁning transcription factor function (40). Such behavior could be explained by a quantitative model in which metastable cooperation of C/EBPα and PU.1 on both granulocytic and macrophage target genes is converted into a stable antagonism and ﬁxed granulocyte or macrophage fate by the activation of the crossinhibitory Gﬁ-1 and Egr/Nab downstream regulators (36). C/EBPα thus activates Gﬁ-1, which is required for granulocytic but not monocytic differentiation (41, 42), whereas PU.1 activates the monocyte/macrophagedetermining Egr transcription factors and their cofactor Nab. Egr1 can selectively induce macrophage differentiation (43, 44), and although egr1 deﬁciency by itself does not prevent macrophage development (45), composite egr1−/− egr2+/− bone marrow progenitors show a defect in Csf-1-dependent macrophage differentiation (36). Although compelling, this model is certainly not complete and may involve additional transcription factors to determine monocyte fate. For example, ICSBP/IRF-8 (IFN consensus sequence binding protein/IFN regulatory factor 8) can also drive monocytic differentiation, at the expense of granulocytic differentiation, in ICSBP/IRF-8-deﬁcient progenitors (46). It is tempting to speculate that this may involve its direct interaction with PU.1 (47). Similarly, the Krueppel-like factor KLF4 can induce macrophage fate, and, as a downstream target of PU.1, KLF4 can selectively rescue monocyte differentiation of PU.1−/− progenitors, whereas KLF4 deﬁciency biases myeloid progenitor differentiation toward the granulocytic fate (48). Finally, the MafB and c-Maf transcription factors are highly expressed in monocytes and macrophages (49–52) and can selectively drive monocyte fate in myeloid progenitors (2, 52, 53). Similar to the cell fate choices described above involving an expression level–dependent

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switch from cooperative to antagonistic interactions, moderate expression of PU.1 is compatible with MafB and macrophage fate, whereas higher levels antagonize MafB in the macrophage-to-DC choice. In addition, MafB plays a critical role in integrating monocytic differentiation and cell cycle arrest (see below).

Integration of Cytokine Signaling and Transcription Factor Activity As indicated above, the growth factor Csf-1 and its receptor Csf-1R/c-fms are critical for monocyte differentiation from bone marrow progenitors. Dissection of the human and mouse c-fms proximal promoters has revealed that c-ets-1, c-ets-2, and PU.1 trans-activate the c-fms proximal promoter (54). Consistent with this, PU.1-deﬁcient myeloid progenitors do not express c-fms (55). Already in early progenitor cells with low c-fms levels, PU.1 is assembled in a primed chromatin conformation on both the proximal promoter and an fms intronic regulatory element (FIRE) enhancer (56), which is required for recruitment of Egr transcription factors that then mediate high-level expression in more mature cells. These observations have led to the suggestion that cell intrinsic commitment events induce the upregulation of the c-fms receptor, which allows proliferation and survival of monocytic cells (57). However, c-fms expression cannot rescue macrophage differentiation in PU.1-deﬁcient cell (55), indicating that, in the absence of PU.1, c-fms signaling is not sufﬁcient to drive macrophage differentiation. Furthermore, c-fms is already expressed at low levels in early multipotent stem and progenitor cells (58). Understanding commitment to the monocytic lineage requires determining which transcriptional events control the sensitivity of c-fms signaling in these cells.

MONOCYTES AND THE MONONUCLEAR PHAGOCYTE SYSTEM (MPS) Macrophages and DCs form networks of phagocytic cells throughout most

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tissues—sometimes referred to as the MPS (23)—and play major roles in development, scavenging, inﬂammation, and antipathogen defenses (59, 60). The MPS was initially deﬁned as a population of cells, derived from a bone marrow progenitor, that differentiate and enter the blood as monocytes and then enter tissues to become resident tissue macrophages and antigen-presenting cells (61). However, it was soon recognized that DCs and macrophages have a remarkable heterogeneity related to their origin, phenotype, tissue localization, proliferative potential, and function (59, 62). Macrophages and DCs can be divided into three main groups according to their half-life, to their replacement after bone marrow graft, and to whether their differentiation is elicited by inﬂammation or not. The potential mechanisms for the renewal of individual subsets include (a) self-renewal of resident postmitotic cells; (b) migration, homing, and limited proliferation of adult bone marrow–derived progenitor cells in peripheral tissues (13, 63); and (c) the extravasation and differentiation of circulating precursors such as blood monocytes. These mechanisms are not mutually exclusive—they could operate in parallel or sequentially during the life of the animal—and they are likely to depend on environmental cues such as inﬂammation. Langerhans cells (LC) of the epidermis and microglia are macrophages of the central nervous system. Most remain host-derived after syngeneic bone marrow transplantation but can be replaced by bone marrow–derived cells, possibly blood monocytes, in circumstances in which the resident cells are depleted by UV or gamma irradiation (64, 65). A second group of cells is exempliﬁed by conventional DCs (cDCs). The work of Ralph Steinman and colleagues established that DCs represent a distinct family of cells that regulate the immune responses (59). DCs were originally described as the population of cells enriched from mouse spleens that are responsible for so-called mixed lymphocyte reaction activity (66). These splenic cells, now known as

cDCs, are present in all lymphoid organs and can be divided into subsets according to phenotype, location, and function (67). cDCs, such as CD8a+ and CD8a− DCs of the spleen and lymph nodes, have a short half-life and renew in the steady state from a bone marrow precursor without a monocytic intermediate (12, 14, 63) (see below and Figure 1). A third group of cells represents shortlived cells that differentiate from blood monocytes in response to inﬂammation or infection such as monocyte-derived DCs or TNFα- and iNOS-producing (Tip)-DCs (9, 10, 68, 69). Of note, the work of Massberg et al. (13) has shown that hematopoietic stem and progenitor cells (HSPCs) can circulate and proliferate within extramedullary tissues and give rise to tissue-resident myeloid cells, preferentially DCs. HSPC differentiation is ampliﬁed upon exposure to Toll-like receptor (TLR) agonists such as LPS (13). Therefore, the MPS cannot be considered as a simple family of monocyte-derived cells, but must be considered as a more complex cellular system involved in the scavenging of dying cells, pathogens, and molecules via a variety of cellular processes, such as phagocytosis and endocytosis using, for example, membrane pattern-recognition receptors (60). And the contribution of monocytes to this complex cellular system is an area of active research.

cDCs: conventional dendritic cells of the lymphoid organs Tip-DC: TNF-α and iNOS-producing DC

MDP, A COMMON PROGENITOR FOR MONOCYTE MACROPHAGES AND DENDRITIC CELLS Today, investigators accept, on the basis of transplantation studies, the prevalent model that monocytes, many macrophage subsets, most of the cDCs in the secondary lymphoid organs of mice, and at least a fraction of the DCs in the mouse thymus probably originate from a myeloid progenitor (14, 70–72). Among myeloid precursors, the MDP was identiﬁed as a subset of bone marrow–proliferating cells that share the www.annualreviews.org • Blood Monocytes

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Microglia Langerhans cells

Fetal progenitors GMPs CD34+ CD16/32+ HSPC

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Myeloblast Lin– CD117/ckit+ CX3CR1–

HSC

CMP

Lin– CD117/ckit+ Sca-1+

Lin– CD117/ckit+ Sca-1– CD16/32– CD34+

MDP/CDP Lin– CD115/CSF1R+ CD117/ckit int/lo CX3CR1+ CD135/Flk2 (Flt3)+

Bone marrow

Inﬂammatory macrophages and inﬂammatory DCs, TipDCs pDC

Mucosal macrophages Alternatively activated macrophages?

Monocytes DC precursor

Blood

Spleen CDC11chigh DCs

Tissue

Figure 1 Differentiation of the macrophage/DC progenitor and origin of macrophage and DC subsets.

CDP: common DC precursor

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phenotype of GMPs (73), Lin− Sca1− IL7Rα− CD117(cKit)low CD34+ CD16+ , and that speciﬁcally express the Csf-1R (CD115) and the chemokine receptor CX3CR1 (Figure 1) (11, 14; C. Auffray, D.K. Fogg, E. NarniMancinelli, B. Senechal, C. Touillet, et al., manuscript submitted). The MDP gives rise to monocytes, several macrophage subsets, and spleen cDCs (11, 12, 14). Of note, the MDP generates cDCs directly, without a monocytic intermediate (11, 12, 14, 63), whereas monocytes themselves generate other types of DCs, including inﬂammatory DCs or mucosal DCs (Figure 1) (9–11, 16). The MDP has no signiﬁcant granulocytic potential (11, 12, 14). These initial studies did not detect plasmacytoid DC (pDC) potential cells (11, 12, 14), but our recent data indicate that MDPs actually give rise to pDCs in vivo (C. Auffray, D.K. Fogg, E. Narni-Mancinelli, B. Senechal, C. Touillet, et al., manuscript submitted). Therefore, the MDP is a common preAuffray

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cursor that gives rise in vivo to monocytes, macrophages, and the two main subsets of DCs: cDC and pDCs. The chemokine receptor and adhesion molecule CX3CR1 is not expressed on early hematopoietic progenitors, including CMPs and GMPs, but it is ﬁrst detected on MDPs. CX3CR1 is therefore associated with the commitment of myeloid progenitors to the monocyte/macrophage/DC lineage (12, 14), although its role in the development and homeostasis of cells of the MPS remains unknown.

Common Dendritic Cell Precursor (CDP), MDP, and Monocytes However, the controversy on the origin of monocytes and DCs is not completely resolved, and another precursor—the CDP, for common DC precursor—was recently reported to generate cDCs and pDCs, but not monocytes (74, 75). Importantly, the CDP did not respond to

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CSF-1 (74, 75). This result was interpreted as indicating the existence of two pathways for cDC generation: the CDP pathway involved in homeostasy and the MDP pathway involved in inﬂammation. However, the authors did not compare the potential of CDP with that of MDP in their experimental system, and thus another explanation for the discrepancy in the literature is that the differences in differentiation potential may reﬂect differences in experimental protocols rather than in intrinsic properties of the cells. We favor the latter explanation because, as expected from the original papers (14, 75), the MDP and CDP share the same surface phenotype by ﬂow cytometry (Lin− IL7Ra− CD117int/low CD135+ CD115+ CX3CR1+ ) (C. Auffray, D.K. Fogg, E. Narni-Mancinelli, B. Senechal, C. Touillet, et al., manuscript submitted). Furthermore, CDP was reportedly puriﬁed from mouse bone marrow using an antibody against CD115 (AFS98) that very efﬁciently blocks CSF-1 binding to its receptor and CSF-1-dependent proliferation in vitro (76–79). In our laboratory, MDPs puriﬁed in the presence of AFS98 antibody failed to respond to subsequent culture with M-CSF (C. Auffray, D.K. Fogg, E. Narni-Mancinelli, B. Senechal, C. Touillet, et al., manuscript submitted). Therefore, as proposed recently (12), MDP and CDP may represent overlapping populations with a similar differentiation potential, and the puriﬁcation process of CDP likely explains at least in part, its impaired response to M-CSF and its poor macrophage potential.

Homeostasis of cDCs of the Lymphoid Organs Is Independent of Monocytes Although monocytes can generate several subsets of DCs in inﬂammatory conditions, the homeostasis of cDCs of the lymphoid organs is independent of blood monocytes and dependent on the rate of DC progenitor (MDP) input from blood and its proliferation within the spleen (12, 63). Flt3 (Fms-like tyrosine kinase 3,

Flk2, CD135) is closely related to cFms/Csf1-R and is broadly expressed on early hematopoietic precursors (28). At physiological levels, MDPs do not require Flt3-mediated signals for their generation, but precursors that have entered the spleen undergo cell division locally under the control of Flt3 while they differentiate into cDCs, and thus Flt3 controls homeostatic cDC division in the periphery in vivo (12). Another study identiﬁed lymphotoxin-α as a critical mechanism in maintaining the size of the CD8α− cDC pool in the spleen via local homeostatic expansion (27). Flt3 and lymphotoxin-α therefore control the local homeostatic replenishment of cDCs of the peripheral lymphoid organ.

HOMEOSTATIC CONTROL OF THE MONOCYTE POOL AND RELEASE FROM THE BONE MARROW Control of Monocyte Proliferation In general, besides being associated with the suppression of alternative developmental pathways, macrophage differentiation is also tightly associated with cell cycle withdrawal, and it is believed that monocytes do not proliferate. Whereas myeloid progenitor cells both differentiate and proliferate in response to Csf-1, terminally differentiated cells become refractory to proliferative signals (80), despite their continued ability to sense Csf-1 (81). Proliferative Csf-1 signaling involves the activation of c-myb and c-myc target genes via Ets-1/2 transcription factors (82, 83). Consistent with this, constitutively active alleles of c-myb and c-myc or their overexpression induce continued cycling of myelo-monocytic progenitor cells and macrophages, respectively (84, 85). MafB not only induces macrophage differentiation but also inhibits progenitor proliferation (86). It is tempting to speculate that this may involve MafB’s ability to repress Ets-1 (49), www.annualreviews.org • Blood Monocytes

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particularly as inhibitory Ets factor complexes participate in cell cycle arrest during terminal macrophage differentiation (80). Furthermore, MafB directly engages in SUMO modiﬁcation– dependent physical cross-inhibitory interactions with Myb proteins (86), indicating that relative c-Myb and MafB activities can shift the homeostatic balance between progenitor proliferation and terminal differentiation. As c-Maf can also inhibit Ets-1 and c-Myb transactivation (87) and MafB deﬁciency causes compensatory c-Maf upregulation (88), MafB and c-Maf may cooperate in this process. However, investigators have suggested that a fraction of blood monocytes can be induced to proliferate in vitro after exposure to M-CSF and GM-CSF, and recent evidence shows that M-CSF-driven monocyte-to-macrophage differentiation is associated in vitro with transcription of positive regulators of cell proliferation, such as cell cycle–associated cyclin A2, B1 and B2, D1 and D3, and E2 genes (89). Our own results suggest that a similar phenomenon occurs in vivo in monocytes that extravasate during infection with Listeria monocytogenes (Lm) (C. Auffray and F. Geissmann, unpublished results). However, we could not directly detect proliferation of monocytes in vivo so far. This may suggest that postmitotic blood monocytes that extravasate and enter tissues and differentiate into macrophages or DCs could be induced to proliferate in response to homeostatic or inﬂammatory stimuli within their microenvironment. Local homeostatic control of monocyte, macrophage, and DC proliferation is in fact an area of active investigation. Local proliferation appears to be sufﬁcient for the renewal of microglia (65) and LCs (64) throughout life in the steady state as well as during the course of a variety of diseases. Only under deﬁned conditions are both microglia and LCs replaced with bone marrow–derived cells. However, the molecular mechanisms that control LC and microglial homeostasis remain to be understood. The molecular basis for this is unknown, and it would be interesting to determine in these cases whether the mechanisms that assure cell

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cycle withdrawal at terminal differentiation can be temporarily suspended.

Roles of p21cip and Fas Although the homeostatic control of the monocyte pool in the periphery is relatively poorly understood beyond the role of Csf1-R, PU.1, and Maf family transcription factors, studies of gene-targeted mice have revealed some new aspects of monocyte biology. Mice deﬁcient in the cyclin-dependent kinase inhibitor p21cip have a decreased number of blood monocytes and are resistant to serum transfer–induced arthritis, a phenotype reversed by the transfer of wild-type monocytes, suggesting a role for p21cip in regulating the development and/or differentiation of monocytic populations (90). The Fas pathway is also likely to play a role in vivo in governing monocyte/macrophage homeostasis, perhaps via the control of monocyte survival. Compared with congenic control C57BL/6 mice, Fas-deﬁcient mice display increased numbers of circulating monocytes in the steady state and in a model of systemic inﬂammatory arthritis (91).

Control of Monocyte Emigration from the Bone Marrow by Inflammation and Chemokine Receptors The mechanisms controlling monocyte emigration from the bone marrow niche where they are generated is an area of active investigation and were recently reviewed (16). Inﬂammation, owing either to infection or to a high-fat diet, clearly has a profound effect on the number of blood monocytes, most likely by increasing their egress from the bone marrow (16, 92, 93). Important studies have demonstrated that the chemokine receptor CCR2 and its ligands CCL7 and CCL2 are required for the emigration of the inﬂammatory Ly6c+ (Gr1+ ) subset of monocytes (see below) from the bone marrow and determine their frequency in the circulation (8, 92, 94, 95). The role of CCR2 and its ligands has recently been reviewed elsewhere (16).

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Interestingly, the simultaneous inactivation of CCR2 (or its ligands) and of CX3CR1 and CCR5, two other chemokine receptors expressed on monocytes, has a synergistic effect in decreasing monocyte numbers in the blood, monocytosis induced by a high-fat diet, and atherosclerosis (93, 95). This suggests either that each of these chemokine receptors has additive effects on the same monocytes and/or that different monocyte subsets are dependent on distinct chemokine receptors for their egress from bone marrow and their recruitment into tissues. The mechanisms by which CCR5 and CX3CR1 contribute to controlling the number of monocytes in the periphery are not known.

