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Contributors

Numbers in parenthesis indicated the pages on which the authors’ contributions begin.

Frederick W. Alt (43), Howard Hughes Medical Institute, The Children’s Hospital Boston, CBR Institute for Biomedical Research, and Harvard Medical School, Boston, Massachusetts 02115 Craig H. Bassing (43), Howard Hughes Medical Institute, The Children’s Hospital Boston, CBR Institute for Biomedical Research, and Harvard Medical School, Boston, Massachusetts 02115 Brian Becknell (209), Medical Scientist Program and Integrated Biomedical Graduate Program, Ohio State University, Columbus, Ohio 43210 Michael R. Blackburn (1), Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, Texas 77030 Michael A. Caligiuri (209), Medical Scientist Program, Integrated Biomedical Graduate Program, Department of Internal Medicine, Division of Hematology/Oncology, and Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210 Michael C. Carroll (137), CBR Institute for Biomedical Research, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 Jayanta Chaudhuri (43), Howard Hughes Medical Institute, The Children’s Hospital Boston, CBR Institute for Biomedical Research, and Harvard Medical School, Boston, Massachusetts 02115 Darryll D. Dudley (43), Howard Hughes Medical Institute, The Children’s Hospital Boston, CBR Institute for Biomedical Research, and Harvard Medical School, Boston, Massachusetts 02115 V. Michael Holers (137), Departments of Medicine and Immunology, University of Colorado Health Sciences Center, Denver, Colorado 80217 Kusumam Joseph (159), Division of Pulmonary/Critical Care Medicine and Allergy/Clinical Immunology, Medical University of South Carolina, Charleston, South Carolina 29425 ix

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c o n tr i b u t o rs

Allen P. Kaplan (159), Division of Pulmonary/Critical Care Medicine and Allergy/Clinical Immunology, Medical University of South Carolina, Charleston, South Carolina 29425 John F. Kearney (113), Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35204 Rodney E. Kellems (1), Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, Texas 77030 To-Ha Thai (113), Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35204 Jose A. Villadangos (241), Immunology Division and Cooperative Research Center for Vaccine Technology, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia Nicholas S. Wilson (241), Immunology Division and The Cooperative Research Center for Vaccine Technology, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia

Adenosine Deaminase Deficiency: Metabolic Basis of Immune Deficiency and Pulmonary Inflammation Michael R. Blackburn and Rodney E. Kellems Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, Texas 77030

1. 2. 3. 4. 5. 6. 7.

Abstract .................................................................................................. Introduction ............................................................................................. ADA Deficiency in Humans .......................................................................... Models of ADA-Deficient Severe Combined Immunodeficiency Disease .................... Metabolic Disturbances in ADA Deficiency ....................... ................................ Pulmonary Consequences of Elevated Adenosine ................ ................................ Additional Physiological Consequences of Elevated Adenosine ................................ Concluding Remarks ................................................................................... References ...............................................................................................

1 2 3 7 12 21 31 33 36

Abstract Genetic deficiencies in the purine catabolic enzyme adenosine deaminase (ADA) in humans results primarily in a severe lymphopenia and immunodeficiency that can lead to the death of affected individuals early in life. The metabolic basis of the immunodeficiency is likely related to the sensitivity of lymphocytes to the accumulation of the ADA substrates adenosine and 20 deoxyadenosine. Investigations using ADA-deficient mice have provided compelling evidence to support the hypothesis that T and B cells are sensitive to increased concentrations of 20 -deoxyadenosine that kill cells through mechanisms that involve the accumulation of dATP and the induction of apoptosis. In addition to effects on the developing immune system, ADA-deficient humans exhibit phenotypes in other physiological systems including the renal, neural, skeletal, and pulmonary systems. ADA-deficient mice develop similar abnormalities that are dependent on the accumulation of adenosine and 20 -deoxyadenosine. Detailed analysis of the pulmonary insufficiency seen in ADA-deficient mice suggests that the accumulation of adenosine in the lung can directly access cellular signaling pathways that lead to the development and exacerbation of chronic lung disease. The ability of adenosine to regulate aspects of chronic lung disease is likely mediated by specific interactions with adenosine receptor subtypes on key regulatory cells. Thus, the examination of ADA deficiency has identified the importance of purinergic signaling during lymphoid development and in the regulation of aspects of chronic lung disease.

1 advances in immunology, vol. 86 0065-2776/05 $35.00

ß 2005 Elsevier Inc. All rights reserved.

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1. Introduction Adenosine deaminase (ADA) is an essential enzyme of purine metabolism (Fig. 1) and is highly conserved throughout phylogeny. The initial clue revealing the importance of ADA to mammalian organisms came with the chance discovery that a form of severe combined immunodeficiency disease (SCID) in humans was associated with ADA deficiency (Giblett et al., 1972). In this serendipitous way ADA deficiency was the first of the immunodeficiency diseases for which the underlying biochemical defect was discovered. Subsequent investigations indicated that ADA deficiency accounts for approximately 20% of cases of human SCID and that it is the most severe of the immunodeficiency diseases, affecting both cell-mediated and humoral immunity (Buckley et al., 1997; Hershfield and Mitchell, 2001). Soon after their discovery that defects in ADA were associated with immunodeficiency, Giblett and colleagues examined other immunodeficient individuals for deficiencies in

Figure 1 Catabolism of adenosine and 20 -deoxyadenosine. Adenosine and 20 -deoxyadenosine are deaminated to inosine and 20 -deoxyinosine by adenosine deaminase (ADA). This is followed by cleavage of the purine base from the ribose or deoxyribose sugar moieties by the enzyme purine nucleoside phosphorylase (PNP) to produce hypoxanthine. Hypoxanthine is salvaged back to inosine monophosphate (IMP) in most tissues by hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or is oxidized first to xanthine and then to uric acid by the enzyme xanthine oxidase (XO). In humans, uric acid is excreted in the urine, whereas in the mouse uric acid can be converted to allantoin by the enzyme uricase before excretion.

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purine catabolic enzymes and found that defects in purine nucleoside phosphorylase (Fig. 1) also result in immunodeficiency disease (Giblett et al., 1975). These findings demonstrate the importance of purine metabolism in development of the immune system. Deciphering the mechanisms by which defects in purine metabolism lead to abnormal lymphopoiesis has proved a difficult task, and although significant progress in this arena has been made, many questions remain. However, efforts to understand the metabolic basis of the immunodeficiency associated with ADA deficiency have led to advances in the treatment of certain leukemias, and the treatment of ADA deficiency in humans has advanced aspects of enzyme replacement and gene replacement therapies. The generation of ADA-deficient mice (Blackburn et al., 1998; Migchielsen et al., 1995; Wakamiya et al., 1995) provided the opportunity to examine the pathways by which disturbances in purine metabolism influence physiological systems in the whole animal. ADA-deficient mice develop a combined immunodeficiency similar to that seen in ADA-deficient humans, and experiments in these mice have provided novel mechanistic information about how the accumulation of the ADA substrates adenosine and 20 -deoxyadenosine impact components of the immune system. In addition, by removing the major enzyme that controls adenosine levels in tissues and cells, these mice have served as biological screens for physiological processes sensitive to chronic elevations in adenosine (Blackburn, 2003). In particular, ADA-deficient mice have served as a useful model for deciphering the role of adenosine signaling in chronic inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). This review discusses the current understanding of how the accumulation of ADA substrates impacts the immune and pulmonary systems by comparing findings in ADA-deficient humans and mice. 2. ADA Deficiency in Humans ADA deficiency in humans arises from naturally occurring mutations in the ADA gene that are inherited in an autosomal recessive manner. Most ADAdeficient humans are diagnosed early in life, when they present with marked lymphopenia; failure to thrive; and opportunistic fungal, viral, and bacterial infections (Buckley et al., 1997; Hershfield and Mitchell, 2001). These patients have little to no detectable ADA activity and severe metabolic disturbances associated with the loss of ADA activity. The thymus is absent or small and dysplastic in ADA-deficient individuals (Borzy et al., 1979), and they have severely reduced numbers of peripheral T, B, and natural killer (NK) cells (Buckley et al., 1997). ADA-deficient SCID is the only immunodeficiency in which all three cell types are severely reduced in number. Without