HETEROGENEITY OF MONOCYTIC CELLS Over the past 25 years, numerous lines of evidence have indicated that the roles of monocytes, both in the control of pathogens and the pathophysiology of inﬂammation, may be attributable to discrete functional subsets. Therefore, as our understanding of monocyte biology improves and these cells appear more and more important in the general ﬁeld of inﬂammation, the issue of monocyte heterogeneity becomes more relevant to human health. It is now recognized that mouse and human, but also rat and pig, blood monocytes can be divided into phenotypic and functional subsets (10, 96–98); however, in the present review we only consider human and mouse cells.

General Features of Monocytes In humans and mice, monocytes have some typical morphological features such as irregular cell shape, oval- or kidney-shaped nucleus, cytoplasmic vesicles, and high cytoplasm-tonucleus ratio. However, they are still very heterogeneous in size and shape and are difﬁcult to distinguish by morphology or by light scatter analysis alone from blood DCs, activated lymphocytes, and NK cells. Human and mouse blood monocytes can be deﬁned by the expression of the Csf-1 receptor

(MCSF-R, CD115) and the chemokine receptor CX3CR1. They are distinct from PMNs, NK cells, and lymphoid T and B cells and do not express Nkp-46, CD3, CD19, or CD15. Monocytes are equipped with a large array of scavenger receptors that recognize lipids and various microorganisms, and stimulated monocytes can produce large concentrations of ROS; complement factors; prostaglandins; nitric oxide (NO) (in mice); cytokines such as TNFα, IL-1β, CXCL8, IL-6, and IL-10; vascular endothelial growth factor; and proteolytic enzymes; and they have been involved in the defense against pathogen, as reviewed recently (16–18, 99). Antigen presentation has been described as a classical feature of monocytes, but since the identiﬁcation of discrete subsets of DCs among monocyte cells, bona ﬁde monocytes have been found in most cases to be far less efﬁcient than DCs for antigen presentation (59). Initial work, performed when separation techniques that allow the investigator to distinguish monocytes from NK cells were less efﬁcient, reported that monocytes have a cytotoxic potential. However, more recent studies have not conclusively addressed this issue. In recent years, investigators have identiﬁed several distinct populations of blood monocytic cells. Two of these populations have been characterized as DCs and are brieﬂy discussed below. In humans, three populations deﬁned by the expression of CD14 and CD16 (CD14+ CD16− , CD14+ CD16+ , and CD14dim CD16+ ) (97, 99–101) have retained the name monocytes and are discussed below. In mice, two main subsets have been characterized (8, 10, 15, 69, 102–104), although additional subsets have been proposed (105). At present, direct comparison of the functions of mouse monocyte subsets with their putative human orthologs is relatively difﬁcult, at least in part because the experimental systems used in human and mouse studies are different.

Blood Dendritic Cells The myeloid blood DC population represents 5% of monocytic-like cells (∼0.5% of www.annualreviews.org • Blood Monocytes

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peripheral blood mononuclear cells) in human (106). These blood DCs stimulate T cell proliferation in vitro, express class II antigens and CD11c, and in human—for most authors—are negative for the monocyte markers CD14 and CD16. A second DC subset corresponds to pDCs and also represents ∼0.5% of peripheral blood mononuclear cells in human. pDCs are the most potent IFN-α-producing cells in response to viral pathogens (107, 108).

Bona Fide Monocytes The remaining 95% of monocytic cells are presently considered by most authors as bona ﬁde monocytes (10, 62, 69, 101, 109, 110). Distinction between monocytes and circulating DCs is easy in the mouse because mouse monocytes do not express MHC class II antigens or the integrin CD11c. In contrast, although they are poor antigen-presenting cells, human monocytes express MHC class II antigens (106) and the integrin CD11c.

THREE SUBSETS OF HUMAN MONOCYTES AS DEFINED BY THEIR PHENOTYPE AND CYTOKINE PRODUCTION Almost 30 years ago, it was shown that human peripheral blood monocytes were not a homogeneous population but rather differ in their phenotype and functions (reviewed in 99). In the early 1980s, Yasaka et al. (112), Weiner et al. (115), Figdor et al. (111), Akiyama et al. (113–114), and Elias et al. (116) described the existence of two functional subsets of monocytes in human. On the basis of size and density, these authors distinguished a major population of regular or large monocytes with higher phagocytic and myeloperoxydase activity and higher superoxide release, and a minor population of intermediate or small monocytes with low peroxydase activity but with a higher capacity to elaborate and release IL-1 and to mediate antibody-dependent cytotoxicity. Although they were limited by the puriﬁcation of monocytes using density gradient alone, 678

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and the likely presence of contaminating NK cells and DCs, many results from these early studies were conﬁrmed in more recent studies, where prospective monocyte subsets have been deﬁned on the basis of their difference in surface marker expression. The work by the group of ZieglerHeitbrock (97) has revealed that the small monocytes could be identiﬁed by the expression of CD16 (FcγR-III). Accordingly, the major subset of monocytes that express CD14 but lack CD16 has higher phagocytic activity but lower cytokine production than does the minor subset of small monocytes that express CD16 (101). The CD14+ CD16− monocytes represent 80% to 90% of blood monocytes, express high levels of the chemokine receptor CCR2 and low levels of CX3CR1, and produce IL-10 rather than TNF and IL-1 in response to LPS in vitro (117, 118) (10, 109). Their phenotype resembles that of mouse Ly6c+ (Gr1+ ) monocytes, although the latter are very efﬁcient at producing inﬂammatory cytokines (16, 104). In contrast to this major subset, human CD16+ monocytes express high levels of CX3CR1 and low levels of CCR2 (10, 109, 118), are responsible for the production of TNF-α in response to LPS stimulation, and were called proinﬂammatory (101, 119). Several studies have reported that CD16+ monocytes are found in larger numbers in the blood of patients with acute inﬂammation (120) and infectious diseases (121, 122). Of interest, CD16+ monocyte numbers are reduced in the blood after treatment with glucocorticoids (121). However, work by the group of GrageGriebenow has shown that CD16+ monocytes are composed of at least two populations with strikingly distinct functions (99). Monocytes that express CD16 and CD14 (CD14+ CD16+ ) also express the Fc receptors CD64 and CD32, have phagocytic activity, and are entirely responsible for the production of TNF-α and IL1 in response to LPS (123). In contrast, monocytes that express CD16 but very low levels of CD14 (CD14dim CD16+ ) lack the expression of other Fc receptors, are poorly phagocytic

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and do not produce TNF-α or IL-1 in response to LPS (124). The actual function of the CD14dim CD16+ monocytes remains elusive, but they may be expanded in the blood of septic patients (121).

by several antibodies, including AL-21 (which is speciﬁc for Ly6C) and RB6-8C5 antibody (Gr1) (which also recognizes Ly6G). Ly6G is only expressed by granulocytes. Therefore, the Gr1 antibody and Ly6C-speciﬁc antibodies label the same cells in the mouse blood, i.e., a subset of monocytes, granulocytes, pDCs, and NK cells, whereas pDCs and NK cells do not express CD115, and granulocytes express CD115 at low level (18, 129).

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MONOCYTE SUBSETS IN MICE During the past few years, several teams have developed strategies for studying in vivo the differentiation, recruitment, and functions of blood monocytes using adoptive transfer and intravital studies (15, 23). They have generated mouse models by inserting green ﬂuorescent protein (GFP) into the Lysozyme-M gene (125), into the Cx3cr1 gene (126), or as a transgene driven by the c-fms promoter (23). Monocytes have also been labeled using latex bead injection (102, 127) and 111 Indium (104). Mouse monocytes are identiﬁed in blood based on the expression of CD115; based on their FSC SSC proﬁle; based on the expression of F4/80, CD11b, Dectin-1 (the beta glucan receptor); and based on the variable expression of the Gr1/Ly6C antigen and 7/4 antigen (10, 18, 62). All circulating monocytes express the GFP reporter in Cx3cr1gfp/+ mice (126), and several teams, including us, have studied blood monocytes in some detail in this model (10, 15, 128). The Ly6C antigen is a glycosylphosphatidylinositol-anchored molecule also expressed by granulocytes, 40% of NK cells, and pDCs. Ly6C is recognized

MURINE CD115+ Ly6C+ (Gr1+ ) INFLAMMATORY MONOCYTES The main subset of CD115+ monocytes expresses Ly6C (Gr1+ ), the chemokine receptor CCR2, the adhesion molecule L-selectin (CD62L), and a low level of the chemokine receptor CX3CR1. As discussed above, they are a phenotypic equivalent to human CD14+ monocytes (10). Murine Ly6C+ (Gr1+ ) monocytes are selectively recruited to inﬂamed tissues and lymph nodes in vivo, produce high levels of TNF-α and IL-1 during infection or tissue damage, and were termed inﬂammatory monocytes (8, 10, 16, 69, 104, 128). A number of studies using either adoptive transfer of monocytes or latex bead–labeled monocytes strongly support the conclusion that at least a proportion of TNF-α-producing inﬂammatory DCs are the progeny of Ly6C+ (Gr1+ ) monocytes (9, 10, 16). These inﬂammatory DCs either uptake antigen in peripheral tissues and then migrate into lymphoid organs or, in the case of Tip-DCs, may migrate from the red pulp to the white pulp of the spleen (8, 68, 130). Ly6C+ (Gr1+ ) monocytes can also replenish macrophages and DC resident cell compartments in the skin (e.g., LCs) (131), digestive tract (mucosal DCs) (11), and lung (127, 132, 133) (see Figure 1).

Roles of CD115+ Gr1+ Monocytes During Microbial Infection In Vivo The role of monocytes during microbial infection has been reviewed recently (16, 17). Seminal studies from the groups of Pamer www.annualreviews.org • Blood Monocytes

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(8, 68, 130) and of Drevets and Leenen (69) have characterized the role of these cells in vivo following microbial infection, using mice infected with the intracellular bacteria Lm. Following infection, Ly6C+ (Gr1+ ) blood monocytes egress massively from bone marrow to the bloodstream in a CCR2-dependent fashion and differentiate via a MyD88-dependent mechanism into cells that produce TNF-α and NO and that upregulate MHC class II antigens, CD80, CD86, and CD11c (8, 94, 130). These cells were therefore termed TipDCs (for TNF-α/iNOS-producing DCs) (68) (Figure 2). The severe reduction of Tip-DCs in CCR2-deﬁcient mice was associated with a

reduced control and clearance of Lm following primary infection (68). Inside infected spleens, Tip-DCs secrete high levels of TNF-α and rapidly migrate to T cell zones of splenic follicles, where they also express high levels of the inducible nitric oxid synthase (iNOS) that generates NO radicals. However, although Tip-DCs efﬁciently stimulate a mixed lymphocyte reaction in vitro, their marked reduction in the spleen of CCR2-deﬁcient mice did not lead to a defective T cell priming and proliferative response or to an impaired differentiation into IFN-γ/TNF-α-secreting T cell effectors (68). This suggests that the main function of these M2-type or alternatively activate dmacrophage

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monocyte-derived inﬂammatory DCs is to kill bacteria rather than to regulate T cell functions. However, these data do not rule out such a role for these cells. They are found in very close proximity with Lm-speciﬁc cells and participate in the inﬂammatory environment by releasing high levels of inﬂammatory mediators such as TNF-α (130). Ly6C+ (Gr1+ ) monocytes have also been reported in other infectious models in vivo and contribute to the control of pathogen growth during infections with other bacteria, such as Brucella melitensis (134), or with parasites, such as Toxoplasma (135). A recent study by the Dubois group (136) indicates that CD115+ Gr1+ monocytes are recruited to the inﬂamed dermis via the chemokine receptor CCR6 and its ligand CCL20 and that depletion of monocytes prevents in vivo priming of CD8+ cytotoxic T lymphocytes against the model protein antigen ovalbumin administered with adjuvant. Transfer of CCR6-sufﬁcient Gr1+ monocytes was enough to restore CD8+ T cell priming in CCR6−/− mice via a direct antigen presentation mechanism (136). This work identiﬁed a mechanism for the recruitment of CD115+ Gr1+ monocytes to the skin and suggested that these monocytes are required for efﬁcient cross-priming of CD8+ cytotoxic T lymphocytes after mucosal or skin immunization in this model. Lauvau and colleagues (137) have recently described cells similar to the Tip-DCs that can play a critical role in protection against secondary Lm infection, suggesting that these cells may be important for primary and secondary protective immunity.

CD115+ Ly6C+ (Gr1+ ) Monocytes in Tumor-Bearing Mice In Vivo It is remarkable and intriguing that cells with the very same phenotype, expressing CD115, Gr1, and CD11b, also expand in the spleen of a tumor-bearing host (138, 139). These cells have been characterized as part of the myeloid-derived suppressor cell (MDSC) population that mediates the development of

tumor-induced T regulatory cells and T cell anergy. Tumors induce the expansion of these MDSCs, in both animal models and human patients. MDSCs impair antigen-speciﬁc T cell responses and, particularly, CD8+ T cell responses via molecular mechanisms that involve NO and/or reactive oxygen intermediate production (140–143). This suggests that CD115+ Ly6C+ (Gr1+ ) monocytes could be expanded and polarized toward MDSCs that inhibit T cell–mediated immunity and toward Tip-DCs that strengthen T cell immunity by signals associated with tumor and infection, respectively, or that the CD115+ Gr1+ monocyte population contains two distinct functional subsets that can be expanded by signals associated with tumor and infection. Investigating the relationship between MDSCs and Tip-DCs may be of interest both for basic understanding of monocyte biology and for the potential clinical applications.

MDSCs: myeloidderived suppressor cells

MURINE CD115+ Ly6C− (Gr1− ) MONOCYTES The second subset of monocytes is characterized by a smaller size; high expression of the chemokine receptor CX3CR1, of LFA1 (lymphocyte-function associated antigen 1), and of CD43; and by the lack of expression of Ly6c (Gr1− ), CCR2, or L-selectin (10, 69). This subset has been initially termed resident in mice because these monocytes have a longer half-life in vivo and are found in both resting and inﬂamed tissues after adoptive transfer (10). We and others initially proposed that they may be involved in the renewal of resident macrophage and DC populations (10, 62). However, there is not yet strong supporting evidence for this hypothesis. Investigators had suggested—by analogy with human CD16+ monocytes—that Ly6C− (Gr1− ) monocytes are the main producers of TNF-α (144), but, as discussed above, this is not the case because Ly6C+ (Gr1+ ) monocytes are clearly the main producers of TNF-α during infection. Progress came from the use of adoptive transfer and of intravital microscopy, a powerful www.annualreviews.org • Blood Monocytes

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method to monitor in vivo dynamic parameters of innate or adaptive immune responses (145).

Murine Ly6C− (Gr1− ) Monocytes Patrol Blood Vessels in the Steady State

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Intravital microscopy observation, using Cx3cr1gfp/+ mice as reporters, revealed that Ly6C− (Gr1− ) monocytes exhibited a constitutive long-range crawling on the luminal side of the endothelium, in steady-state condition, within most blood vessels in the dermis and within branches of the mesenteric vein and the mesenteric artery (15). Ly6C− (Gr1− ) monocytes crawl with an average velocity of 12 μm/min. Crawling requires ﬁrm binding to the endothelium mediated by the β2 integrin LFA-1 (CD11a/CD18, αL β2 ) and by the chemokine receptor CX3CR1. Surprisingly, most crawling monocytes stay within blood vessels in the steady state and appear to patrol the endothelium, independently of the direction of the blood ﬂow, for extended periods of time (i.e., 30 min to several hours). In the absence of overt inﬂammation, extravasation is a rare event observed in less that 1% of crawling cells (15). Therefore, we hypothesize that murine Ly6C− (Gr1− ) monocytes constitutively patrol blood vessels and may play important functions in scavenging oxidized lipids, dead cells, and potential pathogens. Patrolling Ly6C− (Gr1− ) monocytes are ideally located to survey endothelial cells and surrounding tissues. CX3CR1, as well as TNF-α and LFA-1, have been implicated in the pathogenesis of atherosclerosis (146–148). It will be important to investigate whether Ly6C− (Gr1− )-patrolling monocytes may contribute to the pathogenesis of inﬂammatory disorders and could represent a target for their treatment. Besides the involvement of LFA-1 and CX3CR1, the molecular mechanisms that control the apparently random crawling of Ly6C− (Gr1− ) monocytes and their potential roles as scavenger are yet to be characterized. 682

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Patrolling Ly6C− Gr1− Monocytes Extravasate and Are Responsible for a Very Early Inflammatory Response During Infection with Listeria monocytogenes However, in response to tissue damage (irritants, aseptic wounding, and peritoneal infection with Lm), Ly6C− (Gr1− ) monocytes extravasate rapidly within 1 h and invade the surrounding tissues (Figures 2 and 3). Global gene expression of Ly6C− (Gr1− ) monocytes puriﬁed from the peritoneum of mice at an early time (2 h) after infection with Lm revealed that, when exposed to this pathogen in vivo, Ly6C− (Gr1− ) develop a very early but transient inﬂammatory response that includes the transcription of genes coding for cytokines, lysozyme, defensins, and complement and includes Lm-associated patternrecognition receptors and phagocytosis such as TLRs (TLR1, TLR2), scavenger receptors (SrA, Cd36, dectin-2, and MDL1), IgFc receptors, and genes associated with antigen presentation (15) (see also Figure 3). This early response also included numerous chemokines involved in the recruitment and activation of other effector cells such as granulocytes, Ly6C− (Gr1− ) monocytes, NK cells, and T cells. At this time, 1 and 2 h after infection, Ly6C− (Gr1− ) monocytes are the main blood cell type extravasated into the peritoneum and are the only producers of TNF-α, a cytokine central to macrophage-mediated inﬂammation and the innate immune response (15). However, this inﬂammatory response is only transient, and at 8 h after infection, Ly6C+ (Gr1+ ) monocytes are the main producers of inﬂammatory cytokines (Figure 3).