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intervention, ADA-deficient individuals die from overwhelming infections within the first year of life. A smaller population of ADA-deficient patients presents later in life with a less severe form of immunodeficiency that coincides with less severe loss of ADA enzymatic activity and associated metabolic disturbances (Santisteban et al., 1993). Specific mutations within the ADA gene have been identified for both ‘‘early’’ and ‘‘late’’ onset ADA deficiency (reviewed in Hershfield and Mitchell, 2001), and the severity of the mutations, regarding the loss of ADA enzymatic function, correlates well with the severity of the ensuing disease (Hershfield, 2003). The most successful treatment for ADA deficiency is histocompatible bone marrow transplantation from an HLA-matched sibling. Because this treatment option is seldom available, alternative treatments have been identified, including T cell–depleted haploidentical bone marrow transplantation from a parent. However, these approaches have met with limited success. A successful biochemical approach for the treatment of ADA deficiency involves the use of enzyme replacement therapy wherein a polyethylene glycol–modified form of bovine ADA (PEG–ADA) is provided to patients by twice weekly intramuscular injection (Hershfield et al., 1993). Polyethylene glycol appears to protect the bovine ADA from proteolytic and immunologic attack, hence increasing the circulating half-life of this exogenous enzyme. ADA replacement therapy is effective in reducing the metabolic impact of ADA deficiency and has prolonged the life of individuals who have in some cases been treated for more than 8 years (Hershfield, 1995). Relatively few complications have been reported with respect to allergic reactions or immunogenicity to PEG–ADA, and it appears to be the best option for the prolonged treatment of ADA-deficient patients who lack an HLA-identical marrow donor. ADA deficiency has received considerable attention as the testing ground for the development of gene therapy protocols. Several features of ADA deficiency make it an attractive candidate for gene replacement therapy: bone marrow or cord blood stem cells are relatively accessible cell populations; individuals with as little as 5% normal ADA activity have normal immune function, suggesting a high degree of replacement may not be necessary; and evidence exists to suggest that T cells with ADA activity can be selected for and enriched in an ADA-deficient environment. The latter is supported by observations in ADA-deficient individuals where spontaneous clinical remissions occurred in association with mosaicism for ADA expression (Hirschhorn et al., 1994, 1996), which in one instance was associated with the reversion of a mutation in the ADA allele to normal in lymphoid cells (Hirschhorn et al., 1996). Thus, the hope is that efficient transfer of a recombinant ADA gene into hematopoietic cells will result in the outgrowth of a genetically repaired immune system. For these and other reasons, ADA gene therapy studies

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were the first to use ex vivo approaches to stably introduce new genetic information into patients (Blaese et al., 1995; Bordignon et al., 1995). Protocols have been initiated in patients in whom peripheral blood T lymphocytes (Blaese et al., 1995; Bordignon et al., 1995), bone marrow– derived stem cells (Bordignon et al., 1995; Hoogerbrugge et al., 1996), or cord blood stem cells (Kohn et al., 1998) have been transduced with retroviral vectors carrying recombinant ADA. However, the successes of ADA gene therapy attempts have been limited. Although it appears that transduced cells remain viable in ADA-deficient individuals, the transduction efficiency and ultimately the expression levels of ADA in transduced cells are not high enough to provide clinical benefit. Patients receiving ADA gene therapy are also maintained on ADA enzyme replacement therapy. PEG–ADA therapy no doubt complicates the evaluation of the benefits of ADA gene therapy in these patients and may even affect the ability of transduced cells to expand. However, the cessation of PEG–ADA therapy in patients receiving ADA gene therapy results in the return of metabolic disturbances and a decrease in certain lymphoid cell populations (Kohn et al., 1998), suggesting PEG–ADA therapy provides much better protection than ADA-transduced cells. Thus, until methods for achieving higher levels of transduction and expression are achieved, PEG–ADA therapy will likely remain the treatment of choice for this disorder in the absence of a compatible bone marrow donor. Most immunodeficiency diseases are associated with defects in genes that encode proteins that play obvious roles as signaling components involved in immune cell development and function. A well-known example is the X-linked form of SCID that is associated with defects in the gc chain component of important cytokine receptors such as interleukin (IL-2) (Noguchi et al., 1993). Other examples include defects in adaptor protein kinases involved in T-cell responses, such as ZAP70 (Elder, 1998) and Lck (Goldman et al., 1998); enzymes involved in the rearrangement of B- and T-cell receptor chain genes, such as recombination-activating proteins 1 and 2 (RAG-1 and RAG2) (de Saint-Basile et al., 1991; Schwarz et al., 1996); transporter associated with antigen processing 1 or 2 (TAP-1 or TAP-2) (Donato et al., 1995; Furukawa et al., 1999), which are important in processing peptide antigens for presentation to MHC class I molecules; and transcription factors that regulate the production of MHC class II molecules (Klein et al., 1993). It is reasonable to expect that defects in these molecules that are important in immune cell development and function would lead to immunodeficiency disease. However, it is less clear why defects in a widely distributed enzyme of purine metabolism, such as ADA, would cause such a robust form of SCID. ADA interacts with the cell surface protein CD26/dipeptidyl-peptidase (Kameoka et al., 1993), and it has been proposed that this interaction may

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play an important role in immune cell function (Morimoto and Schlossman, 1998). The involvement of this interaction in the immunodeficiency seen in ADA deficiency seems unlikely (Richard et al., 2000) but needs to be pursued further. ADA is highly expressed in lymphoid cells and in the human thymus (Hirschhorn et al., 1978), suggesting that a special need for the regulation of adenine nucleosides is required for proper lymphoid development and function. Substantial research has been devoted to investigating the link between the purine metabolic disturbances and the immunologic consequences associated with ADA deficiency. In this regard, most attention has focused on the impact of the substrates of the ADA-catalyzed reaction, adenosine and 20 deoxyadenosine (Fig. 1; and see Fig. 4), on aspects of T-cell development. Both nucleosides are elevated in ADA-deficient patients (Hirschhorn, 1993), and each possesses distinct cellular activities that can impact lymphocyte development and function (see Fig. 4). ADA enzyme replacement therapy in humans serves to lower circulating levels of adenosine and 20 -deoxyadenosine in association with improvement in immune status (Hershfield, 1995; Hershfield et al., 1993), further adding to the notion that the increased concentrations of these substrates are responsible for the immunodeficiency seen. Ex vivo experiments using human cells, as well as investigations in animal models, have led to the discovery of specific cellular pathways that likely account for the deletion of lymphocyte populations. These activities are discussed in detail in Section 3. Immunodeficiency is the most thoroughly studied feature of human ADA deficiency; however, other abnormalities have been reported (Table 1). A large number of ADA-deficient patients have bony abnormalities that include flared costochondral junctions in the ribs and short growth plates with few proliferating and some hypertropic and necrotic chondrocytes (Cederbaum et al., 1976). Neurological abnormalities have been reported (Hirschhorn et al., 1980), as have renal defects that include mesangial sclerosis along with unusual cortical adrenal fibrosis (Ratech et al., 1985). Liver abnormalities (Bollinger et al., 1996) and pulmonary insufficiencies of unknown etiology (Hershfield and Mitchell, 2001) have also been noted. In addition, a higher incidence of the inflammatory lung disease asthma, as well as increases in circulating eosinophils and elevations in serum IgE, have been noted in ADA-deficient individuals (Hirschhorn, 1999; Kawamura et al., 1998; Levy et al., 1988). Autoimmune features have also been documented (Geffner et al., 1986). The degree to which these phenotypes are attributed to the metabolic disturbances seen in ADA-deficient patients is not known; however, analyses of phenotypes in ADA-deficient mice suggest that chronic elevations in adenosine may be involved in some of these features.

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a de n o s i n e de a m i nas e d e fic i e n c y Table 1 Phenotypic and Metabolic Disturbances in ADA-Deficient Humans and Mice ADA-deficient humans . Lymphopenia: peripheral T, B, and

ADA-deficient mice . Lymphopenia: Splenic T, B, and

NK cell numbers reduced to less than 10% of normal . Absent or small thymus

.