Patrolling Ly6C− Gr1− Monocytes Differentiate into Alternatively Activated Macrophages During Infection with Listeria monocytogenes and in the Healing Myocardium Interestingly, following this transient production of inﬂammatory mediators, the balance of

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ventional Ly6C+ (Gr1+ ) monocytes that, when they enter the peritoneum in response to the same Lm infection, initiate a DC differentiation program characterized for example by the upregulation of RelB and PU.1, but not of cMaf and MafB (15) (Figures 2 and 3). Therefore, in the presence of the same pathogen in vivo, the two subsets of monocytes differentiate into distinct cell types: Ly6C− (Gr1− )patrolling monocytes initiate a macrophage differentiation program that resembles that of M2

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transcription factors that specify the alternative macrophage or DC fate of monocytes (51, 149) indicates that extravasated Ly6C− (Gr1− ) monocytes initiate a typical macrophage differentiation program, characterized by upregulation of cMaf, MafB, egr1, egr2, and egr3 and of genes such as Arginase, Fizz1, MR, Mgl2, and IL-4Rα, markers of alternatively activated, also termed M2-like, macrophages (89, 150) (Figures 2 and 3). This ﬁnding is in contrast with the differentiation of the con-

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macrophages, while Ly6C+ (Gr1+ ) monocytes differentiate into DC-like cells that resemble Tip-DCs (15). These ﬁndings are consistent with three other recent studies. Nahrendorf et al. (104) have shown that the healing myocardium sequentially mobilizes Ly6C+ (Gr1+ ) monocytes, with phagocytic, proteolytic, inﬂammatory functions that can digest damaged tissue, and Ly6C− (Gr1− ) monocytes that have attenuated inﬂammatory properties, express vascular endothelial growth factor, and may promote healing via myoﬁbroblast accumulation, angiogenesis, and deposition of collagen. Landsman et al. (133) have reported that adoptively transferred CD115+ Gr1− monocytes, but not CD115+ Gr1+ monocytes, were able to generate macrophages in the lung of recipient mice, whereas both monocyte subsets could generate pulmonary DCs. Arnold et al. (151) studied the phenotype of monocytes in the tibialis anterior muscles after local injection of a drug that induces muscle necrosis followed by a regeneration process. Using latex bead labeling of circulating monocyte subsets, they concluded that during the ﬁrst days after injury, the muscle recruited only F4/80low CX3CR1low Ly6C+ (Gr1+ ) nondividing monocytes that express mRNA for TNFα and IL-1, whereas after day 4 their numbers decreased, and CX3CR1high Ly6C− (Gr1− ) cells increased in numbers and exhibited features of antiinﬂammatory macrophages. Arnold et al. (151) concluded that Ly6C+ (Gr1+ ) monocytes had differentiated into Ly6C− (Gr1− ) monocytes, whereas the studies by Auffray et al. (15), Nahrendorf et al. (104), and Landsman et al. (133) concluded that distinct populations of monocytes are recruited from the blood.

Together, these observations reveal an unsuspected dichotomy (depicted in Figure 4) of the differentiation potential and functions of blood monocyte subsets during Lm infection and myocardial infarction.

CONCLUDING REMARKS As our understanding of monocyte biology improves, monocytes appear more and more important in the general ﬁeld of inﬂammation and inﬂammatory diseases, including atherosclerosis, and the issues of monocyte differentiation and heterogeneity become relevant. There is accumulative evidence that blood monocytes actually consist in several functional subsets. Open questions remain: the numbers of these subsets, the similarities between human and mouse subsets, and whether the differentiation of monocyte subsets represents an early commitment, at the stage of the MDP for example, akin to the division of lymphocytes into B, T CD4+ , and T CD8+ cells, or whether it involves a potentially reversible polarization of more mature cells akin to the polarization of mature T cells. These questions are being actively investigated by a growing number of laboratories. The putative roles of Ly6C+ (Gr1+ ) monocytes in the regulation of lymphocyte-mediated responses and their relationship with MDSCs are of particular interest for tumor biology. Finally, the analysis of the functions of Ly6C− (Gr1− )-patrolling monocytes and of their potential involvement in vascular inﬂammation should foster new investigations of the role of monocytes inside blood vessels—an already very active area of investigation in the ﬁeld of atherosclerosis— and the mechanisms that control patrolling.

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

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ACKNOWLEDGMENTS Work was supported by a EURopean Young Investigator (EURYI) award and by grants from the Ville de Paris, Agence Nationale de la Recherche (ANR IRAP2005), and Fondation pour la Recherche Medicale (Equipe FRM 2006) to F.G. and grants from the Association for International Cancer Research (AICR 2006, 05-0079), the Association de la Recherche sur le Cancer (ARC 3857), and the Institut National du Cancer (INCa) to M.S. F.G. and C.A. are indebted to past and present members of the laboratory.

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Regulation and Function of NF-κB Transcription Factors in the Immune System Sivakumar Vallabhapurapu and Michael Karin Laboratory of Gene Regulation and Signal Transduction, Departments of Pharmacology and Pathology, Cancer Center, University of California, San Diego, California 93093; email: karinofﬁ[email protected]

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Key Words

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

IKK, IκB, NIK, p100, TRAF

This article’s doi: 10.1146/annurev.immunol.021908.132641

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0693$20.00

The mammalian Rel/NF-κB family of transcription factors, including RelA, c-Rel, RelB, NF-κB1 (p50 and its precursor p105), and NF-κB2 (p52 and its precursor p100), plays a central role in the immune system by regulating several processes ranging from the development and survival of lymphocytes and lymphoid organs to the control of immune responses and malignant transformation. The ﬁve members of the NF-κB family are normally kept inactive in the cytoplasm by interaction with inhibitors called IκBs or the unprocessed forms of NF-κB1 and NF-κB2. A wide variety of signals emanating from antigen receptors, pattern-recognition receptors, receptors for the members of TNF and IL-1 cytokine families, and others induce differential activation of NF-κB heterodimers. Although work over the past two decades has shed signiﬁcant light on the regulation of NF-κB transcription factors and their functions, much progress has been made in the past two years revealing new insights into the regulation and functions of NF-κB. This recent progress is covered in this review.

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INTRODUCTION

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NF-κB was ﬁrst identiﬁed about 20 years ago as a transcription factor that binds to the intronic enhancer of the kappa light chain gene (the κB site) in B cells (1, 2). Soon thereafter, NF-κB emerged as a major regulator of innate and adaptive immunity and inﬂammatory responses (3–6). Subsequent studies focused on how the immune system responds to pathogens by activating NF-κB and on the mechanisms by which NF-κB regulates transcription of inﬂammatory genes. However, the observations that many common diseases, including cancer, atherosclerosis, and diabetes, are associated with dysregulation of NF-κB sparked a broad interest in this transcription factor and in the signaling pathways that control its activity. Importantly, the lessons learned from studying NF-κB signaling in immune and inﬂammatory cells are broadly applicable to other cell types and organ systems. As a large number of excellent reviews have already covered the role of NF-κB transcription factors in immunity and inﬂammation (3, 5–12), this review focuses mainly on recent progress in our understanding of both positive and negative regulatory mechanisms that control NF-κB activity during immune and inﬂammatory responses, the cross talk between the classical and alternative NF-κB activation pathways, and their role in immunity, inﬂammation, lymphocyte differentiation, and lymphoid malignancies. The NF-κB family of transcription factors consists of NF-κB1 (p50 and its precursor p105), NF-κB2 (p52 and its precursor p100), RelA (also called p65), c-Rel, and RelB, all of which are characterized by presence of an N-terminal Rel homology domain (RHD) responsible for homo- and heterodimerization as well as for sequence-speciﬁc DNA binding (Figure 1). RelA, c-Rel, and RelB also contain a C-terminal transcription activation domain (TAD), whereas the p52 and p50 subunits do not and therefore rely on interactions with other factors to positively regulate transcription (12) (Figure 1). Whereas RelB preferentially heterodimerizes with p100 (13)

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as well as its processed form p52 (14, 15), RelA and c-Rel predominantly heterodimerize with p50 (10). Unlike other NF-κB family members, RelB also has a leucine zipper (LZ) motif in its N terminus, which plays an important transcriptional regulatory role (12). However, whether the LZ confers additional functional speciﬁcity to RelB, including its heterodimerization with NF-κB2 or other possible partners, remains to be determined. Most NF-κB dimers have been crystallized and their structures solved, thus establishing that the RHD is composed of two immunoglobulin-like folds, one engaged in dimerization and the other in DNA recognition (16). Small differences in dimer interfaces and solvent-exposed surfaces dictate partner preferences and DNA sequence selectivity (16). A number of posttranslational modiﬁcations at different parts of these molecules, including phosphorylations and acetylations, further modulate DNA binding and transcriptional activities (17) (Figure 1). Recent work that has identiﬁed ribosomal protein S3 (RPS3) as a DNA-binding partner for certain NF-κB dimers suggests that high-afﬁnity DNA binding and sequence speciﬁcity may depend on interaction of NF-κB dimers with auxiliary proteins (18). RPS3, which is a KH domain protein, translocates to the nucleus concomitantly with RelA upon lymphocyte activation and binds to RelA homodimers as well as p50:RelA heterodimers on the DNA to form part of the NF-κB complex at speciﬁc regulatory sites (18).

INHIBITORS OF NF-κB: THE IκB FAMILY The most important NF-κB-interacting proteins are the inhibitors of NF-κB, the IκB proteins (10, 12). IκBs retain NF-κB dimers in the cytoplasm of nonstimulated cells and form a small family including IκBα, IκBβ, and IκBε, which are classical IκBs, as well as a few “novel” IκBs that are discussed below. The IκBs can serve different functions and are characterized by the presence of multiple ankyrin repeats (Figure 2) that mediate binding to NF-κB dimers and can interfere with the function of

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Figure 1 The NF-κB family. Schematic diagram for different NF-κB members is shown. Different domains including Rel homology domain (RHD), transcription activation domain (TAD), leucine zipper motif (LZ), glycine-rich region (GRR), and ankyrin repeats (ANK) are shown. Posttranslational modiﬁcations such as phosphorylation, acetylation, and ubiquitination at indicated amino acid positions are shown by P, Ac, and Ub, respectively. p105 is ubiquitinated at multiple sites.

the nuclear localization signals (NLS) present in the latter. The C-terminal halves of p105 and p100 also harbor multiple ankyrin repeats that allow them to serve an IκB-like function (13, 19). The p105 C-terminal ankyrin repeats (also called IκBγ) selectively bind p50 (the processed form of p105), RelA, and c-Rel and retain them in the cytoplasm (19–21). Proteasomemediated processing removes the C-terminal half of p105 to generate the p50 subunit (22). p105 processing primarily occurs constitutively in nonstimulated cells (22, 23). However, upon cell stimulation with cytokines such as tumor necrosis factor (TNF)-α or interleukin (IL)-1, p105 is phosphorylated and undergoes rapid and complete proteasomal proteolysis without p50 production (24, 25). This results in

the release of p105-bound Rel subunits that can now migrate to the nucleus. The major function of p105, however, appears to be a reservoir for production and regulation of the p50:p50 homodimer (19). Unlike p105, the C terminus of p100 (also called IκBδ) preferentially binds to RelB to keep it in the cytoplasm. Previously, p100 was thought not to have a major effect on other NF-κB family members (3, 13–15, 26). However, recent reports argue that p100 also plays an important role in regulating RelA homodimers (27). Furthermore, in the absence of all three typical IκBs, p100 levels are highly elevated, and most of the NF-κB subunits remain in the cytoplasm, suggesting that p100 may have a role in the inhibition of classical NF-κB dimers as well (28). New insights into

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Figure 2 The IκB family. Schematic diagram showing different IκB proteins that contain several ankyrin repeats (ANK). Phosphorylation and ubiquitination at speciﬁc amino acid residues are indicated. Proline (P), glutamic acid (E), serine (S), and threonine (T) domains are indicated as PEST.

the mechanism of signal-induced p100 processing are discussed below. Although the IκBs are similar in structure (Figure 2), they each have their own binding preferences and are subject to differential transcriptional regulation by NF-κB family members (29, 30). For instance, on the one hand, classical RelA:p50 heterodimers are predominantly regulated by IκBα, the best studied IκB family member (10). On the other hand, IκBε preferentially regulates RelA:RelA as well as cRel:RelA dimers (29, 31, 32). The role of IκBβ is less well understood, although it was shown to bind RelA:p50 heterodimers associated with κB sites on DNA, suggesting it may regulate their nuclear function (33, 34). The three major IκBs undergo signal-induced proteasomal degradation with different kinetics (35). IκBα, for example, is degraded most rapidly in response to inﬂammatory stimuli such as TNFα and lipopolysaccharide (LPS) and is resynthesized in an NF-κB-dependent manner to 696

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constitute a negative feedback loop in which newly synthesized IκBα enters the nucleus to associate with deacetylated RelA:p50 heterodimers and shuttle them back to the cytoplasm (36, 37). Therefore, the absence of IκBα impairs the termination of NF-κB activity following stimulation with TNF-α or LPS (38). By comparison, signal-induced IκBβ and IκBε degradation and resynthesis occur at much slower kinetics (35). These temporal differences in IκB degradation and resynthesis seem to play major roles in determining their functional characteristics in regulation of NF-κB activity. In addition to these classical IκB family members, the novel or atypical IκB-like proteins, which include BCL3 (B cell CLL/ lymphoma 3), IκBζ, and IκBNS, are subject to a different form of regulation and serve very different functions. While a few reports suggest that BCL3 interacts with and participates in the removal of p50 and p52 homodimers from DNA and thereby terminates the

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transcriptional repressive effects of these TADlacking dimers (39–41), other studies indicate that BCL3 interacts with p52 and p50 to confer upon them transcriptional activity (42–45). However, BCL3 may actually facilitate the repressive function of p50 by stabilizing p50 homodimers and thereby preventing the access of TAD-containing dimers to κB sites occupied by p50 homodimers, a process thought to contribute to LPS tolerance (46). One possible explanation for this apparent contradiction is that the ability of BCL3 to form transcriptionally active complexes with p50 and p52 homodimers may depend on its signal-induced phosphorylation (47). Owing to its ability to regulate NF-κB activity, BCL3 has been implicated in cancer development, where it acts as a critical regulator of cyclin D1 transcription (48, 49). BCL3 also inhibits p53 activity by inducing transcription of the Hdm2/Mdm2 gene, which codes for a ubiquitin ligase that targets p53 to proteasomal degradation (50). However, whether BCL3 carries this function through association with p50 or p52 homodimers remains to be determined. BCL3-knockout mice are viable but exhibit impaired humoral immune responses and lack splenic germinal centers, suggesting a critical role for BCL3 in B cell development (51). IκBζ is a recently identiﬁed IκB with a weak homology to other family members and is expressed in an inducible manner in response to LPS (52, 53). IκBζ also acts in the nucleus, but, unlike other IκBs, IκBζ seems to potentiate the transcriptional activity of NFκB dimers, which is surmised because, in the absence of IκBζ, induction of several NF-κB target genes in response to LPS or IL-1 is impaired (54). However, IκBζ also associates with p50 homodimers and may function as a coactivator in a similar manner to BCL3 (54). Yet IκBζ also negatively regulates RelA-containing dimers (54, 55). Thus, the function of IκBζ may be quite similar to that of BCL3. The presence of NF-κB- and c/EBP-binding sites in the target gene promoter appear to be required for rendering the gene promotor subject to IκBζ-dependent regulation (56). Inter-

estingly, expression of c/EBPδ is also induced in response to LPS in an NF-κB-dependent manner (57, 58), and thus the coordinated induction of c/EBPδ and IκBζ may be responsible for expanding or altering the set of NF-κB target genes that are activated during the course of an innate immune response. IκBNS is another unique IκB-like protein that is also rapidly induced, in this case upon ligation of the T cell receptor (TCR) in thymocytes (59). Correspondingly, IκBNS plays an important role in negative selection of T cells (59). Like IκBζ, IκBNS inhibits transcriptional activation by NF-κB dimers during thymocyte negative selection (59). Recent reports have shown that in addition to regulating TCR-induced NF-κB activity, IκBNS also negatively regulates LPS-induced NF-κB activity in macrophages and in dendritic cells (DCs) (60). Interestingly, macrophages lacking IκBNS exhibit prolonged NF-κB activity at speciﬁc promoters in response to LPS stimulation (60), suggesting that IκBNS is a gene-speciﬁc terminator of NF-κB activity. IκBNS-deﬁcient mice are highly susceptible to endotoxic shock and intestinal inﬂammation, presumably owing to hyperexpression of selective Toll-like receptor (TLR)-induced inﬂammatory genes, such as IL-6 and IL-12p40 (60). Further studies are required to understand the mechanism by which IκBNS is targeted to NF-κB subunits at selective gene promoters.