. . . .

Costochondral junction abnormalities Renal abnormalities Hepatic pathology Pulmonary insufficiency (increase in asthma incidence) . Peripheral eosinophilia . Elevated IgE . Neurological abnormalities

. . . .

. Elevated plasma adenosine

.

. Elevated plasma and urine

.

. . .

2-deoxyadenosine levels . Elevated dATP levels in

.

erythrocytes . AdoHcy hydrolase inhibition in erythrocytes

.

NK cell numbers reduced to less than 10–20% of normal Absent or small thymus and spleen (with abnormal germinal centers) Costochondral junction abnormalities Renal abnormalities Hepatic pathology Pulmonary insufficiency (with features of asthma) Peripheral and lung eosinophilia Elevated IgE Neurological abnormalities (ventriculomegaly) Elevated adenosine levels in plasma, spleen, thymus, liver, lung, kidney, and bone marrow Elevated 2-deoxyadenosine levels in plasma, urine,a spleen, thymus, and bone marrow Elevated dATP levels in erythrocytes, spleen, thymus, liver, lung, and kidney AdoHcy hydrolase inhibition in erythrocytes, spleen, thymus, liver, lung, kidney, and bone marrow

a Our unpublished observations. Abbreviations: ADA, adenosine deaminase; AdoHcy, S-adenosylhomocysteine; NK, natural killer.

3. Models of ADA-Deficient Severe Combined Immunodeficiency Disease 3.1. Generation of ADA-Deficient Mice In efforts to generate animal models to investigate the mechanisms by which ADA deficiency affects the immune system and to promote the advancement of novel treatment approaches such as ADA enzyme replacement therapy and ADA gene therapy, two groups generated ADA-deficient mice. Ada null alleles were generated by the targeted insertion of the neomycin gene into the mouse Ada gene (Migchielsen et al., 1995; Wakamiya et al., 1995). Heterozygote intercrosses failed to produce ADA-deficient pups, and further analysis revealed that ADA-deficient fetuses died during the fetal period, just before term or soon after birth. Phenotypic analysis of ADA-deficient fetuses revealed severe hepatocellular damage and loss of liver function (Migchielsen et al.,

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1995; Wakamiya et al., 1995). Abnormal lung pathology was also reported (Migchielsen et al., 1995). These defects were associated with marked elevations of adenosine and 20 -deoxyadenosine in ADA-deficient fetuses, as well as evidence of dATP accumulation and inhibition of S-adenosylhomocysteine (SAH) hydrolase enzymatic activity, which are downstream metabolic pathways affected by increases in 20 -deoxyadenosine (Migchielsen et al., 1995; Wakamiya et al., 1995). These findings demonstrate that ADA plays an essential role in prenatal development and that the prenatal liver and lung are sensitive to elevations in ADA substrates. However, the prenatal death of ADA-deficient fetuses prevented the detailed analysis of the immune system and other physiological systems affected in ADA-deficient humans. ADA is present in virtually all cells, but enzyme concentrations differ as much as 10,000-fold among cell types (Blackburn and Kellems, 1996). In mice, the highest levels of ADA enzyme activity are observed in trophoblast cells of the placenta (Knudsen et al., 1991), epithelial cells lining the gastrointestinal tract (Chinsky et al., 1990; Witte et al., 1991), thymus (Chinsky et al., 1990), and uterine decidual cells (Knudsen et al., 1991). Within these cells and tissues, ADA gene expression is subject to pronounced developmental control, and transgenic mouse studies have identified key gene regulatory elements responsible for directing expression to specific cell types including the thymus (Aronow et al., 1989), forestomach (Xu et al., 1999), small intestine (Dusing et al., 1997), and trophoblast cells of the placenta (Winston et al., 1992). Transgenic mouse studies led to the identification of an enhancer sequence located between 5 and 6 kb upstream of the transcription initiation site of the mouse Ada gene that was capable of directing reporter gene expression in all trophoblast cell lineages in a manner that coincides with endogenous Ada expression. The identification of the gene regulatory elements from mouse Ada responsible for high levels of expression in trophoblast cells (Shi et al., 1997; Winston et al., 1992) provided an approach to rescue ADA-deficient fetuses from prenatal lethality. Trophoblast-specific gene regulatory sequences were used to target the expression of an ADA-encoding minigene specifically to the trophoblast lineage of transgenic mice (Blackburn et al., 1995). These mice were introduced onto the ADA-deficient background to generate mice that contained the ADAencoding minigene and were also homozygous for the null Ada allele. Restoring ADA to trophoblast cells of the placenta was sufficient to prevent the liver damage seen in ADA-deficient fetuses and was able to rescue them from prenatal lethality. When metabolic disturbances were examined in rescued ADA-deficient fetuses, it was found that placental ADA was able to prevent the accumulation of 20 -deoxyadenosine and dATP in ADA-deficient fetuses while having little effect on lowering the levels of circulating adenosine

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(Blackburn et al., 1995). These findings suggest that 20 -deoxyadenosine cytotoxicity (see Fig. 4) likely accounts for the liver injury seen in ADAdeficient fetuses. Once born, and with the loss of the placenta, ADA enzymatic activity was not observed in any of the tissues examined in postnatal ADAdeficient mice (Blackburn et al., 1998). These studies demonstrated the importance of the prenatal expression of ADA in the trophoblast cells of mice, but more importantly, they provided a strategy for the generation of postnatal ADA-deficient mice in which the mechanistic impact of ADA substrate accumulation could be examined. As with ADA-deficient humans, postnatal ADA-deficient mice develop a combined immunodeficiency (Blackburn et al., 1998). In addition, ADA-deficient mice develop a number of nonimmune phenotypes that have also been noted in ADA-deficient humans (Table 1). These include renal, neurological, and bony abnormalities, as well as a severe pulmonary insufficiency. ADAdeficient mice are relatively normal at birth but fail to thrive and die by 3 weeks of age as a result of the severity of the phenotypes mentioned earlier. These animals respond well to ADA enzyme replacement therapy (Blackburn et al., 2000a,b) and have provided the opportunity to examine the metabolic mechanisms underlying both immune and nonimmune phenotypes in the context of the whole animal. 3.2. Impact of ADA Deficiency on the Immune System ADA-deficient mice provided the opportunity to directly assess the effect of ADA deficiency on the thymus and spleen, critical immune organs that are not accessible in ADA-deficient humans. The status of the immune system progressively deteriorates in ADA-deficient mice until the death of the animals at about 3 weeks of age (Blackburn et al., 1998). By postnatal day 18, thymocyte and splenocyte numbers are less than 10% of those seen in control littermates (Fig. 2A). This is associated with a decrease in thymus size and a substantial increase in apoptosis in the cortical and medullary regions of the thymus (Apasov et al., 2001). The severe reduction in thymocyte number was associated with the almost complete loss of CD4þCD8þ double-positive thymocytes. These results indicate that thymocyte development is seriously impaired in ADA-deficient mice in association with a pronounced deficiency in the production or stability of double-positive thymocytes. Spleens are also smaller in ADA-deficient mice, and analysis of splenocytes revealed a severe reduction in T, B, and NK cells (Fig. 2B). This combined lymphopenia was also seen in peripheral blood. Thus, as in ADA-deficient humans, ADA-deficient mice exhibit a combined immunodeficiency with a severe reduction in T, B, and NK cells (Table 1).

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Figure 2 Severe lymphopenia in ADA-deficient mice. (A) Thymus and spleens were removed from 17-day-old control (wild-type or heterozygous) or ADA-deficient mice, and the mean number of cells in each organ (SEM, n ¼ 5) was determined. (B) Splenocytes were collected from 17-dayold control and ADA-deficient mice and subjected to flow cytometry. Specific antibodies to cell surface markers were used to identify T cells (anti-CD3 and anti-TCRb), B cells (anti-CD45R and anti-IgM), and NK cells (anti-DX5). Mean total cell counts SEM were determined, n ¼ 6. Data are adapted from Blackburn et al. (2000a).