ACTIVATION OF NF-κB Many different stimuli activate NF-κB transcription factors to induce their nuclear accumulation (3, 8). The major and most wellstudied activation pathway used by most stimuli is the canonical NF-κB signaling pathway, which mainly impinges upon RelA:p50 and c-Rel:p50 heterodimers. This pathway centers around activation of the trimeric IκB kinase (IKK) complex comprising the catalytic subunits IKKα and IKKβ and the regulatory/ scaffold subunit IKKγ (also called NEMO for NF-κB essential modulator) (10). The mechanism by which the IKK complex is

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activated, as is discussed below, is quite complex and is different from one receptor to another. As mentioned above, binding of IκBs to NF-κB dimers keeps the latter in the cytoplasm. When bound to RelA:p50, IκBα masks the RelA NLS but not the p50 NLS (10). However, owing to the presence of a strong nuclear export signal in IκBα, the IκBα:RelA:p50 trimer is predominantly cytoplasmic, although a constant shuttling of this complex between the cytoplasm and nucleus has been documented (61, 62). Receptor engagement results in IKK activation, and the activated IKK complex phosphorylates IκBα on Ser32 and Ser36, leading to its polyubiquitination at Lys19 by the Skp1, Cdc53/Cullin1, and F-box protein β transducin repeat-containing protein (βTRCP) SCFIκB E3 ubiquitin ligase complex, which catalyzes the formation of degradative Lys48-linked polyubiquitin chains (10). The ubiquitinated IκBα is degraded via the 26S proteasome, thereby exposing the strong NLS on RelA and inducing nuclear translocation of RelA:p50 dimers (10). This pathway also applies to complexes retained by IκBβ and IκBε, which also serve as IKK substrates (63). However, differences in the relative afﬁnity of IKK to IκBs results in degradation of each IκB with distinct kinetics. The IKK complex may also phosphorylate the TADs of RelA and c-Rel while still in the cytoplasm and thus may enhance their transcriptional activity as well as their turnover in the nucleus (17, 64). Of the two catalytic IKK subunits, IKKβ is the one that is responsible for the majority of IκB kinase activity in most cell types. Yet in the absence of IKKβ, IKKα can provide residual IκB kinase activity (65), whereas deletion of IKKα in IKKβ-expressing cells has nearly no affect on classical IKK activity (66). Nonetheless, there are situations, for instance in RANKL-stimulated mammary epithelial cells, in which IκB phosphorylation and degradation are mainly IKKα dependent (67). However, in osteoclast progenitors and macrophages, RANKL activates classical NF-κB dimers mainly via IKKβ (68). As mentioned above, IKKα can also phosphorylate RelA and c-Rel in the cytoplasm of activated

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macrophages, and this phosphorylation contributes to the termination of NF-κB activation by innate immune stimuli (64). The nuclear ubiquitin ligase that recognizes the IKKα-phosphorylated RelA and c-Rel proteins remains to be identiﬁed. An important and unique function of IKKα is activation of the alternative NF-κB pathway, which is based on inducible processing of p100 and activation of RelB:p52 heterodimers (3, 14, 15). Interestingly, the classical IKK signaling pathway described above feeds into the alternative pathway through upregulation of NF-κB2 expression, but the processing of p100 is strictly dependent on its phosphorylation by IKKα and activation of the latter by the NF-κB-inducing kinase (NIK) (14, 69). The alternative NF-κB pathway is activated in response to a small subset of TNF family members, including CD40L, LTαβ, BAFF (B cell– activating factor), RANKL (receptor activator of NF-κB ligand), and TWEAK (TNF-related weak inducer of apoptosis) (14, 15, 70–73), and is regulated quite differently from the classical pathway. Substantial progress has been made in our understanding of the mechanism by which this pathway is regulated both in nonstimulated and receptor-stimulated cells, and this progress is described in detail below. In the alternative pathway, NIK-activated IKKα dimers phosphorylate p100 at speciﬁc serine residues within its C-terminal ankyrin repeat domain (63, 74) (Figure 1). This phosphorylation event allows recognition of p100 by the SCFIκB E3 ligase complex resulting in its polyubiquitination (74, 75). However, because of the presence of a speciﬁc STOP signal located between the p52 N-terminal portion and the p100 C-terminal ankyrin repeat domain, the ubiquitinated p100 molecule undergoes only partial degradation, resulting in release of the N-terminal p52 fragment bound to RelB. The alternative NF-κB pathway has been implicated in lymphoid organogenesis as well as in B cell development and survival (14, 76). Several recent reports indicate that the alternative NF-κB signaling is mainly regulated through the control of NIK turnover. Moreover, aberrant NIK

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turnover appears to be associated with certain B cell malignancies. We discuss these details below.

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NEGATIVE REGULATION OF CANONICAL NF-κB Prompt activation of NF-κB is required for a successful immune response, but this response cannot last forever and needs to be properly terminated to avoid tissue damage and even death owing to shock and organ failure. In addition, uncontrolled inﬂammation can increase the risk of cancer and autoimmune disease (5). A number of distinct mechanisms are involved in terminating NF-κB activation, and they exert their effects at different levels. As discussed above, the major regulators of NF-κB activity are the IκBs, and several of them are also involved in feedback inhibition of NF-κB (36). For instance, one of the earliest NF-κB target genes is NfkBia, which encodes IκBα (77). Newly synthesized IκBα enters the nucleus as a monomer, where it associates with DNA-bound p50:RelA dimers, leading to their inactivation and export into the cytoplasm. This forms a simple negative feedback loop that prevents irreversible activation of NF-κB. However, recent reports suggest that negative regulation of NF-κB activity is much more complicated. First, it was postulated that NF-κB dimers are in an equilibrium on and off the DNA and that IκBα only binds NF-κB dimers that are not in actual contact with the DNA rather than by removing DNA-bound NF-κB dimers from the DNA (78). Second, even without IκBα, classical NF-κB activation can still be terminated (78). Third, proteasome inhibition after IκBα has been degraded and NF-κB has entered the nucleus results in elevated expression of NF-κB target genes (78). These observations suggest the operation of additional mechanisms that terminate the NF-κB response that may involve degradation of DNA-bound NF-κB subunits. Indeed, recent reports demonstrated that after initial activation by inﬂammatory cytokines or innate immune stimuli, promoter-bound RelA is targeted to proteasomal degradation, which

terminates the NF-κB response (78). However, it is not clear whether degradation of promoterbound RelA also depends on NF-κB transcriptional activity or whether this mechanism is orchestrated by posttranslational modiﬁcation of RelA and other NF-κB subunits. In fact, IKKα has an unexpected negative regulatory role in activated macrophages, where it is required for proper termination of the NF-κB response (64). In its negative regulatory capacity, IKKα phosphorylates RelA, while still in the cytoplasm, at Ser236 , a modiﬁcation that accelerates the proteasomal degradation of nuclear RelA and contributes to termination of RelA-dependent transcriptional responses (64). In this case, IKKα is activated by the same stimuli that activate IKKβ and that trigger classical NF-κB signaling. Interestingly, NIK, which has long been thought to act upstream of IKKα in the alternative NF-κB pathway, is also required to terminate the NF-κB response downstream of inﬂammatory signals (S. Vallabhapurapu & M. Karin, unpublished data). Whether NIK functions via IKKα or by a different mechanism to negatively regulate NF-κB signaling remains to be further investigated. Recent reports have described additional players and E3 ubiquitin ligases involved in the nuclear degradation of RelA (78). COMMD1 (copper metabolism gene MURR1 domain), which is a ubiquitously expressed inhibitor of NF-κB (79), can induce degradation of nuclear RelA (80). Furthermore, COMMD1 acts as an important component of an EC2S multisubunit E3 ubiquitin ligase complex (81). EC2S resembles the SCF multisubunit E3 ubiquitin ligase complex and is composed of elongins B and C, cullins 2 or 5, the RING ﬁnger protein Rbx1 (RING box-1), and a suppressor of cytokine signaling (SOCS) protein (78). COMMD1 seems to link SOCS1, which serves as the substrate recognition subunit of the complex, to RelA (81). However, the predominant cytoplasmic localization of SOCS1 raises questions as to whether it is involved in nuclear RelA degradation. Nevertheless, one cannot rule out the possibility that some EC2S translocates to the nucleus upon macrophage activation. It would

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be of interest to determine whether IKKα and/or NIK control the recruitment of the EC2S ubiquitin ligase complex to nuclear RelA. Further adding to the complexity of negative regulation, PDLIM2, a LIM domain protein, promotes degradation of nuclear RelA in response to inﬂammatory signals (82). PDLIM2 has a PDZ domain that has a chaperone function, and it promotes the incorporation of RelA into insoluble promyelocytic leukemia nuclear bodies that are rich in proteasome function (83). The PDLIM2 LIM domain, which is structurally similar to RING ﬁnger domains, causes the polyubiquitination of RelA in promyelocytic leukemia bodies and thereby downregulates nuclear NF-κB activity (82). It is not clear whether both E3 ligases (COMMD1EC2S and PDLIM2) are required to downregulate NF-κB activity in the same cell type in response to the same stimulus. One possibility is that the two ubiquitin ligases are signal and cell type speciﬁc. Indeed, in the absence of PDLIM2, LPS-induced polyubiquitination of nuclear RelA in DCs is completely absent, suggesting that in these cells PDLIM2 cannot be replaced by other E3 ligases (78). In addition to degrading nuclear RelA, other negative regulatory mechanisms target the DNA-binding function of nuclear RelA by a protein called PIAS1 (protein inhibitor of activated STAT1) (84). PIAS1 directly interacts with RelA and prevents it from binding to DNA. In line with the negative regulatory role of PIAS1, mice lacking this factor exhibit elevated levels of proinﬂammatory cytokines and increased NF-κB DNA binding (84). However, PIAS1 appears to effect only a subset of NF-κB target genes, and a recent report showed that another member of the PIAS family, PIASy, also negatively regulates both STAT1 and NF-κB signaling by cooperating with PIAS1 (85). However, the physiological importance of negative regulation of NF-κB by these factors is not completely clear. NF-κB signaling is also regulated by other negative regulatory mechanisms that mainly target receptor proximal events. A major negative regulator of NF-κB signaling is A20, ﬁrst

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discovered in 1990 as an NF-κB-induced feedback regulator. A20 is characterized by the presence of both a deubiquitinase domain (DUB) and a C2/C2 zinc ﬁnger E3 ligase domain (86, 87). Because of the presence of these functionally opposing domains, investigators have proposed that A20 acts via a two-step mechanism. First, the A20 DUB domain removes the K63-linked polyubiquitin chains from RIP1, an adapter protein kinase essential for NF-κB activation by TNF-α (87). Second, the A20 E3 ligase domain promotes K48-linked polyubiquitination of RIP1, leading to its proteasomal degradation, thus terminating the transmission of signals from TNF receptors (TNFR) to the IKK complex (87). Interestingly, it appears that the initial removal of K63-linked polyubiquitin chains from RIP1 is a prerequisite for the K48linked polyubiquitination of the same molecule (87). Additional factors may control the A20mediated inhibition of IKK signaling. Enesa et al. (88) demonstrated that Cezanne (cellular zinc ﬁnger anti-NF-κB) is involved in RIP1 deubiquitination following TNFR activation and thus contributes to attenuation of NF-κB signaling. Moreover, TAXBP1, which ﬁrst was identiﬁed as a protein that interacts with the HTLV TAX protein (89), interacts with A20 and facilitates its interaction with RIP1 (90). However, the HECT domain ubiquitin ligase Itch binds A20 via TAXBP1 and thereby promotes K48-linked RIP1 ubiquitination and proteasomal degradation (91). According to this recent study, A20 acts as an adapter protein that links RIP1 to Itch, which is the actual ubiquitin ligase responsible for K48-linked polyubiquitination of RIP1. Moreover, ABIN proteins, which were previously identiﬁed as inhibitors of NF-κB activation, also interact with A20 (92). However, the mechanism by which ABIN modulates A20 function is not yet clear. Nevertheless, it appears that ubiquitination and deubiquitination of key factors in the NF-κB signaling pathway allow ﬁne-tuning of the NF-κB activation response and may also be important for the resolution of inﬂammation. K63-linked ubiquitination of IKKγ/NEMO and TNFR-associated factors (TRAFs) is an

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important aspect of IKK and NF-κB activation (93–95). The tumor suppressor CYLD (cylindromatosis) may inhibit NF-κB activation by removing K63-linked polyubiquitin chains from IKKγ/NEMO as well as from TRAF2 (96, 97). In the absence of CYLD, in addition to enhanced TRAF2 ubiquitination, TNF-α induces more rapid degradation of IκBα, thereby leading to enhanced NF-κB activity (98). CYLD-mediated removal of K63-linked polyubiquitin chains from BCL3 also inhibits cyclin D1 expression by limiting BCL3:p50- and BCL3:p52-mediated transcriptional activation (98). CYLD-mediated inhibition of BCL3 and cyclin D1 gene expression impairs tumor cell growth (98). Interestingly, a mouse strain has been described that solely expresses a natural splice variant of CYLD that is shorter in length (sCYLD) and lacks both IKKγ/NEMO- and TRAF2-binding domains (99). Owing to the lack of IKKγ/NEMO- and TRAF2-binding domains, sCYLD cannot negatively regulate NF-κB, and B cells from these mice express elevated levels of several NFκB target genes, including IκBα, p100, and RelB (99). Although p100 levels are increased in sCYLD-expressing B cells, p100 processing is not elevated. Despite the high levels of p100, both cytosolic and nuclear RelB levels are increased in these B cells (99), which could probably be due to an overall increase in RelB expression.

ACTIVATION OF IKK AND CLASSICAL NF-κB SIGNALING NF-κB activation depends on the IKK catalyzed phosphorylation of IκB proteins. In addition to its three core subunits—IKKα, IKKβ, and IKKγ/NEMO—the IKK complex was suggested to contain other components, whose functions are either not NF-κB speciﬁc or not fully characterized. One such protein is ELKS, a 105-kDa polypeptide found to coprecipitate with IKKβ (100). siRNA-mediated silencing of ELKS interfered with TNF-α- and IL-1induced IκB phosphorylation (100). ELKS may serve as a substrate-recruiting subunit that fa-

cilitates the phosphorylation of IκBs by IKK (100). However, whether ELKS has an additional role in IKK activation was not investigated, and ELKS-deﬁcient mice are yet to be described. More recent studies suggest that ELKS may participate in IKK activation by the DNA damage-responsive kinase ataxia telangiectasia mutated following genotoxic stress (101). Two other proteins may be part of the IKK complex: Cdc37 and HSP90 (102). However, HSP90 interacts with many other protein kinases and seems to serve primarily as a chaperone that controls proper protein folding. Thus, HSP90 is unlikely to have a speciﬁc function in IKK and NF-κB signaling. By contrast to these IKK-associated proteins, the role of the core IKK subunits has been well established by a variety of genetic experiments, most importantly gene ablations in mice (38). Based on these experiments, it is undisputable that IKKγ/NEMO is obligatory for classical NF-κB signaling, as in its absence the IKK complex can no longer be activated (103, 104). IKKγ/NEMO has no role in activation of the alternative IKKα-dependent signaling pathway, however (3, 71, 105). Of the two catalytic subunits, IKKβ is the major IκB kinase in most cell types, and IKKβ-deﬁcient cells or mice exhibit severe NF-κB activation defects (65). As mentioned above, one exception is mammary epithelial cells stimulated by RANKL, in which IκB degradation is IKKα dependent (67). However, in macrophages and preosteoclasts RANKL signaling to NF-κB is IKKβ dependent (68). By contrast to IKKβ and IKKγ/NEMO, IKKα is essential for activation of the alternative NF-κB signaling pathway (14). IKKα and IKKβ also have NF-κBindependent functions. It was ﬁrst demonstrated that IKKα regulates formation of the mammalian epidermis independently of NF-κB by regulating production of a soluble keratinocyte differentiation factor (kDIF) and expression of terminal differentiation genes (106). The identity of kDIF is still unknown, but recent experiments provide clear insights