3.3. Impaired Intrathymic T-Cell Development Decreased numbers of circulating T cells, and a decrease in the number of CD4þCD8þ double-positive cells, suggest a block at a specific stage in thymocyte development. Determining the specific stage at which mouse thymopoiesis is affected in ADA deficiency came from elegant studies conducted in mouse fetal thymic organ culture (FTOC) (Thompson et al., 2000), an ex vivo system that allows for the monitoring of thymocyte development in a controlled environment. FTOCs performed on thymuses from ADA-deficient mouse fetuses on day 15 of gestation, or from normal fetuses treated with the potent and specific ADA inhibitor 20 -deoxycoformycin, showed a profound inhibition of thymocyte development past the CD4CD8CD44loCD25þ stage. The inhibition of differentiation in ADA-deficient FTOCs was not due

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to a failure of b selection, as T-cell receptor b locus (TCRb) rearrangements and transcription of the T early a locus occurred normally. Rather, the cells appeared to be dying from apoptosis, because treatment of ADA-deficient FTOCs with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone (zVADfmk), overexpression of the antiapoptotic protein Bcl-2, or deletion of apoptotic protease-activating factor 1 (APAF-1) improved cell yield and the extent of differentiation (Thompson et al., 2000; Van De Wiele et al., 2002). These studies are further supported by findings in genetically modified mice treated with the ADA inhibitor 20 -deoxycoformycin. It was shown that the thymocyte apoptosis seen after ADA inhibition in vivo could be prevented by overexpression of Bcl-2 or by the removal of p53 (Benveniste and Cohen, 1995). Thus, a strong case can be built that thymocytes in an ADAdeficient environment die by apoptosis as a consequence of failing developmental checkpoints during thymopoiesis. As is discussed in Section 4, the majority of available evidence suggests that the depletion of T cells in the thymus is due to the consequences of 20 -deoxyadenosine accumulation that culminates in apoptosis. 3.4. Impaired B-Cell Maturation in the Spleen Most studies examining the effects of ADA deficiency on lymphocytes have focused on T-cell development, with relatively little attention being given to the impact of ADA deficiency on B-cell ontogeny and function. Detailed analysis of B-cell development and function in ADA-deficient mice provided the first definitive evidence that there is an intrinsic defect within the B lymphocyte compartment in ADA deficiency (Aldrich et al., 2003). Surprisingly, B-cell development in the bone marrow of ADA-deficient mice was normal; however, spleens were smaller, splenic architecture was abnormal, and splenic B cells showed defects in proliferation and activation. ADAdeficient B cells demonstrated a higher propensity to undergo B cell receptor-mediated apoptosis, suggesting ADA plays a role in the survival of B cells during antigen-dependent responses. IgM production by extrafollicular plasmablast cells was higher in B cells isolated from ADA-deficient mice, indicating that activated B cells accumulate extrafollicularly as a result of poor or nonexistent germinal center formation. This finding was supported by the observation that there was a profound loss in germinal center formation in the spleens of ADA-deficient mice. The altered splenic environment and signaling abnormalities seen may contribute to a block in B-cell antigendependent maturation in ADA-deficient spleens. Most evidence suggests that defects in B-cell development and function are associated with 20 -deoxyadenosine cytotoxicity (see Section 4); however, more work is needed to

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identify the specific mechanisms involved. Similarly, little is known about the nature of the NK cell lymphopenia seen in ADA-deficient humans or mice, and more research is needed to decipher the mechanisms involved in the depletion of this cell type. Thus, specific effects of ADA deficiency on specific lymphocyte populations have emerged from analysis of cellular populations in ADA-deficient mice and mouse FTOCs. By providing sufficient materials to examine subpopulations of T and B cells during various stages of their development, ADA deficiency in mice has provided new insight into the profile of ADA-deficient SCID. 4. Metabolic Disturbances in ADA Deficiency Metabolic disturbances associated with ADA deficiency in humans have been monitored in accessible fluids and cellular components such as blood and urine (Hershfield and Mitchell, 2001; Morgan et al., 1987; Simmonds et al., 1978). Elevated levels of adenosine and 20 -deoxyadenosine are readily detected in these samples. ADA-deficient mice provided the opportunity to examine tissue-specific consequences of ADA deficiency in an animal model with features resembling those seen in ADA-deficient humans. There were marked increases in adenosine and 20 -deoxyadenosine concentrations in the serum of ADA-deficient mice (Blackburn et al., 1998). In addition, the ability to examine metabolic disturbances in a variety of tissues revealed a widespread accumulation of adenosine, whereas marked 20 -deoxyadenosine accumulation was predominantly limited to lymphoid tissues such as the bone marrow, thymus, and spleen (Aldrich et al., 2003). Furthermore, treatment of ADAdeficient mice by ADA enzyme therapy was able to prevent the accumulation of 20 -deoxyadenosine in the thymus and spleen in association with the prevention of lymphopenia (Fig. 3) (Blackburn et al., 2000a). These findings provide in vivo precedence for the accumulation of 20 -deoxyadenosine in lymphoid organs being associated with the lymphopenia seen in ADA deficiency. 4.1. Deoxyadenosine Metabolism: Origin and Consequences 20 -Deoxyadenosine can be cytotoxic to T cells through a number of pathways (Fig. 4). Increases in 20 -deoxyadenosine can lead to the inhibition of S-adenosylhomocysteine (SAH) hydrolase, which carries out the hydrolysis of SAH to adenosine and homocysteine (Hershfield et al., 1979; Kredich and Hershfield, 1979). Such inhibition can lead to an accumulation of SAH that in turn can inhibit key transmethylation reactions that utilize S-adenosylmethionine (SAM) as a methyl donor. Alternatively, 20 -deoxyadenosine can be phosphorylated to dATP by nucleoside and nucleotide kinases (Ullman et al., 1981), and

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Figure 3 Effects of ADA enzyme therapy on the thymus and spleen of ADA-deficient mice. (A) 20 -Deoxyadenosine levels were quantified in the thymuses and spleens of 17-day-old control mice (wild-type or heterozygous) and ADA-deficient mice, and in those of ADA-deficient mice treated with a high dose of PEG –ADA, for up to 7 weeks. Data are presented as mean 20 -deoxyadenosine levels SEM, n ¼ 3. nd, not detectable. (B) Splenocytes were collected from 17-day-old control and ADA-deficient mice, and from ADA-deficient mice treated with a high dose of PEG –ADA, and subjected to flow cytometry. Specific antibodies to cell surface markers were used to identify T cells, B cells, and NK cells. Mean total cell counts SEM were determined, n ¼ 6. Data are adapted from Blackburn et al. (1998, 2000a).

elevations in dATP can in turn lead to the inhibition of ribonucleotide reductase (Ullman et al., 1979) and in so doing deplete the cell of deoxynucleotides needed for DNA synthesis or repair. dATP has also been shown to be important in the activation of apoptotic protease-activating factor 1 (APAF-1) (Leoni et al., 1998), an important protein in the apoptotic cascade, whereas other studies suggest dATP can facilitate the loss of cytochrome c from the mitochondria (Yang and Cortopassi, 1998), which is a key step in the apoptotic pathway. In nondividing lymphocytes, dATP has been proposed to interfere

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Figure 4 Proposed mechanisms by which ADA deficiency can affect cell viability and function. (1) Loss of ADA activity by genetic mutation or pharmacologic inhibition leads to elevations in adenosine (Ado) and 20 -deoxyadenosine (dAdo) and loss of inosine (Ino) and 20 -deoxyinosine (dIno) production through this pathway. (2) Elevations in Ado can lead to aberrant adenosine receptor (AdoR) signaling that can affect cellular viability or function through mechanisms that are not well characterized. (3) Elevations in dAdo can lead to inhibition of the enzyme S-adenosylhomocysteine (SAH) hydrolase, which is responsible for the hydrolysis of SAH to homocysteine (Hcy) and Ado. SAH hydrolase inhibition can lead to elevations in SAH that can in turn inhibit cellular transmethylation reactions (X) that utilize S-adenosylmethionine (SAM) as a methyl group donor. Inhibition of transmethylation reactions can lead to activation of apoptosis or disruption in key pathways that are necessary for cell differentiation and function. These pathways are largely uncharacterized as they pertain to ADA deficiency. (4) dAdo can be phosphorylated to dATP through pathways that involve various deoxynucleoside and deoxynucleotide kinases. Two that have been shown to be important in the initial phosphorylation of dAdo to dAMP are adenosine kinase and deoxycytidine kinase. Increases in cellular dATP pools have been associated with the induction of apoptosis through pathways that include the inhibition of ribonucleotide reductase (RNR), which can decrease deoxynucleotide pools needed for DNA synthesis and repair; the activation of apoptosis by activation of APAF-1 or other mechanisms that could include activation of poly(ADA-ribose) polymerase; NAD depletion; the induction of double-strand breaks; or the release of cytochrome c from the mitochondria. Whereas specific mechanisms are still being worked out, most evidence suggests that elevations in dAdo leading to increases in dATP and the subsequent induction of apoptosis are likely responsible for the lymphoid depletion seen in ADA-deficient humans and mice.

with DNA repair by the activation of poly(ADP-ribose) polymerase, which can deplete cellular NAD pools (Seto et al., 1985). Thus, the expansion of dATP pools in lymphoid cells caused by elevations in 20 -deoxyadenosine may directly or indirectly access apoptotic pathways.