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into the mechanism by which IKKα controls epidermal differentiation as well as skeletal and craniofacial morphogenesis (107). Both functions of IKKα require its nuclear accumulation in epidermal keratinocytes, which depends on an NLS unique to IKKα and absent in IKKβ (107). Defective skeletal and craniofacial morphogenesis in Ikkα−/− mice stems from elevated levels of FGF8 produced by IKKαdeﬁcient keratinocytes (107). Exactly how nuclear IKKα controls FGF8 expression is still unknown, but recent studies have identiﬁed a number of important IKKα target genes in keratinocytes (108). These genes encode negative regulators of Myc function and expression, namely Mad1, Mad2, and Ovol1. The negative regulation of these genes requires the transforming growth factor (TGF)-β-induced interaction of IKKα with the TGF-β-responsive Smad2 and Smad3 transcription factors (108). The TGF-β-induced repression of both Mad1 and Ovol1 does not depend on the protein kinase activity of IKKα but does require its nuclear accumulation and promoter recruitment (108). Whether IKKα is also involved in TGF-β signaling in immune cells remains to be seen. The kinase function of nuclear IKKα is important for controlling expression of other genes, however. It was ﬁrst suggested that nuclear IKKα controls gene transcription by phosphorylating histone H3 (109, 110). Although IKKα can phosphorylate histone H3 in vitro, its effects on histone H3 phosphorylation in vivo are most likely indirect and exerted through recruitment of other histone modiﬁers. IKKα kinase activity is also required in the nucleus for repression of the antimetastatic gene Maspin (111). The IKKα-mediated repression of Maspin transcription strongly enhances the metastatic activity of prostate cancer cells. Interestingly, the nuclear accumulation of IKKα in these cells correlates with inﬁltration of T cells into advanced prostate tumors (111). The kinase activity of nuclear IKKα also regulates transcription by derepressing the silencing mediator for retinoic acid and thyroid hor-

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mone receptor (SMRT)-HDAC3 corepressor complex (112). In response to TNF-α, IKKα simultaneously phosphorylates RelA at S536 and SMRT at S2410 and thereby displaces the SMRT-HDAC3 corepressor complex and allows acetylation of RelA by p300, which results in efﬁcient RelA-mediated transactivation (112). Interestingly, a recent report indicated that in colorectal tumor cells IKKα associates with target sites on chromatin and causes removal of SMRT-mediated repression by phosphorylating SMRT. This results in elevated expression of Notch target genes, such as Hes1, Hes5, and Herp2, which enhances colorectal tumorigenesis (113). Curiously, the same phosphorylation site (S536) used by IKKα to stimulate RelA transcriptional activity in cancer cells is also the site through which IKKα accelerates RelA turnover in activated macrophages (64). IKKβ also has NF-κB-independent functions. IKKβ physically interacts with TSC1, a tumor suppressor and a repressor of the mTOR pathway (114). This interaction results in suppression of TSC1:TSC2 activity and may activate mTOR signaling, resulting in enhanced angiogenesis and tumor development (114). IKKβ may also enhance tumorigenesis by direct phosphorylation of the tumor suppressor FOXO3a, leading to its ubiquitination and degradation (115). Although neither of these functions has been demonstrated in IKKβdeﬁcient mice and their physiological relevance remained ambiguous, a recent study provided a more physiological NF-κB-independent role for IKKβ in IgE-mediated anaphylaxis as well as in IgE-mediated mast cell degranulation by phosphorylating SNAP-23 (116). Despite their distinct functions, IKKα and IKKβ are very similar in structure and exhibit 50% sequence identity overall (Figure 3). A notable difference between the two subunits, other than the NLS that is unique to IKKα, lies in their C-terminal NEMO-binding domain, which in the case of IKKβ exhibits much greater afﬁnity to IKKγ/NEMO (117). Thus, in the absence of IKKβ, IKKα may not interact very

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effectively with IKKγ/NEMO, thus explaining its weak IκB kinase activity. IKKγ/NEMO is a 48-kDa regulatory subunit that contains a kinase-binding domain (KBD) at its N terminus and a ubiquitin-binding domain (UBD) as well as LZ and ZF motifs at its C terminus (Figure 3) (11, 63). Different regions within the KBD of IKKγ mediate interactions with IKKα and IKKβ (11, 63). Although the exact biochemical function of IKKγ in regulating IKK activity is still enigmatic, its major roles appear to be the assembly of a hexameric IKK complex containing four catalytic subunits and two IKKγ molecules, as well as linking this complex to upstream regulators (11, 63). This connection most likely relies on the UBD of IKKγ, which interacts with the K63-linked polyubiquitinated form of RIP1 (118, 119). Furthermore, a core fragment of IKKγ is sufﬁcient for assembly of the large holo-IKK complex (120). Targeting of this fragment to the membrane using a myristoyl group was sufﬁcient for constitutive IKK signaling in Jurkat T cells (120). As discussed below, this membrane recruitment may mimic the natural process of TCR-mediated IKK activation. Recently, IKKγ was found to have IKKindependent functions. It is required for activation of the MAP kinases (MAPK) JNK and p38 by members of the TNF family (121). This function is mediated through the participation of IKKγ in formation of a large multisubunit signaling complex required for recruitment of the MAPK kinase kinases (MAP3K) MEKK1 and TAK1 to activated TNFR family members (122). IKKγ also has a role in activation of the interferon (IFN) response, where it acts through a poorly deﬁned mechanism to promote activation of the IFN response factors (IRFs) following viral infection (123). Thus, IKKγ/NEMO may serve a scaffold or adapter function in several signaling pathways, and the pathophysiological effects of its deﬁciencies in mice (103, 104) or humans (124–126) may be due to inactivation of these pathways in addition to the well-documented defects in NF-κB signaling.

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Figure 3 IKK subunits. Schematic representation of individual IKK subunits. Structural and functional motifs that are common and unique to these proteins are shown as indicated. Abbreviations: KD, kinase domain; LZ, leucine zipper motif; HLH, helix loop helix motif; NBD, NEMO-binding domain; NLS, nuclear localization signal; KBD, kinase-binding domain; UBD, ubiquitin-binding domain; ZF, zinc ﬁnger motif.

CLASSICAL NF-κB SIGNALING BY MEMBERS OF THE TNFR FAMILY Much of our current but still incomplete understanding of the classical NF-κB pathway comes from studies of TNF-α signaling. TNFα is the prototypic member of a large family of trimeric cytokines and is one of the most potent activators of inﬂammation and classical NF-κB signaling. TNF-α binds to two receptors, TNFR1 and TNFR2, of which TNFR1 plays a much broader role in NF-κB activation. TNFR1 ligation induces receptor trimerization and recruitment of the adapter protein TNFR1-associated DEATH domain protein (TRADD) that binds to a speciﬁc region in the cytoplasmic domain of TNFR1 (127, 128). Recent studies of TRADD-deﬁcient mice have demonstrated its important role in IKK activation (129). Initially, TRADD was thought to be required for the recruitment of RIP1, TRAF2, TRAF5, and the activated TNFR1 (130). However, TRADD ablation studies revealed that in the absence of TRADD a residual amount of RIP1 is still recruited to the ligated TNFR1, and this amount may sufﬁce for a low level of signaling in cells, such as macrophages, that express high amounts of RIP1 (129). Although

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RIP1 is a member of a small family of related protein kinases, only RIP1 is required for NF-κB activation downstream of TNFR1 (131, 132). Curiously, however, reconstitution of RIP1-deﬁcient cells with a kinase-inactive variant of RIP1 restores NF-κB signaling, indicating that RIP1 does not function as a protein kinase in the NF-κB activation process (132). Most likely (and as we discuss below), RIP1 is an adapter that enhances IKK recruitment to the activated receptor through an interaction between polyubiquitinated RIP1 and IKKγ (11, 118, 119). Receptor engagement results in two types of RIP1 polyubiquitination: a rapid formation of K63-linked polyubiquitin chains and a delayed formation of K48-linked polyubiquitin (87, 119). The initial K63-linked ubiquitination of RIP1 seems to be important in IKK and NF-κB activation as the K63-linked polyubiquitin on RIP1 stabilizes the association with the UBD-containing IKKγ subunit (118, 119). However, not all TNFR family members engage RIP1, and in the case of CD40 or BAFFR the recruitment of IKK to the receptor complex may be mediated by TRAF2 and TRAF6 (133, 134). The ubiquitin ligase responsible for RIP1 K63-linked ubiquitination has not been formally identiﬁed, but TRAF2 is a good candidate (119). All TRAF proteins, with the exception of TRAF1, contain a RING ﬁnger at their N-terminal region, followed by a variable number of ZF motifs that may be involved in binding of polyubiquitin chains (135, 136). At their C-terminal half, all TRAF proteins, including TRAF1, contain a TRAF domain that mediates binding to adapter proteins, such as TRADD, or direct binding to the cytoplasmic segments of those TNFR family members that do not rely on adapter proteins, such as CD40 or BAFF-R (135, 136). How the binding of IKKγ to the K63-linked polyubiquitin chains of RIP1 triggers IKK activation is unknown. While we proposed that receptor or membrane recruitment of IKK complexes can result in their activation through trans-autophosphorylation (137), others have suggested that upstream kinases may be responsible for IKK activation. One such ki-

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nase, MEKK3, interacts with RIP1, and ﬁbroblasts lacking MEKK3 exhibit defective NF-κB activation in response to TNF-α or IL-1 (131, 132, 138). It was therefore suggested that RIP1 functions as an adapter that recruits MEKK3 to the activated TNFR1 signaling complex, where MEKK3 phosphorylates IKKα and IKKβ at their activation loops (139). However, MEKK3 is not unequivocally an essential IKK kinase in immune cells, and it remains to be shown that MEKK3-deﬁcient mice have major defects in IKK and NF-κB activation. Another putative IKK kinase is TAK1 (140, 141). TAK1, with its regulatory subunits TAB1, TAB2, and TAB3 (140, 141), appears to be recruited to TNFR signaling complexes via the TRAF2dependent polyubiquitination of RIP1 and to phosphorylate the activation loops of the IKK catalytic subunits (142, 143). Receptors that do not rely on RIP1 may recruit TAK1 more directly via TRAF2 or TRAF6. However, the analysis of TAK1- and TAB1/2-deﬁcient mice has also not provided unequivocal support for a ubiquitous and general role for TAK1 in IKK activation (144). TAK1 is a critical MAP3K responsible for activation of JNK and p38 MAPK cascades downstream of TNFR family members and other inﬂammatory/immune receptors, however (122, 144). The involvement of TAK1 or MEKK3 in IKK activation may therefore be cell type speciﬁc, and these kinases may be responsible for amplifying IKK signaling after the initial activation of IKK via trans-autophosphorylation (Figure 4). Consistent with this proposal, engagement of TNFR1 or CD40 results in the very rapid recruitment of IKK to the activated receptor, where IKKβ undergoes rapid activation detectable by phosphorylation of its activation loop (122, 145, 146). In the case of TNFR1, receptor recruitment of IKK may be mediated by TRAF2/5 and RIP1 (11, 146) and, in the case of CD40, by TRAF2/6 (133, 134). Although MEKK1 and TAK1 are also recruited to CD40, their activating phosphorylation occurs in the cytoplasm and not on the receptor, and it is considerably delayed relative to IKK activation

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(122). Thus, it is not certain whether TAK1 or any other upstream kinase is involved in IKK activation, rather than IKK itself.

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Members of the TLR/IL-1R family are also potent activators of classical NF-κB signaling. The cytoplasmic regions of TLR/IL1R family members share a common motif called the TIR domain (147, 148). Similar to TNFRs, TIR-containing receptors do not have intrinsic catalytic activity and rely on recruitment of adapter proteins, ubiquitin ligases, and protein kinases to generate and transduce signals that broadcast their activation. Two major TIR domain–containing adapters, MyD88 (myeloid differentiation primary response gene 88) and TRIF (TIR domain– containing adapter-inducing IFN-β), play important roles in TLR/IL-1R signaling (147). While TLR2 and TLR4 bind to MyD88 or TRIF via intermediary adapters such as TIRAP (Toll/interleukin-1 receptor adapter protein, also referred to as MAL) or TRAM, other TLRs, such as TLR9 and TLR3, appear to bind their respective adapters, MyD88 and TRIF, directly (147, 149). Whereas TLR4 uses both TRIF and MyD88, TLR3 and TLR9 signal exclusively through TRIF and MyD88, respectively (147, 150). The recruitment of different adapters to distinct TLRs is probably determined by the folding pattern and the surface properties of TIR domain–containing adapters. It appears that the BB and DD loops present in the TIR domains of TLRs and the TIRcontaining adapters allow them to interact with each other (151, 152). Supporting this notion, the BB of TLR2 and the DD loop of MyD88 are required for the two proteins to interact (151, 152). In addition, receptor dimerization occurs via interaction of the BB loop of one TLR with the DD loop of another. In the case of MyD88, investigators recently demonstrated that, in addition to the BB and DD loops, a region called the Poc site (Ile179)

K1 TA

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ACTIVATION OF THE CLASSICAL NF-κB PATHWAY BY TLR/IL-1R SIGNALING

? ?

P

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Figure 4 Activation of classical NF-κB signaling by TNF-α. Ligation of TNFR1 results in TRADD-dependent TRAF2/TRAF5 and RIP1 recruitment. TRAF2 causes K63-linked ubiquitination of RIP1 and also recruits IKK to the receptor complex, where binding of IKKγ to ubiquitinated RIP1 stabilizes IKK interaction with the receptor complex. This may also lead to a conformational change of the IKK complex. TAB2 and TAB3 interact with TRAF2 and TAK1, leading to TAK1 activation that may then phosphorylate IKKβ. Alternatively, MEKK3, which is brought near the receptor complex presumably by RIP1 may also phosphorylate and activate IKK. On the other hand, IKK may also be activated by autophosphorylation. Activated IKK phosphorylates IκBα at speciﬁc serine residues, leading to the proteasome-mediated degradation of the latter. Degradation of IκBα releases the NF-κB heterodimers, which then migrate to the nucleus and regulate gene expression.

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(named after the phenotype Pococurante) is critical for the interaction of MyD88 and the BB loop of the interacting TLRs. Thus, a mutation in the Poc site impairs the signaling by receptors that interact with MyD88 (153). However, TLR2:TLR6 dimers, which interact with MyD88, seem to require either a BB loop or the Poc site of MyD88. As described above, some TLRs can directly interact with MyD88 or TRIF, whereas others, such as TLR2 and TLR4, depend on TIRAP or TRAM. This is presumably due to the electrostatic surfaces of the TIR domains present in the receptors and different adapters. For instance, for both TLR4 and MyD88, the TIR domain is highly electropositive, and therefore direct interaction is not possible. The TIR domain of TIRAP, on the other hand, is highly electronegative, allowing efﬁcient interaction with the electropositive TIR domain of TLR4. TIRAP thereby forms a bridge between TLR4 and MyD88 (152). Similar to TIRAP, TRAM, which is specifically required for TLR4 signaling, may function as a bridging factor between TLR4 and TRIF (154, 155). However, LPS signaling is more impaired in TRAM-deﬁcient cells than in TRIF-deﬁcient cells, indicating that TRAM has roles beyond being a bridging factor. The function of TRAM depends on two important biochemical modiﬁcations: (a) N-terminal myristoylation, which is required for membrane tethering; and (b) phosphorylation by PKCε at a site proximal to the myristoylation. Mutation of either of these sites impairs TRAM function, although how phosphorylation regulates signaling through TRAM is unclear (156, 157). The MyD88-dependent pathway leads to activation of IKK and NF-κB via TRAF6, which is recruited to MyD88 in a manner dependent on IRAK1 (IL-1R-associated kinase 1) (147, 158, 159). The related protein kinase IRAK4 is also involved in TLR/IL-1Rmediated IKK and NF-κB activation (147), and its absence causes severe immunodeﬁciency in humans (160). The IRAKs and RIP1 share structural features, and, like RIP1, the kinase functions of IRAK1 and IRAK4 are dispens-

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able for NF-κB activation (161). The IRAKs also undergo K63-linked ubiquitination following receptor activation (162), and it appears that TRAF6 is the ubiquitin ligase responsible for IRAK1/4 ubiquitination (162). However, the IRAKs are not only substrates for TRAF6 but are also involved in its recruitment to the activated receptor (163, 164). Following recruitment by IRAKs, TRAF6 binds to the TAB1/2/3 complex, leading to TAK1-mediated IKK activation (Figure 5) (140, 141, 163–165). However, as discussed above, the role of the TAK1:TAB1/2/3 complex in IKK activation remains controversial, although it is generally agreed that this complex is needed for JNK and p38 activation (144). Indeed, a TAK1:TAB1 fusion protein is constitutively active and capable of stimulating AP-1 activity (166). Another TRAF protein that is recruited to MyD88 and TRIF signaling complexes upon TLR/IL-1R engagement is TRAF3 (167, 168). TRAF3 is not involved in classical IKK activation (11), but it is required for activation of the IKK-related kinases TBK1 and IKKε, which regulate IFN gene induction (167, 168). However, IFN induction plays an important role in activating the full NFκB transcriptional response in IFN-producing cells because of transcriptional cooperation between the IRFs and NF-κB (169–171). Moreover, during the response to LPS, the interaction between IRF3 and NF-κB dimers enhances NF-κB-mediated activation of select gene promoters (172). Subsequent studies have shown that IRF3 and IRF5 are involved in TLRinduced production of TNF-α that may lead to secondary and delayed activation of NF-κB (173, 174). Thus, functional cross talk between IFN and NF-κB signaling appears to amplify and prolong the NF-κB activity. A full discussion of IFN signaling, however, is beyond the scope of the current review. Despite signiﬁcant advances in understanding the role of TLR family members in pathogen recognition (175, 176), detailed insights into their biochemical mode of action are lacking. Part of the complexity in understanding the biochemistry of TLR signaling relates