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The inhibition of SAH hydrolase and the accumulation of dATP are features commonly monitored in red blood cells of ADA-deficient patients (Coleman et al., 1978; Hershfield et al., 1979). Similarly, SAH hydrolase inhibition and dATP elevations are seen in tissues of ADA-deficient mice (Aldrich et al., 2003; Blackburn et al., 1998). SAH hydrolase is inhibited in all tissues examined in ADA-deficient mice, with the greatest degree of inhibition seen in the thymus, spleen, and bone marrow (Fig. 5A). The accumulation of dATP is also abundant, with the highest levels being found in red blood cells, thymus, and spleen

Figure 5 S-Adenosylhomocysteine hydrolase enzymatic activity and dATP levels in immune compartments of ADA-deficient mice. (A) S-Adenosylhomocysteine (SAH) hydrolase enzymatic activity was determined in red blood cells (RBC), bone marrow (BM), thymus, and spleen of 17-day-old control and ADA-deficient mice. SAH-specific activity is given as nanomoles of adenosine converted to SAH per minute per milligram protein SEM, n ¼ 4. (B) HPLC analysis was used to quantify dATP levels in RBC, BM, thymus, and spleen of 17-day-old control and ADAdeficient mice. Values are presented as mean nanomoles of dATP per milligram protein SEM, n ¼ 5. Data are adapted from Aldrich et al. (2003) and Blackburn et al. (1998, 2000a).

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(Fig. 5B). Interestingly, dATP accumulations are not seen in the bone marrow of ADA-deficient mice (Aldrich et al., 2003). Thus, as with the accumulation of 20 -deoxyadenosine, the most severe disturbances in SAH metabolism and dATP accumulation are seen in components of the immune system, suggesting these pathways may be involved in driving the immunodeficiency seen in association with ADA deficiency. 4.2. Potential Importance of S-Adenosylhomocysteine Hydrolase Inhibition and dATP Accumulation What then are the relative contributions of SAH hydrolase inhibition and dATP accumulation to the depletion of certain lymphocyte populations in ADA deficiency? Treatment of mice with a specific SAH hydrolase inhibitor caused a block in T-cell development similar to that seen in ADA deficiency (Benveniste et al., 1995). In addition, SAH accumulation has been implicated in the enhancement of apoptosis (Ratter et al., 1996). These studies suggest that elevations in 20 -deoxyadenosine and the subsequent inhibition of SAH hydrolase can lead to a block in T-cell development either by inhibiting yet unidentified transmethylation reactions that are essential for T-cell development or by contributing to apoptosis. In contrast, other studies suggest that inhibition of SAH hydrolase is not involved and implicate the accumulation of dATP and the subsequent induction of apoptosis in the block in T-cell development seen in ADA deficiency. Experiments in mouse FTOCs demonstrated that 20 -deoxyadenosine levels are generated as a by-product of apoptosis in the thymus (Thompson et al., 2000). In addition, FTOC studies demonstrated that the depletion of T cells in an ADA-deficient environment could be prevented by treatment with an adenosine kinase inhibitor that prevented the accumulation of dATP (Van De Wiele et al., 2002). In these experiments, SAH hydrolase enzymatic activity continued to be inhibited despite rescue of the T-cell phenotype, suggesting that SAH inhibition is not involved and implicating dATP accumulation in the induction of apoptosis. Additional evidence implicating dATP comes from studies in ADA-deficient mice, where it was shown that there was not a block in B-cell development in the bone marrow of ADAdeficient mice, but defects were noted in the spleen (Aldrich et al., 2003). In these studies, SAH hydrolase was significantly inhibited in both the bone marrow and spleen, whereas dATP accumulation was not found in the bone marrow, suggesting that dATP accumulation contributes to B-cell defects in ADA-deficient mice. The mechanisms by which dATP is toxic to B cells is not known, but may involve the activation of apoptotic pathways as appears to be the case in developing T cells.

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The mechanisms behind the lack of dATP accumulation in the bone marrow of ADA-deficient mice are not understood but may relate to the inability of 20 deoxyadenosine to be phosphorylated to dATP in developing B cells of the bone marrow. This addresses a key feature to understanding the sensitivity of certain cell types to 20 -deoxyadenosine. The relative levels of the enzymes that are responsible for the phosphorylation of 20 -deoxyadenosine to dATP, as well as the catabolism of dATP, may dictate the vulnerability of certain cell types to 20 -deoxyadenosine (Carson et al., 1980). Mutations in components that phosphorylate 20 -deoxyadensoine to dATP protect cells from 20 -deoxyadenosine (Hershfield et al., 1982; Ullman et al., 1978, 1981). A high incidence of apoptosis, which can generate 20 -deoxyadenosine, occurs in the thymus. This, combined with increased levels of deoxynucleoside kinases, could provide the rationale for the sensitivity of thymocytes to 20 -deoxyadenosine, in that they may have a greater capacity to accumulate dATP. This hypothesis is far from confirmed but provides attractive avenues for continued efforts to understand the mechanisms underlying the lymphoid toxicity associated with ADA deficiency. Additional studies are needed to examine the levels of deoxynucleoside kinases and deoxynucleotide catabolic enzymes, not only in various lymphoid and nonlymphoid organs but also in different lymphoid precursors during different stages of development. These efforts will help clarify why certain cells, such as developing T cells, are sensitive to elevations in 20 -deoxyadenosine, whereas others, like developing B cells in the bone marrow, are not. 4.3. Adenosine Signaling and Immune Development and Function Adenosine is a ubiquitous and potent signaling nucleoside. It influences cell function by engaging G protein-coupled adenosine receptors that access a variety of intracellular signaling pathways (Fig. 6) (Fredholm et al., 2001). Four adenosine receptors have been identified (A1, A2A, A2B, and A3 adenosine receptors), and each receptor has a unique affinity for adenosine and a distinct cellular and tissue distribution that can vary among species. A2A and A2B adenosine receptors are commonly coupled to adenylate cyclase by the stimulatory G protein (as) and serve to increase intracellular cAMP levels (Londos et al., 1980; Stiles, 1997), whereas A1 and A3 adenosine receptors are coupled to adenylate cyclase by the inhibitory G protein (ai) and hence serve to lower intracellular levels of cAMP (Londos et al., 1980; Stiles, 1997). In addition, evidence exists to suggest that adenosine receptors can couple to other effector molecules such as phospholipase C and phosphatidylinositol-3-kinase (PI3 kinase) (Olah and Stiles, 1995; Stiles, 1997). Therefore, adenosine receptor signaling can access pathways that regulate intracellular cAMP and Ca2þ and

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Figure 6 Adenosine metabolism and signaling. In response to cellular stress or damage, ATP is released into the extracellular space by mechanisms that are not fully understood. ATP is itself a potent signaling molecule via its interaction with P2 purinergic receptors. ATP is rapidly dephosphorylated by extracellular nucleotidases to form extracellular adenosine (Ado). Ecto-50 -nucleotidase (ecto50 NT) is one such enzyme that plays an important role in regulating local adenosine production for receptor signaling. Extracellular Ado can interact with adenosine receptors (AdoR) that are coupled to heterotrimeric G proteins, which in turn couple adenosine receptor activation to various effector molecules that can regulate second-messenger systems to influence cell function. Extracellular Ado can also be deaminated by ADA that can exist extracellularly, or it can be transported into cells via facilitated nucleoside transporters. Intracellular Ado is generated from the dephosphorylation of AMP by a cytosolic form of nucleotidase (cyto50 NT), or the hydrolysis of S-adenosylhomocysteine (SAH), in a reaction that also produces homocysteine (Hcy). Intracellular Ado can be transported out of the cell or it can be phosphorylated back to ATP. The first enzyme in this step is adenosine kinase (AK). Alternatively, intracellular Ado is deaminated to inosine (Ino) by ADA.

can therefore influence cellular physiology through a variety of signaling pathways. Adenosine signaling plays important roles in regulating homeostasis in a number of physiological systems including the cardiovascular (Belardinelli et al., 1989), nervous (Fredholm and Dunwiddie, 1988), renal (Churchill, 1982), and immune (Huang et al., 1997) systems. In addition, signaling through adenosine receptors can affect inflammatory processes that help regulate the severity of certain diseases or pathological conditions. Perhaps the best characterized inflammatory aspects of adenosine signaling are the antiinflammatory and protective features of A2A adenosine receptors. Studies have shown that A2A receptor engagement can promote antiinflammatory

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effects that help to regulate the severity of inflammation associated with reperfusion injury (Linden, 2001; Okusa et al., 1999) or infectious challenges (Ohta and Sitkovsky, 2001). These protective functions likely involve the engagement of adenosine receptors on neutrophils, lymphocytes, and macrophages (Cronstein and Hasko, 2004). Adenosine has also been shown to protect organs such as the heart (Lasley et al., 1990) brain (Rudolphi et al., 1992), and liver (Day et al., 2004) from ischemic damage and has been shown to exhibit chemoprotective properties (Fishman et al., 2000). In contrast to these prominent protective and antiinflammatory aspects, adenosine signaling can also promote or exacerbate tissue injury (Blackburn, 2003). Such is the case with adenosinemediated mast cell degranulation, which can influence inflammation, tissue injury, and physiological alterations seen in chronic lung diseases such as asthma (Forsythe and Ennis, 1999; Fozard and Hannon, 1999). Whether adenosine signaling serves protective or harmful functions is likely dictated by the cell type-specific expression of adenosine receptors and the effector systems they couple to, together with the concentration and duration of adenosine produced in the local environment. The ability of adenosine receptor engagement to influence so many aspects of disease suggests that they will be laudable targets for the development of selective therapeutic compounds. This is no doubt the rationale behind the efforts of the many pharmaceutical and biotechnology companies with active programs in adenosine-based therapeutics. T cells express various adenosine receptors (Van De Wiele et al., 2002) and adenosine has been shown to induce apoptosis in T lymphocytes in vitro in a receptor-dependent manner (Kizaki et al., 1990). Thus, the accumulation of adenosine in the thymus could lead to T-cell apoptosis, which could account for the depletion of developing T cells in ADA deficiency. Studies have addressed the involvement of specific adenosine receptors during T-cell development in mouse FTOCs (Van De Wiele et al., 2002). In these studies, fetal thymuses from A2A adenosine receptor- and A3 adenosine receptor-deficient mice were subjected to conditions of ADA deficiency, and T-cell development was monitored. Removal of the A2A and A3 adenosine receptors was not able to prevent the block in T-cell development seen in association with ADA inhibition. Similarly, treatment with the nonselective adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA) or the nonselective antagonist XAC (xanthine amine congener) had no effect on T-cell development in this model system. These findings provide evidence that signaling through adenosine receptors is not responsible for the block in T-cell development seen in ADA deficiency. However, it is not clear from these studies whether or not adenosine signaling may play a role in peripheral T-cell function. Studies of mature cells recovered from the spleens of ADA-deficient animals revealed that ADA deficiency is accompanied by T-cell receptor

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activation defects in T cells in vivo (Apasov and Sitkovsky, 1999; Apasov et al., 2001). Ex vivo experiments with ADA-deficient thymocytes and peripheral T cells suggested that elevated adenosine and abnormal adenosine receptor signaling may be responsible for impaired T-cell receptor signaling. These findings suggest that adenosine signaling pathways may regulate thymocyte function in the periphery. In support of this view, ex vivo ADA-deficient thymocytes demonstrated inhibited tyrosine phosphorylation of T-cell receptor–associated signaling molecules and inhibited T-cell receptor-triggered calcium increases. These studies emphasize the potential importance of adenosine signaling in impaired T-cell immunobiology. Thus, it is likely that accumulation of 20 deoxyadenosine in the thymus is responsible for the depletion of the majority of thymocytes through dATP accumulation and apoptosis, and those cells that survive this insult may encounter defects in proper T-cell receptor signaling that are mediated by widespread accumulations of adenosine. The specific adenosine receptors involved in this process are not clear; however, evidence points to the involvement of the A2A adenosine receptor (Apasov et al., 2000). More research into the specific impact of adenosine receptor signaling on T, B, and NK cell function is needed to help clarify the role of these signaling pathways in ADA deficiency, as well as normal immune homeostasis. 4.4. Broader Relevance of ADA Deficiency Efforts to understand the metabolic mechanisms underlying the lymphopenia associated with ADA deficiency have provided novel approaches for the treatment of certain lymphoid neoplasms (Beutler and Carson, 1993). Initially, it was found that 20 -deoxycoformycin, a selective suicide inhibitor of ADA, showed efficacy in the treatment of hairy cell leukemia, a malignancy with a relatively low proliferation rate. Subsequently, researchers found that the nonhydrolyzable 20 -deoxyadenosine analog, 20 -chlorodeoxyadenosine (2CdA; cladribine [Leustatin]), was highly effective in the treatment of hairy cell leukemia (Beutler and Carson, 1993). The mechanism of action of 2CdA involves its phosphorylation to 2CdATP in lymphoid cells. This feature is attributed to the relatively high levels in lymphoid cells of deoxycytidine kinase, which promotes conversion to 2CdATP, and to the relatively low levels of nucleotidase, which can break 2CdAMP down. Increases in 2CdATP are in turn thought to induce apoptosis by coactivating two key apoptosis-regulating factors: poly(ADP-ribose) polymerase and APAF-1 (Carson and Leoni, 2003; Leoni et al., 1998). The effectiveness of 2CdA in the treatment of hairy cell leukemia suggests that the activation of this pathway may provide an effective means of destroying nonproliferating malignant cells. In addition to the

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treatment of malignancies, studies have also shown the potential usefulness of 2CdA in the treatment of autoimmune disorders (Beutler and Carson, 1993). Thus, the efforts to understand the metabolic mechanisms of ADA deficiency have provided novel and far-reaching approaches for the treatment of more common disorders not associated with ADA deficiency. 5. Pulmonary Consequences of Elevated Adenosine In addition to immunodeficiency, ADA-deficient individuals often develop pulmonary insufficiencies (Hirschhorn, 1999; Stephan et al., 1993). It is generally assumed that these pulmonary insufficiencies are associated with opportunistic infections inherent to the compromised immune system of these patients. However, the frequent appearance of pulmonary insufficiencies in ADA-deficient patients suggests that the metabolic disturbances associated with ADA deficiency may have a direct impact on lung development, repair, or function. ADA-deficient mice develop many phenotypes other than immunodeficiency, of which the most prominent is pulmonary insufficiency (Blackburn et al., 1998). This has provided the opportunity to examine more closely the impact of the ADA-associated metabolic disturbances on the lung. ADA-deficient mice develop outward signs of respiratory distress during the second week of life. This respiratory distress is progressive and is believed to contribute to the death of these animals by 3 weeks of age (Blackburn et al., 2000b). Analysis of the lungs revealed that the pulmonary insufficiency is not due to opportunistic infections that may result from the compromised immune system in these animals. Instead, it was found that a specific pattern of lung inflammation and damage is induced by the accumulation of adenosine in the lungs (Blackburn et al., 2000b). Accumulation of adenosine has long been noted in the lungs of individuals suffering from chronic lung disease (Driver et al., 1993); however, the contribution of adenosine to the regulation of lung inflammation and the mechanisms involved are not clear. The serendipitous finding of adenosine-dependent lung inflammation and damage in ADA-deficient mice has provided an opportunity to examine specific aspects of adenosine signaling and lung inflammation in the context of the whole animal. 5.1. Adenosine Signaling in Asthma and Chronic Obstructive Pulmonary Disease Asthma and chronic obstructive pulmonary disease (COPD) are lung diseases that afflict millions of individuals and result in billions of dollars in annual health care costs. Persistent pulmonary inflammation and airway remodeling