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to the presence of different TLRs in different intracellular compartments with distinct lipid and protein compositions (175). It was recently suggested that the lipid microenvironment surrounding each receptor may have major effects on its mode of signaling (175, 177). In addition to TLRs, other microbial pattern-recognition receptors (PRRs), including nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) and retinoic acid–inducible gene I (RIG-I)-like receptors also provide innate immune responses via activation of NF-κB (178–180). NLRs are a family of intracellular PRRs that are characterized by presence of the conserved NOD domain. NLRs are found in both mice and humans, and their general domain organization includes either (a) an N-terminal CARD domain, (b) a pyrin domain, or (c) a baculovirus inhibitor repeat domain, plus a NOD domain in the middle and a C-terminal leucine-rich repeat motif, which detects microbial patterns (178, 180). Different NLRs vary in their N-terminal domains, but the NOD domain and the leucine-rich repeat are conserved among all the NLRs. Although the major function of the NLRs appears to be activation of the inﬂammasomes (178–180), some NLRs such as NOD1 and NOD2 activate the NF-κB pathway (181, 182). Upon binding to γ-D-glytamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MDP), respectively, NOD1 and NOD2, via CARD interactions, recruit RIP2, a CARD-containing serine-threonine kinase (179, 183, 184). RIP2 directly binds and facilitates K63-linked polyubiquitination of NEMO/IKKγ and activates TAK1, which might directly phosphorylate and activate IKKβ and thereby activate the classical NF-κB pathway (185–187). RIG-like helicases are involved in sensing the viral dsRNA. Three members of this family were identiﬁed that include two CARDcontaining proteins, RIG1 and MDA5, and the third member, LGP2, is devoid of a CARD domain (179). Upon activation by dsRNA or viral infection, both RIG1 and MDA5 bind dsRNA and trigger NF-κB activation by recruiting a

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Figure 5 Ligand binding by TLRs results in the recruitment of receptor-speciﬁc adapters and induces activation of NF-κB. In the case of TLR4, ligand binding results in NF-κB activation via both TRIF- and MyD88-dependent pathways. TIRAP (also known as MAL) and TRAM are recruited and presumably serve as bridging factors to recruit MyD88 and TRIF, respectively. MyD88 recruits TRAF6 and members of the IRAK family, leading to oligomerization and self-ubiquitination of TRAF6 as well as recruitment of TAB2 and TAB3, which in turn activate TAK1. Activated TAK1 may then directly phosphorylate IKKβ to activate the IKK complex, resulting in IκBα degradation and NF-κB activation. TRIF also recruits TRAF6 by direct interaction. TRAF6 then activates TAK1, culminating in IKK and NF-κB activation in a manner similar to the MyD88-dependent pathway. In addition to recruiting TRAF6, TRIF also recruits RIP1, which might cooperate with TRAF6 to facilitate TAK1 activation.

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mitochondrial-anchored protein called Cardif, which in turn recruits and activates IKK, leading to NF-κB activation (188).

ACTIVATION OF CLASSICAL NF-κB SIGNALING BY ANTIGEN RECEPTORS

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Engagement of antigen receptors on B and T lymphocytes also results in IKK and NF-κB activation. Recently, signiﬁcant progress has been made in understanding the mechanism of IKK activation by both the TCR and BCR. The protein kinase C isozymes PKCθ in T cells (189) and PKCβ in B cells (190) play central roles in recruiting additional factors and in IKK activation. Genetic studies have identiﬁed several additional factors that play important roles in TCR-mediated NF-κB activation, including kinases of the Src (Lck and Fyn) and Syk (ZAP70) families, as well as adapters such as LAT and SLP-76, and intracellular signaling components including phospholipase C (PLC) γ1, Vav1, BCL10, 3-phosphoinositidedependent kinase 1 (PDK1), CARMA1 [caspase recruitment domain (CARD) membraneassociated guanylate kinase (MAGUK) protein 1], and MALT1 (mucosa-associated lymphoid tissue 1) (191, 192). Recent biochemical studies led to the postulation that TCR activation results in sequential recruitment of these factors to the immunological synapse (IS) as well as PKCθ-dependent formation of a complex between CARMA1, BCL10, and MALT1 (reviewed in 11, 192, 193). This complex, called CBM, promotes the K63-linked polyubiquitination of IKKγ/NEMO and subsequent IKK activation (194). Triggering the TCR/CD3 complex results in the recruitment of the Src family kinases Fyn and Lck, and the latter (Lck) causes phosphorylation of the conserved immunoreceptor tyrosine activation motifs (ITAMs) on the CD3 subunit of the TCR (192). Subsequent recruitment of the Syk tyrosine kinases ZAP70 and Syk results in phosphorylation of the adapter molecules LAT and SLP-76, leading to their interaction via the adapter GADS and 708

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recruitment of other adapters, such as Grb2, the p85 subunit of PI3K PLCγ1, the Tec family tyrosine kinase Itk, and the nucleotide exchange factor Vav1 (192, 193). Phosphorylation of SLP-76 upon TCR triggering induces its binding to Vav1, and their interaction facilitates NF-κB activation in response to TCR activation (195). The Vav1-SLP6-Itk complex also activates PLCγ1, leading to the release of diacylglycerol (DAG), which stimulates PKCs (196). Costimulatory signals from CD28 also play an important role in PI3K activation, which induces recruitment to the plasma membrane of Pleckstrin homology domain proteins such as PDK1 and AKT, as well as PDK1mediated phosphorylation and translocation of PKCθ to the IS (196, 197). Vav1 is also involved in this recruitment in a PI3K- and AKT-dependent manner (191, 196). Vav1 may be required for effective PI3K activation and sustained Ca2+ signaling, and it may also be involved in NFAT and JNK activation (198, 199). Upon translocation to the IS, PKCθ undergoes conformational changes, presumably owing to phosphorylation by Lck as well as by PDK1, allowing PKCθ to interact directly with IKK (191, 200, 201). Eventually, a large signaling complex is formed at the engaged receptor within the lipid rafts, leading to activation of the PI3K-AKT and the Ras-MAPK pathways (192). As stated above, activation of PI3K results in activation of PDK1, which in turn activates PKCθ. Activated PKCθ was proposed to phosphorylate CARMA1, leading to a conformational change that allows CARMA1 to associate with BCL10 (192). In addition to activating PKCθ, PDK1 also interacts with CARMA1, which is now bound to MALT1-associated BCL10 (192). The active CBM complex thus formed leads to IKK activation through an ill-deﬁned mechanism, which may involve IKK trans-autophosphorylation, as suggested for TNFR and TLR/IL-1R signaling pathways (Figure 6). Recently, TRAF2 and TRAF6, which were thought to be predominantly involved in TNFR and TLR/IL-1Rmediated IKK activation, were shown to be involved in TCR-mediated IKK activation (202).

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Figure 6 Stimulation of the TCR results in recruitment and activation of the Src (Lck and Fyn) and Syk (ZAP70) family kinases. ZAP70 then phosphorylates adapter proteins LAT and SLP-76, resulting in formation of a multimolecular complex containing PLCγ1 and nucleotide exchange factor Vav1. Activation of PLCγ1 results in generation of IP3 and Ca2+ , as well as DAG, which in turn stimulates PKCθ. Signals from TCR and CD28 costimulation result in activation of PI3K, which facilitates recruitment of PKCθ to the immunological synapse (IS). Vav1 also plays a role in the recruitment of PKCθ to the membrane. Phosphorylation of the phosphoinositides by PI3K leads to membrane recruitment of PDK1, which phosphorylates and activates PKCθ to control the recruitment of IKK and CARMA1 into the signaling complex. Phosphorylation of CARMA1 by PKCθ results in the recruitment of BCL10 and MALT1, thus leading to formation of a stable CBM complex. A poorly deﬁned mechanism involving ubiquitination of IKKγ and activation of IKKβ presumably by TAK1 then leads to activation of IKK complex, which phosphorylates IκBα, leading to its degradation and activation of NF-κB.

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MALT1 was found to interact with TRAF6 and stimulate its E3 ubiquitin ligase activity toward IKKγ, resulting in K63-linked polyubiquitination of the latter and IKK activation (202). Moreover, TRAF6 autoubiquitination activates TAK1, which may directly phosphorylate IKKβ. However, the role of TAK1 in TCR-mediated NF-κB activation seems to be important only in thymocytes and not in peripheral T cells (11, 203). Interestingly, IKK recruitment to the CBM complex appears also to depend on caspase 8, and T lymphocytes lacking caspase 8 display defective NF-κB activation in response to antigen receptor engagement (204). However, whether caspase 8 participates in proteolytic events that are required for IKK activation by these receptors remains to be determined. It appears that several of the intracellular signaling molecules and adapters involved in TCR-mediated NF-κB activation are rapidly recruited into spatially and temporally organized structures called supra-molecular activation clusters (SMACs) (192). Although the temporal compositions of SMAC and the IS are not clear, it is evident that PKCθ, which is central to TCR-mediated NF-κB activation, as well as the CBM complex are both rapidly recruited to the SMAC when the TCR is triggered (201, 205, 206). Costimulatory signals from CD28 are postulated to facilitate translocation of these factors to the SMAC (192, 205). In support of the argument that the components of TCR signaling need to localize to these specialized structures, investigators showed that artiﬁcial tethering of NEMO to the IS is sufﬁcient to activate NF-κB (120). Furthermore, CARMA1 is essential for recruitment of IKK to the SMAC (205, 207). TCR triggering might also induce NFκB activation via AKT, which phosphorylates Cot/Tpl2, a serine/threonine kinase involved in IKK activation (208). AKT-mediated phosphorylation of Cot/Tpl2 at serine 400 is critical for the latter to stimulate IKK (208). Interestingly, Cot/Tpl2-mediated IKK activation seems to depend on NF-κB-inducing kinase (NIK), which normally is not involved in the

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activation of the classical pathway (15, 209). In addition to activating the classical NF-κB pathway, AKT may also phosphorylate p65, resulting in enhanced transcription by NF-κB (210). However, other studies using Jurkat T cells revealed that AKT does not have a signiﬁcant effect on p65 (211). Although PKCθ is central for TCRmediated NF-κB activation, some recent studies from two groups using mice lacking the PKCθ gene revealed contradictory results: Whereas the ﬁrst group clearly showed that lack of PKCθ impairs NF-κB activation, the second group showed that absence of PKCθ resulted in complete abrogation of NFAT activation, while activation of NF-κB was only partially impaired (212). Whether these differences were due to the strategies used to inactivate the PKCθ gene or to the genetic background of the mice needs to be clariﬁed by further studies. Finally, activation of NF-κB by the TCR and BCR is downregulated by targeting BCL10 to cIAP2 and Itch-mediated ubiquitination and degradation in the lysosomes followed by IKKβ-mediated phosphorylation (213). This results in the disruption of BCL10MALT1 complex leading to downregulation of NF-κB activation. In addition, phosphorylation of TCR by PKCθ results in its internalization, which in turn leads to reduction of NF-κB activity (214).

ACTIVATION OF IKKα AND THE ALTERNATIVE NF-κB PATHWAY VIA NIK STABILIZATION NIK was ﬁrst identiﬁed as a protein kinase whose overexpression results in NF-κB activation (215). However, the analyses of aly/aly mice, which carry an inactivating point mutation in the Nik gene (216) and Nik−/− mice (217) revealed that NIK is mainly involved in activation of the IKKα-dependent alternative pathway, as the phenotypes of these mice are very similar to those of IkkαAA mice, in which IKKα cannot be activated because of replacement of its phosphoaccepting serines with alanines (14). Subsequently, NIK was found

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to be responsible for activation of IKKα and induction of p100 processing (14, 15, 69). Additional work revealed that NIK is an unstable protein that is subject to rapid turnover in nonstimulated cells (218). Stimulation of B cells with certain TNF family members, such as CD40L, that are capable of inducing NF-κB2/ p100 processing also causes NIK stabilization, leading to the proposal that accumulation of stabilized NIK results in its activation via transautophosphorylation and subsequent IKKα activation (218). The mechanism of receptorinduced NIK stabilization has remained a mystery for quite some time, but it was noted to correlate with receptor-induced TRAF3 degradation, whose mechanism was not understood either (218). Traf3−/− mice exhibit NIK stabilization and constitutive activation of alternative NF-κB signaling, which accounts for postnatal inﬂammation and mortality (219). Inactivation of either the Nik or NfkB2 genes in these mice prevents the inﬂammatory response and postnatal lethality (219, 220). TRAF3 overexpression in HEK293T cells induces NIK ubiquitination and proteasomal degradation, leading to the proposal that TRAF3 may be a NIK-targeting ubiquitin ligase (218). A TRAF3-binding site is present within the N terminus of NIK, and a NIKbinding site is located in the TRAF domain of TRAF3, and both of these sites are important for TRAF3-induced NIK degradation (218). Like other TRAFs, TRAF3 harbors a RING ﬁnger at its N terminus, leading to the hypothesis that its E3 ubiquitin ligase activity targets NIK to proteasomal degradation. However, the RING ﬁnger of TRAF3 is not required for induction of NIK degradation, which depends on the TRAF domain of TRAF3 (220). Interestingly, the RING ﬁnger of TRAF3 is essential for preventing constitutive p100 processing in Traf3−/− cells (221). Despite the contradictory nature of these ﬁndings, other studies support the concept that TRAF3 functions as an adapter that links NIK to another E3 ligase responsible for the K48-linked ubiquitination of NIK and is not a NIK ubiquitin ligase itself (222–225).

Some of the new insights into the mechanism of NIK turnover come from studies on the plasma cell cancer multiple myeloma (MM). Many MM cell lines harbor mutations in the TRAF3 and NIK loci that prevent the interaction between their products, thereby resulting in NIK accumulation (222, 223). Other MM cell lines were found to have large deletions that encompass the linked cIAP1 and cIAP2 loci (BIRC2 and BIRC3), and these mutations were also linked to NIK accumulation (222). The products of these loci, cIAP1 and cIAP2, were previously known as NF-κB-inducible antiapoptotic proteins that interact with TRAF2 (226). Another protein that binds to cIAP1 and cIAP2 is the proapoptotic mediator SMAC, which is released from damaged mitochondria and can induce the rapid degradation of cIAP1 and cIAP2 (227). This ﬁnding led to development of small molecule SMAC mimics that bind both cIAP1 and cIAP2 and also induce their ubiquitin-dependent degradation (224, 225, 228). Curiously, SMAC mimics that were developed for induction of apoptosis in cancer cells also induce NIK stabilization and activation of the alternative NF-κB pathway (224, 225), an effect also seen upon the cIAP1/2 deletions in MM (222). These results suggest that cIAP1/2 and TRAF3 somehow induce the rapid turnover of NIK and thereby inhibit alternative NF-κB signaling in resting cells. TRAF3 is not the only TRAF protein involved in NIK turnover, however. TRAF2 also negatively regulates p100 processing and alternative NF-κB signaling in B cells (229, 230). Furthermore, deletion of TRAF2 also results in elevated NIK expression in B cells and ﬁbroblasts (220, 225). Similar to TRAF3, overexpression of TRAF2 also induces NIK degradation, but unlike TRAF3, the RING ﬁnger of TRAF2 is required for this process (220). Interestingly, both Traf2−/− and Traf3−/− mice exhibit an almost identical phenotype, characterized by perinatal inﬂammation, atrophy of lymphoid organs, and early mortality (104, 231), which in the case of Traf3−/− mice is due to uncontrolled activation of alternative NF-κB signaling (219). Correspondingly,

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cIAP1/2 (220). siRNA-mediated knockdown of TRAF3 prevented formation of this complex, suggesting that TRAF3 functions as an adapter that links NIK to a TRAF2:cIAP1/2 E3 ligase complex (Figure 7). This adapter function is provided by the TRAF domain of TRAF3, and, as mentioned above, its N-terminal portion is

inactivation of the Nik gene prevents the perinatal lethality and splenic atrophy of both Traf2−/− and Traf3−/− mice (220). Consistent with the involvement of TRAF2, TRAF3, and cIAP1/2 in NIK turnover, NIK is associated in nonstimulated B cells with a ubiquitin ligase complex containing TRAF2, TRAF3, and

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Figure 7 Activation of alternative NF-κB signaling by CD40. In resting cells (left), p100, via its C-terminal ankyrin repeats, binds and keeps RelB in the cytosol. NIK, which is an essential kinase involved in the phosphorylation and proteasome-mediated C-terminal processing of p100, is maintained at very low levels owing to rapid proteasome-dependent degradation. TRAF3 links NIK to an E3 complex containing TRAF2 and cIAP1/2, thereby promoting cIAP1/2-mediated K48-linked NIK polyubiquitination and proteasomal degradation. TRAF3 also undergoes a low level of K48-linked polyubiquitination under resting conditions. Activation of CD40 by CD40L (right) leads to recruitment of the cIAP1/2:TRAF2:TRAF3 complex to the receptor, where cIAP1/2 undergoes TRAF2dependent K63-linked polyubiquitination. TRAF2 also undergoes K63-linked self-ubiquitination. K63-linked ubiquitination of cIAP1/2 enhances their K48-speciﬁc E3 ubiquitin ligase activity toward TRAF3, leading to proteasomal degradation of the latter. As a result, TRAF3 levels in the cell drop below a critical threshold, and NIK can no longer be recruited to the cIAP1/2:TRAF2 complex. This leads to stabilization and accumulation of newly synthesized NIK and its activation presumably via autophosphorylation, resulting in activation of IKKα. Activated IKKα phosphorylates p100, leading to proteasome-mediated processing of p100 to p52. C-terminal truncation of p100 releases the p52:RelB heterodimer, which migrates to the nucleus and regulates transcription of its target genes. 712

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fully dispensable for NIK turnover and complex formation (220). In contrast to TRAF3, TRAF2 does not interact with NIK directly, and its ability to induce NIK degradation depends both on its C-terminal TRAF domain, which interacts with TRAF3, and on its N-terminal RING ﬁnger. However, TRAF2 is not directly responsible for the degradative K48-linked polyubiquitination of NIK. Instead, this task is carried out by cIAP1/2, which are potent K48-speciﬁc NIK E3 ubiquitin ligases (224, 225). Moreover, cIAP1/2 interact with TRAF2 and are recruited to NIK in a TRAF2-dependent manner (224, 232). Although these ﬁndings explain how cIAP1/2, TRAF2, and TRAF3 induce NIK degradation in resting cells, a few questions remain: (a) What is the exact role of TRAF2 in this process? (b) How is basal NIK turnover modulated in response to receptor engagement? Varfolomeev et al. (224) suggested that, in response to TWEAK stimulation of cancer cells, cIAP1/2 undergoes degradation in a manner analogous to the action of SMAC mimics and that cIAP1/2 degradation is the critical event that accounts for NIK stabilization. However, neither CD40L nor BAFF, both of which induce NIK stabilization and p100 processing in B cells, can induce substantial cIAP1/2 degradation (220). Instead, the most dramatic protein degradation event induced by these cytokines was TRAF3 degradation (220). The engagement of either CD40 or BAFF-R causes K48-linked polyubiquitination of TRAF3, leading to its degradation in a cIAP1/2-dependent manner (122, 220). Although TRAF2 is required for CD40-induced TRAF3 degradation (233), TRAF2 can only catalyze the formation of nondegradative K63-linked polyubiquitin chains (142, 234). Indeed, the requirement for TRAF2 can be explained by its ability to induce the K63-linked ubiquitination of cIAP1/2, resulting in strong enhancement of their K48-speciﬁc ubiquitin ligase activity toward TRAF3 (220). TRAF2 also recruits TRAF3 to cIAP1/2, and it is needed for recruitment of cIAP1/2 to CD40 and other receptors

(122). These ﬁndings illustrate the existence of a unique protein ubiquitination cascade in which TRAF2 ubiquitinates cIAP1/2 through a K63-linkage and thereby leads to activation of their K48-speciﬁc ubiquitin ligase activity toward TRAF3 (Figure 7). Interestingly, stabilization of NIK also induces classical NF-κB signaling by activating IKKβ (223, 235). Moreover, p100 processing regulates nuclear localization of RelA in addition to RelB (27). Consistent with these reports, TRAF3-deﬁcient cells, which exhibit elevated NIK levels and constitutive p100 processing, also exhibit increased nuclear RelA as well as RelB amounts. However, TRAF2deﬁcient cells, despite elevated levels of NIK and constitutive p100 processing, exhibit elevated nuclear levels of RelB but not RelA. This is puzzling because, if p100 processing regulates RelA nuclear translocation, how is TRAF2 involved in this process? One possible explanation is that activation of the RelB:p52 complex upon p100 processing might induce expression and release of cytokines that might activate TRAF2dependent classical NF-κB signaling, culminating in the nuclear translocation of RelA. Further studies are required to clearly address how this cross talk between the alternative and classical pathway is regulated. Another factor that may be involved in NIK turnover is NLR family member MONARCH-1 (236, 237). MONARCH1 is a myeloid-speciﬁc protein (238), and whether MONARCH-1 impacts the cIAP1/2:TRAF2:TRAF3:NIK complex or induces NIK degradation through some other means is unclear. The physiological role of MONARCH-1 in the alternative pathway and whether MONARCH-1 regulates alternative NF-κB signaling in a cell type– and signal-speciﬁc manner remain to be studied.

NF-κB IN LYMPHOCYTE AND MYELOID DIFFERENTIATION Many of the receptors that activate either the classical or the alternative NF-κB pathways are expressed on hematopoietic cell types,

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suggesting that NF-κB might play an important role in their development and activation. Most of our current knowledge regarding the role of NF-κB in hematopoietic cell survival and differentiation comes from analysis of mice deﬁcient for different NF-κB family members as well as mice lacking different NF-κB inhibitors or IKK subunits (38). For instance, the absence of both c-Rel and RelA affects erythrocyte differentiation, and as a result lethally irradiated wild-type mice that were reconstituted with fetal liver cells from c-Rel−/− Rela−/− doubleknockout embryos suffered from severe anemia (239). In addition, the combined absence of c-Rel and RelA causes impaired monocyte differentiation in vitro (38). However, lack of either c-Rel or RelA alone does not signiﬁcantly impair differentiation of major lymphocyte or myeloid cell types (38). Likewise, the absence of either IKKβ or IKKγ does not have a direct effect on differentiation of such cells, although these IKK deﬁciencies impact the formation of B cells by compromising the survival of B cell progenitors (240, 241). A recent study revealed that the loss of IKKβ in the myeloid lineage can result in massive neutrophilia (231). However, this neutrophilia is probably not due to a differentiation defect per se and is likely to be related to the paradoxical increase in IL-1β production that is associated with the IKKβ-NF-κB deﬁciency in myeloid cells (231). Nonetheless, the granulocyte macrophage-colony stimulating factor receptor (GM-CSF-R), which controls the differentiation of myeloid progenitor cells into granulocytes and macrophages, interacts with IKKβ, suggesting that NF-κB may be more intimately involved in myeloid differentiation (242). However, this involvement may be due mainly to induction of cell survival, with only a small contribution to cell maturation, as suggested for osteoclast formation (68).

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NF-κB IN T CELL DIFFERENTIATION Early thymocyte precursors enter the thymus via the blood stream, where they differentiate into either CD4+ or CD8+ mainstream 714

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αβ T cells. These early precursors, which are CD4− CD8− double negative (DN), undergo a differentiation process that can be separated into four stages, characterized by expression of surface markers such as CD44 and CD25. Once they reach a CD4+ CD8+ double-positive (DP) stage, they then undergo further maturation upon interaction with MHC to reach a CD4+ or CD8+ stage (243). Within the thymus, positive selection of developing thymocytes occurs in the cortex, whereas negative selection occurs in the medulla (243). Interestingly, NF-κB family members are differentially expressed between the cortex and the medulla, with RelA being predominantly expressed in the cortex, whereas RelB and c-Rel are primarily expressed in the medulla (244). These ﬁndings suggest that different NF-κB family members may function during different stages of T cell development. Subsequent studies in several knockout and transgenic mouse models revealed stage-speciﬁc regulation of T cell development by NF-κB signaling. Although mice lacking individual NF-κB subunits did not exhibit gross T cell developmental defects (38), Relb−/− mice show severely impaired negative selection (245). This defect stems from atrophy of the thymic medulla and lack of thymic DCs and is not a T cell–intrinsic defect (246, 247). Investigators have suggested that a cell autonomous requirement for classical NF-κB signaling activated by the pre-TCR during thymocyte development may allow the survival of those T cell precursors that express functional pre-TCR (248, 249). However, NFκB was also reported to have a proapoptotic function in DP thymocytes (250, 251). Yet a different study suggested that NF-κB has an antiapoptotic function during peptide-induced negative selection (59), further complicating the issue of whether NF-κB provides proor antiapoptotic function during thymocyte development. As mentioned above, mainstream CD4+ or CD8+ αβ T cell development is not significantly impaired in the absence of individual NF-κB proteins. However, the thymus is also the site for development and maturation

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of unconventional T cells such as γδ T cells and natural killer T (NKT) cells that bear invariant TCR reactive to lipid antigens (252– 255). Proper development of γδ T cells may depend on LTβ receptor signaling, which activates both the classical and alternative NFκB pathways (15, 256, 257). Recent reports indicate that different NF-κB family members in distinct thymic compartments have important roles during early and late stages of NKT cell development (209, 258). NKT cells have unique maturation requirements. First, they express an invariant TCR formed by the rearrangement of the Vα14 and Jα18 gene segments and are therefore termed Vα14i NKT cells (253). Second, they are positively selected by the MHC class I–like molecule CD1d, which presents lipid antigens (253). Moreover, unlike mainstream T cells, NKT cells are positively selected by CD4+ CD8+ DP thymocytes instead of by thymic epithelial cells (253). Interestingly, classical NF-κB signaling is required within developing NKT cells for postselection maturation, whereas alternative NF-κB signaling is required in thymic stromal cells to support the early stages of NKT cell development (209, 258). Further studies have demonstrated that p50:RelA, but not p50:c-Rel, heterodimers are required for NKT cell maturation, as is IL15- and/or IL-7-induced proliferation of NKT cell precursors (209, 259). Signaling through the cytokine receptor common γ chain (γc) has been implicated in terminal NKT maturation (characterized by acquisition of NK markers such as NK1.1) (260). Transgenic mice in which classical NF-κB was inhibited in a T cell–speciﬁc manner as well as mice that lack RelA in T cells have highly reduced numbers of NK.1.1+ NKT cells, while containing normal numbers of NK1.1− NKT cell precursors, suggesting that classical NF-κB regulates terminal NKT cell maturation (209, 259). Continuous TCR signaling is required for postpositive selection maturation of NKT cells to the NK1.1+ stage (261), raising the possibility that NF-κB might link TCR signaling to γc expression or activation. Indeed, TCR activation results in increased γc expression on NKT cells in an

NF-κB-dependent manner (259). NF-κB binds to an intronic site within the γc gene in a TCRinducible manner and may regulate its expression directly (259). Elevated γc on the NKT cell surface may increase the signal threshold through which γc facilitates maturation to the NK1.1+ stage. These ﬁndings suggest a molecular mechanism by which NF-κB regulates terminal NKT cell maturation (259). There are other factors such as T-bet (262) involved in the terminal maturation of NKT cells to the NK1.1+ stage, and whether those factors cooperate with classical NF-κB signaling in this process remains to be studied. Studies in mice lacking IKKβ indicated that NF-κB signaling might also play a role in the proper development of regulatory T cells as well as of memory T cells (263). Moreover, mutations in the IKKγ/NEMO gene in human patients have revealed an important role for NF-κB signaling in human T cell development, as these patients suffer from a severe immunodeﬁciency (124). Defects in Fas or its ligand (Fas-L) lead to development of abnormal B220+ Thy1+ T cells that accumulate in secondary lymphoid organs. Defects in Fas or Fas-L cause lethal autoimmune disorders called lymphoproliferative disorder (lpr), and generalized lymphoproliferative disorder (gld), respectively (264). Interestingly, T cell–speciﬁc inhibition of NF-κB impaired the development of these abnormal T cells and prevented their accumulation in peripheral lymphoid tissues, thereby rescuing gld/gld mice (265). However, the mechanism by which NF-κB supports development of these abnormal T cells remains to be determined.

NF-κB IN NK CELL DEVELOPMENT NK cells are involved in killing infectious agents by perforin- and granzyme-mediated mechanisms as well as by secreting inﬂammatory cytokines such as TNF-α and IFN-γ (266). NK cells are also involved in killing tumors (266). NF-κB plays a critical role in the development of NK cells (267). Although NK cell

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precursors exhibit constitutive NF-κB activity, the combined deletion of IκBα and IκBε results in reduced numbers of NK cells (267). However, further studies are required to explain how NF-κB regulates NK cell maturation. Studies on human patients with IKKγ/NEMO mutations revealed that in addition to its role in NK cell maturation, IKKγ-dependent classical NF-κB activity is also required for their killer function (268).

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NF-κB IN B CELL DEVELOPMENT Development of B cells depends on a series of transcriptional regulatory events (269–271) that control progression of B cell progenitors in the bone marrow via the pro-B and preB stages into immature B cells that leave the bone marrow to undergo terminal maturation in peripheral lymphoid organs (271–273). Immature B cells are divided into the T1 and T2 stages (274, 275). Activation of classical NF-κB signaling by the pre-BCR appears to provide a survival signal that is critical during early B cell development in the bone marrow (273). In contrast, BAFF-activated alternative NF-κB signaling mainly affects late B cell development in the spleen during the T1 to T2 transition (273, 275). In addition, alternative NF-κB activation provides a survival signal to mature B cells (273). Moreover, NIK and RelB, which are the major players in alternative NF-κB signaling, are required for proper development of marginal zone B (MZB) cells (276, 277). In accordance with the role of this alternative pathway in MZB cell development, BAFF transgenic mice accumulate huge numbers of MZB cells in their spleens, resulting in autoimmunity (278–280). The development or survival of these autoreactive B cells requires that the marginal zone exist, so removal of the spleen, whose follicles are the only ones that contain a proper marginal zone, prevents autoimmunity (278). In addition, the alternative NF-κB pathway is required for expression of B cell integrins that retain MZB cells in the marginal zone (278). Interestingly, expression of the ligands for these integrins, VCAM1 and 716

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ICAM1, in splenic stromal cells is controlled by the classical pathway (278, 281). B cell–speciﬁc deletion of IKKγ/NEMO also results in impaired survival of T1 B cells, resulting in reduced numbers of transitional and mature B cells (241). Moreover, the combined deletion of RelA and c-Rel also results in highly reduced numbers of B cells owing to increased apoptosis (282), suggesting that both the classical and the alternative NF-κB signaling pathways coordinately regulate B cell survival and maturation. Curiously, however, uncontrolled activation of the alternative NF-κB pathway in mice that lack the C-terminal region of p100 culminates in aberrant early B cell development in the bone marrow because of a defective pro-B to preB (CD19− to CD19+ ) transition (283), which is regulated by the B cell–speciﬁc transcription factor Pax5 (271). Pax5 expression is highly reduced in bone marrow B cells from these mice, and deletion of one RelB allele rescued Pax5 expression as well as normal B cell development (283). However, because the defects described above could be due to elevated production of glucocorticoids, which kill B cell progenitors, it is unclear whether RelB is a direct regulator of Pax5. Mice lacking TRAF2 or TRAF3 also exhibit constitutive alternative NF-κB signaling (219, 230) and display similar B cell developmental defects that are rescuable by deletion of NIK (220, 232). Traf2−/− and Traf3−/− mice as well as mice lacking the C-terminal region of p100 all have very high levels of serum cortisol (219, 284), and cortisol is known to kill B cell precursors in the bone marrow (285). Thus, the impaired B cell development in all these mutant mice could be the consequence of excessive glucocorticoid production. In addition to the defects in early bone marrow B cell development, Traf2−/− and Traf3−/− mice exhibit abnormalities in peripheral B cells. Splenic B cells from both these mice have a survival advantage, and, similar to BAFF transgenic mice, the Traf2−/− and Traf3−/− mice accumulate large numbers of MZB cells and exhibit autoimmune features (229, 230, 286). Mice lacking the C-terminal region of p100 also accumulate large numbers of MZB cells

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(287). These results suggest that uncontrolled activation of the alternative NF-κB pathway results in accumulation of MZB cells and results in autoimmunity.