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responses are prominent features of these disorders (Elias et al., 1999). The inflammation associated with asthma and COPD is driven in part by cytokine and chemokine signaling networks, whereas the chronic remodeling and destruction of the airways are associated with the activation of growth factor signaling pathways and disruption of protease/antiprotease balances (Bradding et al., 1997; O’Byrne and Postma, 1999). In contrast to most injury and repair responses, the inflammation seen in these disorders is chronic and may last throughout the life of the affected individual. Although signaling pathways associated with the genesis of inflammation and the control of tissue remodeling have been described, little is known about signaling pathways that serve to regulate the chronic nature of these diseases. Adenosine is rapidly generated as a net result of ATP catabolism that occurs in situations of cellular stress or damage (Fig. 6). Therefore, it is not surprising that pathologic conditions leading to cellular stress and damage, such as the inflammation and tissue damage seen in chronic lung diseases such as asthma, are associated with increases in adenosine levels (Driver et al., 1993; Huszar et al., 2002). The production of adenosine in the lungs of patients with asthma suggests that it may play a role in regulating aspects of the disease. There is substantial clinical and scientific evidence to support this hypothesis. Adenosine can directly influence cellular and physiological processes in the lungs of patients with asthma or COPD (Fozard and Hannon, 1999; Jacobson and Bai, 1997). Exogenous adenosine can elicit acute bronchoconstriction in patients with asthma (Cushley et al., 1983) or COPD (Oosterhoff et al., 1993), while having no effect on normal individuals, suggesting a fundamental difference regarding adenosine signaling in these patients. In addition, adenosine signaling can influence the activity of a number of cell types that play a central role in chronic lung disease including mast cells (Marquardt et al., 1978), eosinophils (Walker et al., 1997), macrophages (Hasko et al., 1996), neuronal cells (Bai et al., 1989), epithelial cells (Johnson and McNee, 1985), and smooth muscle cells (Ali et al., 1994). Adenosine receptor levels are elevated in the lungs of patients with asthma and COPD, which further suggests that increased adenosine signaling may be an important feature of these diseases (Walker et al., 1997). Despite these lines of evidence, the significance of adenosine accumulation in the lung is still not clear nor are the underlying cellular and molecular mechanisms. Part of the challenge of addressing these issues is to obtain adequate animal models with which to address the impact of elevations in endogenous lung adenosine. In this regard, the development of ADA-deficient mice and mice deficient in the various adenosine receptors has proved useful in uncovering specific cellular pathways in the lung that are activated in response to chronic elevations in adenosine.

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5.2. Pulmonary Inflammation in ADA-Deficient Mice Adenosine concentrations in the lungs of ADA-deficient mice are estimated to reach 100 mM (Blackburn, 2003), which is comparable to those measured in fluid collected from the lungs of patients with asthma (Driver et al., 1993). Interestingly, 20 -deoxyadenosine levels are not markedly elevated in the lungs of ADA-deficient mice (Blackburn et al., 2000b), suggesting any local influences of metabolic disturbances are due to the effects of adenosine. In association with elevations in lung adenosine, ADA-deficient mice develop severe pulmonary inflammation and airway remodeling (Blackburn et al., 2000b). Pulmonary features noted include mast cell degranulation (Zhong et al., 2001), increases in activated alveolar macrophages and eosinophils, mucus metaplasia, fibrosis, airway enlargement (Blackburn et al., 2000b), and airway hyperreactivity (Chunn et al., 2001). In addition, there are marked lungspecific elevations of key regulatory cytokines, chemokines, and proteases in ADA-deficient mice (Banerjee et al., 2002). Many of these pulmonary features are also seen in patients with asthma and/or COPD, suggesting that these animals may be useful for examining the role of specific aspects of adenosine signaling in these disorders. Furthermore, the finding that merely elevating adenosine levels can access pathways that lead to the promotion of lung inflammation and damage suggests that adenosine signaling is playing an active role in the exacerbation of chronic lung disease. 5.3. Enzyme Therapy Reversal of Many of the Pulmonary Abnormalities Associated with ADA Deficiency ADA enzyme therapy was used to determine which of the phenotypes seen in the lungs of ADA-deficient mice were dependent on elevations in lung adenosine. As mentioned earlier, bovine ADA that is covalently linked to polyethylene glycol (PEG–ADA) provides an effective means of lowering systemic and tissue levels of ADA substrates in both ADA-deficient humans (Hershfield et al., 1993) and mice (Blackburn et al., 2000a). Injecting ADA-deficient mice with PEG–ADA at a stage when lung disease is established rapidly lowers lung adenosine levels and reverses most aspects of lung inflammation and airway remodeling (Fig. 7). Most notable is the reduction of lung eosinophilia and mucus production in airway epithelium (Blackburn et al., 2000b). ADA enzyme therapy is also able to prevent mast cell degranulation in the lungs of ADA-deficient mice (Zhong et al., 2001), as well as reverse airway hyperreactivity (Chunn et al., 2001). Moreover, lowering lung adenosine levels by ADA enzyme therapy is able to promote the survival of these mice (Blackburn et al., 2000b). ADA-deficient mice treated by ADA enzyme therapy recover rapidly

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Figure 7 Effects of ADA enzyme therapy on pulmonary adenosine levels and pulmonary pathologies in ADA-deficient mice. (A) Adenosine and 20 -deoxyadenosine levels were quantified in the lungs of 18-day-old control (wild-type or heterozygous) and ADA-deficient mice, and in ADAdeficient mice 72 h after ADA enzyme therapy. Values are presented as mean nanomoles per milligram protein SEM, n ¼ 4. nd, not detectable. (B) Eosinophil numbers in the bronchial alveolar lavage fluid of 18-day-old control and ADA-deficient mice, and in ADA-deficient mice 72 h after ADA enzyme therapy, were quantified after staining cytospun cells with Diff-Quik. Data are presented as total cells SEM, n ¼ 5. (C) The degree of mucus production in the bronchial airways was determined in 18-day-old control and ADA-deficient mice and in ADA-deficient mice 72 h after ADA enzyme therapy. Mucus production was determined by quantifying the degree of mucus staining in the airways. Data are adapted from Blackburn et al. (2000b) and are presented as mean mucus score SEM, n ¼ 5.

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from features of respiratory distress and remain normal as long as they are maintained on treatment. Removal of treatment results in a rapid rise in adenosine levels and renewed pulmonary complications. These findings demonstrate a strong correlation between elevations in lung adenosine concentrations in ADA-deficient mice and the activation of cell populations such as mast cells, eosinophils, macrophages, and airway epithelial cells, all of which play central roles in chronic lung diseases such as asthma and COPD. Pulmonary features in ADA-deficient mice, such as mast cell degranulation, eosinophilia, mucus metaplasia, and airway hyperresponsiveness, resemble pulmonary phenotypes seen in allergic asthma. However, there is no indication of allergen immunization or challenge in ADA-deficient mice. This suggests that in this model, adenosine may directly access pathways downstream of adaptive immune responses typically seen in allergic lung disease. Evidence to support this comes from studies in ADA-deficient mice that were mated onto a Rag1-deficient background. In these studies, it was shown that the pulmonary phenotypes seen in ADA-deficient mice persist in the absence of T and B lymphocytes (Chan et al., 2003), suggesting that adenosine is directing its effects in the lung in a lymphocyte-independent manner. These observations raise the possibility that adenosine is serving to modulate the aspect of innate immune responses that promote the development of pulmonary pathologies. Examining the mechanisms through which adenosine directs such responses would greatly increase our knowledge of adenosine actions in normal and diseased lungs. 5.4. Signaling Through Adenosine Receptors Most of the physiological effects of adenosine are attributed to signaling through adenosine receptors (Fredholm et al., 2001). Examination of adenosine receptor transcript levels in whole lungs of ADA-deficient mice by realtime reverse transcription-polymerase chain reaction (RT-PCR) shows that transcripts for the A1, A2B, and A3 adenosine receptors are significantly elevated, whereas transcript levels for the A2A adenosine receptor are not altered (Chunn et al., 2001). Increased expression of adenosine receptors in lung tissue, or the influx of inflammatory cells expressing these receptors, likely represents an increased capacity for adenosine signaling in the adenosine-rich environment of ADA-deficient mice. Evidence to support this has come from studies examining the cellular localization and functionality of adenosine receptors in the lungs of ADA-deficient mice.