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NF-κB AND LYMPHOID MALIGNANCIES Physiological activation of NF-κB during lymphocyte maturation and activation mediates expression of genes involved in proliferation and survival as well as genes involved in immunity to infection (6). However, dysregulated NFκB activation results in aberrant expression of its target genes that regulate cell proliferation or survival, including cyclin D1, cyclin D2, cMyc, c-Myb (288–290), BCL2, and BCL-XL (8), as well as cytokines such as IL-2, IL-6, and CD40-L that regulate growth and proliferation of lymphocytes. Not surprisingly, constitutively active NF-κB has been implicated in various lymphoid malignancies. The ﬁrst link between NF-κB and lymphoid malignancies came from studies on the viral oncogene v-Rel (homolog of the cellular c-Rel) that can cause aggressive leukemia and lymphoma (291). Subsequent studies revealed a number of genetic alterations affecting NF-κB activity in different forms of B and T cell malignancies (292, 293). Constitutively active NF-κB has been reported in malignant cells from patients with acute lymphocyte leukemia, MM, chronic myelogenous leukemia, as well as in myelodysplastic syndromes (292). In several forms of Hodgkin’s lymphoma, which is characterized by Hodgkin and Reed-Sternberg (HRS) cells, constitutive NF-κB mainly composed of RelA was also seen (294, 295). In some HRS cells, transcriptionally active forms of p50 homodimers complexed with BCL3 were also detected (296). Other studies suggest that elevated NF-κB in HRS cells is due to constitutive activation of cell surface receptors such as CD30, CD40, RANK, and Notch1 (297). Surprisingly, in large B cell lymphoma, CD40 was found to form complexes with c-Rel in the nucleus and thereby enhance expression of NF-κB target genes, including CD154, Blys/BAFF, and Bﬂ-1/A1, indicating

that cooperation between nuclear CD40 and c-Rel is important in regulating growth, survival, and proliferation of lymphoma cells (298). Curiously, CD40 contains an NLS, and its nuclear localization was found in normal and neoplastic B cells (299). However, whether association of CD40 with c-Rel is restricted to malignant cells remains to be studied. Although CD30, CD40, and RANK are well-known NFκB activators, Notch1 also regulates the expression of RelB and NfkB2 and activates IKK by direct interaction leading to NF-κB activation; it thereby induces T cell leukemia (300). Moreover, pre-TCR signaling that provides survival signals to maturing thymocytes can result in aberrant NF-κB activation in cooperation with Notch3 upon dysregulation in T cell lymphoma (301, 302). Antigen receptor signaling (which normally activates NF-κB) if dysregulated could lead to a malignant transformation of T and B cells. Triggering the TCR or BCR, as discussed above, recruits the CBM complex (composed of CARMA1, BCL10, and MALT1) and activates the IKK complex. In cases of lymphomas such as activated B cell–like diffuse large B cell lymphoma (ABC-DLBCL) and MALT lymphoma, these signaling complexes are constitutively activated. In the case of MALT lymphoma, which is normally associated with Helocobacter pylori infection and is predominantly seen in the gastric mucosa, NF-κB activation is due to chromosomal translocations, resulting in the expression of IAP2-MALT1 fusion protein as well as misregulated expression of MALT1 or BCL10 genes under the control of immunoglobulin heavy chain enhancer (293, 303). These chromosomal translocations cause the constitutive activation of the BCL10MALT1 complex independently of signals from the antigen receptor and lead to constitutive IKK activation (293, 304). Similarly, the IAP2MALT1 fusion protein also causes constitutive IKK activation, presumably by NEMO/IKKγ ubiquitination (305). In the case of ABCDLBCL, based on a siRNA-mediated knockdown approach, constitutive NF-κB activation is due to activation of components of the CBM

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complex, CARMA1, BCL10, and MALT1 (306). However, it is not clear how CARMA1 is activated in ABC-DLBCL. Other forms of DLBCL, such as germinal center–like DLBCL (GC-DLBCL) and unclassiﬁed group of DLBCLs, also exhibit constitutive activation of NF-κB. Many DLBCLs respond to pharmacological IKKβ inhibitors and undergo apoptosis. The exception is GCDLBCL, in which activation of NF-κB is due to ampliﬁed expression of c-Rel (293, 307). Given that ampliﬁed expression of c-Rel results in its IKK-independent activation, GCDLBCL cells are refractory to IKKβ inhibitors. Although most of these mutations cause malignancies owing to the activation of the canonical pathway, in some cases DLBCL deletion mutations in the NFkB2 gene leading to deletion of its 3 end result in the expression of a truncated form of p100 lacking its ankyrin repeats, leading to constitutive activation of the alternative NF-κB pathway (308). Several oncogenic viruses, such as EpsteinBarr virus (EBV), induce NF-κB activation and thereby lead to lymphomagenesis. Speciﬁcally, latent membrane protein 1 (LMP1) of EBV activates both the classical and alternative NFκB pathways (309). Accordingly, LMP1 alone is sufﬁcient to induce lymphomagenesis in B cell–speciﬁc LMP1 transgenic mice (310). Malignant cells from these mice exhibit elevated nuclear c-Rel. LMP1 can also induce NIKdependent processing of p100 to p52 (311– 313), but it is not clear whether LMP1-induced activation of alternative NF-κB signaling is critical in lymphoma development in EBVinfected individuals. Nonetheless, NFkB2 gene deletion that results in expression of truncated p100 variants were observed in several lymphomas (314), suggesting that activation of alternative NF-κB signaling may be a key oncogenic event, as recently found in MM (see below). Future studies in animal models by crossing LMP1 transgenic mice to Nik−/− or mice lacking different subunits of IKK will address whether classical or alternative NF-κB signaling or both play a critical role in LMP1-induced lymphomagenesis.

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In addition to EBV, two other viruses— Kaposi sarcoma-associated herpesvirus (KSHV) and the human T lymphotropic virus (HTLV)— cause primary effusion lymphoma (PEL) and adult T cell lymphoma (ATL), respectively (315, 316). In the case of PEL, which is a B cell malignancy caused by KSHV infection, IKK is constitutively activated by interacting with a viral protein, vFLIP, a homolog of the cellular FLIP protein (317). Constitutive activation of IKK by vFLIP thus appears to be an important step in the malignancy caused by KSHV because siRNA-mediated knockdown of vFLIP induces death of KSHV-infected cells (318). In the case of ATL caused by HTLV, a viral expressed protein called Tax binds directly to NEMO/IKKγ and activates the IKK complex, resulting in the activation of the classical NF-κB pathway (319). Curiously, Tax can activate the alternative NF-κB pathway by interacting with p100 and inducing its processing (320). Although in many of these cases treatment with IKK inhibitors, which mainly target the classical pathway, results in apoptosis of infected cells, genetic studies are required to precisely address whether the classical or alternative pathway or both are essential for tumorigenesis by HTLV and other viruses. In addition to mutations that affect IKK activity, in several cases of Hodgkin’s lymphoma, NF-κB was found to be activated independently of IKK activity. This is because mutations in the IκBs lead to inactivation or truncation of their C-terminal repeats, resulting in constitutive activation of classical NF-κB pathway (321, 322). However, as discussed above, recent reports indicate that even upon deletion of IκBα, IκBβ, and IκBε, RelA is still largely cytoplasmic, presumably because of elevated levels of p100 and its interaction with RelA (27, 28). Thus, it remains to be studied whether the elevated NFκB activity seen in Hodgkin’s lymphoma associated with IκB mutations is complemented by additional dysregulations in the NF-κB pathway. Although many studies have reported aberrations in the classical NF-κB pathway as being the cause for several forms of lymphoid

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malignancies, some recent reports shed light on the role of alternative signaling in B cell malignancies. While mutations in the NFkB2 gene resulting in truncation of p100 (as described above) are known to be associated with some lymphomas, several recent studies have shown that malignant cells from MM patients harbor mutations in several genes whose products are involved in NIK turnover in nonstimulated B cells (222, 223). All these mutations result in NIK stabilization and constitutive p100 processing. Some of these mutations in either TRAF3 or NIK disrupt the interaction between TRAF3 and NIK (222), which, as mentioned above, results in stabilization of NIK owing to impaired recruitment of the cIAP2:TRAF2 complex. In addition, deletions of the closely linked cIAP1 and cIAP2 loci leading to NIK stabilization were observed in other cases of MM (222, 223). Despite the constitutive activation of alternative p52:RelB heterodimers in such cells, which is due to NIK stabilization, the key oncogenic event may actually be the activation of classical NF-κB signaling. This activation has also been observed in MM, which harbors mutations that lead to NIK stabilization (223). Although prolonged activation of alternative NF-κB signaling may result in production of cytokines that activate classical NFκB signaling, a more likely scenario is that the high levels of NIK that are expressed in such cells may result in direct activation of the IKK complex through phosphorylation of IKKβ. Indeed, IKKβ inhibitors inhibit the proliferation and induce the death of MM cells, whereas depletion of IKKα has no affect on survival of MM cells expressing high levels of NIK (223, 323). These ﬁndings further indicate that activation of classical NF-κB signaling is the major oncogenic event in MM. Consistent with the high oncogenic activity of classical NF-κB signaling in MM, deletion of CYLD, which is a known negative regulator of NF-κB signaling, was also observed in MM, leading to high NF-κB activity (223). The actual utility of IKKβ inhibitors in the treatment of MM and other lymphoid malignancies will have to await the completion of carefully planned clinical

trials. However, in other cases of lymphoid malignancies, speciﬁc inhibition of alternative NF-κB signaling might be required to induce death of the malignant cells. Interestingly, a recent report showed that RelB, whose activity is regulated by the alternative NF-κB pathway, is required in radiation-resistant stromal cells to promote T cell leukemia (324). Thus, in addition to its role in cancer cells, the alternative NF-κB signaling also plays a critical pro-oncogenic role in nonhematopoietic stromal cells by regulating their cross talk with the leukemic cells. Therefore, it is important to develop NIK inhibitors to target alternative NF-κB signaling speciﬁcally as well as to target NIK-mediated activation of IKKβ. Such speciﬁc targeting will test the inhibitors’ utility in the treatment of lymphoid malignancies associated with NIK activation.

CONCLUDING REMARKS NF-κB signaling is regulated by different mechanisms in a cell type– and stimulus-speciﬁc manner. Although signiﬁcant advances have been made in our understanding of classical as well as alternative NF-κB signaling, many aspects are still not clear. For instance, activation of IKK is still an enigma, and the factors that are involved in the receptor proximal IKK activation are not fully understood. Similarly, although NIK turnover by cIAP2:TRAF2:TRAF3 seems to be a critical step in the regulation of alternative NF-κB signaling, further studies are required to address how cIAP1/2 target TRAF3 instead of NIK to proteasomal degradation upon receptor ligation. Although TRAF2-mediated K63-linked ubiquitination of cIAP1/2 might be the regulatory step in this process, additional factors are likely involved. Moreover, the E2 enzymes involved in the ubiquitination of NIK and TRAF3 are still not known. As discussed above, NFκB signaling is both positively and negatively regulated, predominantly via various posttranslational modiﬁcations. Dysregulation of positive or negative NF-κB activation is involved in several genetic disorders, including lymphoid

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malignancies. Thus, it is important to thoroughly understand each step of NF-κB signal regulation. Perhaps detailed insight into the proteomics of both classical and alternative

NF-κB signaling by different stimuli will enhance our understanding of this master regulator of cell survival, inﬂammation, immunity, and cancer.

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS Annu. Rev. Immunol. 2009.27:693-733. Downloaded from arjournals.annualreviews.org by Karolinska Institutet on 03/29/09. For personal use only.

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307. Rosenwald A, Wright G, Chan WC, Connors JM, Campo E, et al. 2002. The use of molecular proﬁling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N. Engl. J. Med. 346:1937–47 308. Neri A, Chang CC, Lombardi L, Salina M, Corradini P, et al. 1991. B cell lymphoma-associated chromosomal translocation involves candidate oncogene lyt-10, homologous to NF-κB p50. Cell 67:1075–87 309. Eliopoulos AG, Young LS. 2001. LMP1 structure and signal transduction. Semin. Cancer Biol. 11:435–44 310. Thornburg NJ, Kulwichit W, Edwards RH, Shair KH, Bendt KM, Raab-Traub N. 2006. LMP1 signaling and activation of NF-κB in LMP1 transgenic mice. Oncogene 25:288–97 311. Luftig M, Yasui T, Soni V, Kang MS, Jacobson N, et al. 2004. Epstein-Barr virus latent infection membrane protein 1 TRAF-binding site induces NIK/IKK α-dependent noncanonical NF-κB activation. Proc. Natl. Acad. Sci. USA 101:141–46 312. Atkinson PG, Coope HJ, Rowe M, Ley SC. 2003. Latent membrane protein 1 of Epstein-Barr virus stimulates processing of NF-κ B2 p100 to p52. J. Biol. Chem. 278:51134–42 313. Eliopoulos AG, Caamano JH, Flavell J, Reynolds GM, Murray PG, et al. 2003. Epstein-Barr virusencoded latent infection membrane protein 1 regulates the processing of p100 NF-κB2 to p52 via an IKKγ/NEMO-independent signalling pathway. Oncogene 22:7557–69 314. Kim KE, Gu C, Thakur S, Vieira E, Lin JC, Rabson AB. 2000. Transcriptional regulatory effects of lymphoma-associated NFKB2/lyt10 protooncogenes. Oncogene 19:1334–45 315. Cesarman E, Chang Y, Moore PS, Said JW, Knowles DM. 1995. Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N. Engl. J. Med. 332:1186–91 316. Poiesz BJ, Ruscetti FW, Reitz MS, Kalyanaraman VS, Gallo RC. 1981. Isolation of a new type C retrovirus (HTLV) in primary uncultured cells of a patient with Sezary T-cell leukaemia. Nature 294:268– 71 317. Liu L, Eby MT, Rathore N, Sinha SK, Kumar A, Chaudhary PM. 2002. The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the IκB kinase complex. J. Biol. Chem. 277:13745–51 318. Keller SA, Schattner EJ, Cesarman E. 2000. Inhibition of NF-κB induces apoptosis of KSHV-infected primary effusion lymphoma cells. Blood 96:2537–42 319. Carter RS, Geyer BC, Xie M, Acevedo-Suarez CA, Ballard DW. 2001. Persistent activation of NF-κ B by the tax transforming protein involves chronic phosphorylation of IκB kinase subunits IKKβ and IKKγ. J. Biol. Chem. 276:24445–48 320. Xiao G, Cvijic ME, Fong A, Harhaj EW, Uhlik MT, et al. 2001. Retroviral oncoprotein Tax induces processing of NF-κB2/p100 in T cells: evidence for the involvement of IKKalpha. EMBO J. 20:6805–15 321. Krappmann D, Emmerich F, Kordes U, Scharschmidt E, Dorken B, Scheidereit C. 1999. Molecular mechanisms of constitutive NF-κB/Rel activation in Hodgkin/Reed-Sternberg cells. Oncogene 18:943– 53 322. Cabannes E, Khan G, Aillet F, Jarrett RF, Hay RT. 1999. Mutations in the IκBα gene in Hodgkin’s disease suggest a tumour suppressor role for IκBα. Oncogene 18:3063–70 323. Hideshima T, Neri P, Tassone P, Yasui H, Ishitsuka K, et al. 2006. MLN120B, a novel IκB kinase β inhibitor, blocks multiple myeloma cell growth in vitro and in vivo. Clin. Cancer Res. 12:5887–94 324. dos Santos NR, Williame M, Gachet S, Cormier F, Janin A, et al. 2008. RelB-dependent stromal cells promote T-cell leukemogenesis. PLoS ONE 3:e2555

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Annu. Rev. Immunol. 2009.27:693-733. Downloaded from arjournals.annualreviews.org by Karolinska Institutet on 03/29/09. For personal use only.

Contents

Volume 27, 2009

Frontispiece Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Translating Molecular Insights in Autoimmunity into Effective Therapy Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Structural Biology of Shared Cytokine Receptors Xinquan Wang, Patrick Lupardus, Sherry L. LaPorte, and K. Christopher Garcia p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 Immunity to Respiratory Viruses Jacob E. Kohlmeier and David L. Woodland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 61 Immune Therapy for Cancer Michael Dougan and Glenn Dranoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 83 Microglial Physiology: Unique Stimuli, Specialized Responses Richard M. Ransohoff and V. Hugh Perry p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p119 The Liver as a Lymphoid Organ Ian Nicholas Crispe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Immune and Inﬂammatory Mechanisms of Atherosclerosis Elena Galkina and Klaus Ley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p165 Primary B Cell Immunodeﬁciencies: Comparisons and Contrasts Mary Ellen Conley, A. Kerry Dobbs, Dana M. Farmer, Sebnem Kilic, Kenneth Paris, Soﬁa Grigoriadou, Elaine Coustan-Smith, Vanessa Howard, and Dario Campana p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 The Inﬂammasomes: Guardians of the Body Fabio Martinon, Annick Mayor, and Jürg Tschopp p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p229 Human Marginal Zone B Cells Jean-Claude Weill, Sandra Weller, and Claude-Agn`es Reynaud p p p p p p p p p p p p p p p p p p p p p p267

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Aire Diane Mathis and Christophe Benoist p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p287 Regulatory Lymphocytes and Intestinal Inﬂammation Ana Izcue, Janine L. Coombes, and Fiona Powrie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p313 The Ins and Outs of Leukocyte Integrin Signaling Clare L. Abram and Clifford A. Lowell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p339

Annu. Rev. Immunol. 2009.27:693-733. Downloaded from arjournals.annualreviews.org by Karolinska Institutet on 03/29/09. For personal use only.

Recent Advances in the Genetics of Autoimmune Disease Peter K. Gregersen and Lina M. Olsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p363 Cell-Mediated Immune Responses in Tuberculosis Andrea M. Cooper p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p393 Enhancing Immunity Through Autophagy Christian Munz ¨ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p423 Alternative Activation of Macrophages: An Immunologic Functional Perspective Fernando O. Martinez, Laura Helming, and Siamon Gordon p p p p p p p p p p p p p p p p p p p p p p p p451 IL-17 and Th17 Cells Thomas Korn, Estelle Bettelli, Mohamed Oukka, and Vijay K. Kuchroo p p p p p p p p p p p p p p485 Immunological and Inﬂammatory Functions of the Interleukin-1 Family Charles A. Dinarello p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p519 Regulatory T Cells in the Control of Host-Microorganism Interactions Yasmine Belkaid and Kristin Tarbell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p551 T Cell Activation Jennifer E. Smith-Garvin, Gary A. Koretzky, and Martha S. Jordan p p p p p p p p p p p p p p p591 Horror Autoinﬂammaticus: The Molecular Pathophysiology of Autoinﬂammatory Disease Seth L. Masters, Anna Simon, Ivona Aksentijevich, and Daniel L. Kastner p p p p p p p p p621 Blood Monocytes: Development, Heterogeneity, and Relationship with Dendritic Cells Cedric Auffray, Michael H. Sieweke, and Frederic Geissmann p p p p p p p p p p p p p p p p p p p p p p p p669 Regulation and Function of NF-κB Transcription Factors in the Immune System Sivakumar Vallabhapurapu and Michael Karin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p693

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