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5.4.1. Mast Cells One approach to begin to address the functionality of adenosine receptors in ADA-deficient mice is to examine the effects of adenosine receptor antagonism in these environments of elevated endogenous adenosine. This approach has revealed an important role for adenosine signaling in mast cell degranulation (Zhong et al., 2001, 2003). Treatment of ADA-deficient mice with the nonselective adenosine receptor antagonists theophylline and MRS 1220 is able to attenuate mast cell degranulation (Zhong et al., 2001) and airway hyperresponsiveness (Chunn et al., 2001) caused by elevations in endogenous adenosine. Further analysis using the adenosine receptor antagonist MRS 1523, which has relative selectivity for the rodent A3 adenosine receptor (Li and Krilis, 1999), suggests that the A3 adenosine receptor is responsible for adenosine-mediated mast cell degranulation in the lungs of ADA-deficient mice (Zhong et al., 2003). The specific adenosine receptors responsible for adenosine-mediated airway hyperreactivity in ADA-deficient mice have not been determined. The ability of A3 receptor antagonism to prevent adenosinemediated mast cell degranulation in ADA-deficient mice is consistent with studies in A3 receptor-deficient mice (Salvatore et al., 2000; Tilley et al., 2000; Zhong et al., 2003) and other rodent systems (Hannon et al., 1995; Jin et al., 1997; Ramkumar et al., 1993; Reeves et al., 1997) that demonstrate a role for the A3 receptor in mast cell degranulation. Whether A3 receptor-mediated mast cell degranulation is the major route of adenosine-mediated mast cell degranulation in humans is not clear. Studies have indicated that the A2B adenosine receptor is responsible for mast cell degranulation in other species including humans (Auchampach et al., 1997; Feoktistov et al., 2001). Clarifying these species-specific differences will be an important step toward the design of adenosine receptor antagonists for regulating adenosine-mediated mast cell degranulation in asthma. 5.4.2. Eosinophils In addition to mast cell degranulation, pharmacological and genetic studies have provided evidence that the A3 adenosine receptor plays a role in regulating eosinophil migration and mucus production in the lungs of ADA-deficient mice (Young et al., 2004). The A3 receptor is expressed in eosinophils and mucus-producing cells in the airways of ADA-deficient mice. Treatment of ADA-deficient mice with MRS 1523, a selective A3 adenosine receptor antagonist, prevents airway eosinophilia and mucus production. Similar findings are seen in the lungs of ADA/A3 double-knockout mice (Young et al., 2004). Interestingly, these studies demonstrate that although eosinophils are decreased in the airways of ADA-deficient mice after antagonism or removal of

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the A3 receptor, elevations in circulating and lung interstitial eosinophils persist, suggesting that signaling through the A3 receptor is needed for the migration of eosinophils into the airways. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus production in an environment of elevated adenosine. 5.4.3. Overview of Receptor Signaling in the Lungs of ADA-Deficient Mice The above-described findings demonstrate the utility of using the ADA-deficient mouse model to examine the specific roles of the A3 receptor in adenosine-dependent phenotypes. Continued efforts using pharmacological and genetic approaches to examine the function of other adenosine receptors in this model will provide useful information about the overall picture of adenosine signaling and chronic lung disease. Doing so will help guide the process of deciding which adenosine receptors, cell types, and signaling pathways to focus on for eventual development of adenosine-based therapeutics. Furthermore, the efficiency of ADA enzyme therapy in reversing aspects of lung inflammation and damage in ADA-deficient mice suggests that there may be reason to consider the use of exogenous ADA to lower the elevated adenosine levels in the lungs of patients with asthma or COPD. Research designed to extend the observations seen in ADA-deficient mice to other established models of asthma and COPD will be needed before such hypotheses can be tested in humans. 5.5. Adenosine in Other Models of Lung Disease There is substantial evidence that adenosine signaling plays a role in animal models of asthma and COPD other than the ADA-deficient model. In a number of well-controlled studies using allergen-sensitized and challenged Brown Norway rats, Fozard and colleagues examined the mechanisms of adenosine-induced bronchoconstriction (Ellis et al., 2003; Hannon et al., 2001; Tigani et al., 2002). Their findings suggest that the bronchonstrictive effects of adenosine are associated with mast cell degranulation and are adenosine receptor dependent; however, the specific adenosine receptor(s) involved have not been determined. Interestingly, treatment of sensitized and challenged rats with ADA enzyme therapy, or an ADA inhibitor, did not prevent or enhance, respectively, bronchoconstriction or inflammation (Ellis et al., 2003), suggesting that endogenous adenosine accumulation has little effect on allergen-induced responses in their model. The reason for this is not clear but may be related to the absolute levels of adenosine that accumulate in the lung. The degree of lung inflammation and damage in this model may not

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be severe enough to allow for abundant and persistent adenosine generation locally in the lung. In addition to the rat, studies in an allergic mouse model have demonstrated that adenosine can promote bronchoconstriction (Fan and Mustafa, 2002). These findings are consistent with those in the ADA-deficient mouse; however, the specific adenosine receptors involved in adenosinemediated airway hyperresponsiveness are not known. This physiological parameter might be directly associated with mast cell degranulation, as is seen in human asthma patients (Cushley et al., 1984) and rat models (Hannon et al., 2001), or it may represent effects on airway nerves or direct effects on airway smooth muscle as has been demonstrated in rabbit models of asthma (Ali et al., 1994; Nyce and Metzger, 1997). Perhaps the most convincing evidence that adenosine is playing a role in the exacerbation of lung inflammation and damage has come from studies in mice overexpressing the helper T cell type 2 (Th2) cytokine IL-13 in the lungs (Blackburn et al., 2003). IL-13–overexpressing mice represent a wellestablished model of chronic lung inflammation and damage (Zhu et al., 1999, 2002). Investigation of the adenosine signaling pathway in these mice demonstrated that with the progression of lung disease, there is a progressive increase in lung adenosine concentrations (Fig. 8A). Interestingly, the levels of ADA transcripts and activity decrease selectively in the lungs of IL-13– overexpressing mice, suggesting there is an orchestrated trend favoring adenosine accumulation in the lungs of these mice. In addition to alterations in adenosine metabolism, there is augmented expression of the A1, A2B, and A3 adenosine receptors, but not the A2A adenosine receptors, in the lungs of IL-13–overexpressing mice, a pattern that is also seen in ADA-deficient mice (Chunn et al., 2001). Remarkably, ADA enzyme therapy diminished the IL-13–induced increases in lung adenosine in association with preventing IL13–induced inflammation (Fig. 8B), chemokine elaboration, tissue fibrosis, and alveolar destruction (Blackburn et al., 2003). These findings suggest that adenosine signaling is playing a role in IL-13–induced lung injury and adds support to the notion that ADA enzyme therapy may provide protection from the detrimental effects of adenosine in chronic lung disease in general. The inflammation and damage seen in IL-13–overexpressing mice is remarkably similar to that seen in the lungs of ADA-deficient mice (Blackburn et al., 2000b). Investigation into the link between ADA-deficient mice and IL-13– overexpressing mice demonstrated that IL-13 is strongly induced in an adenosine receptor-dependent manner in the lungs of ADA-deficient mice. These findings demonstrate that IL-13 and adenosine stimulate one another in an amplification pathway that may contribute to the nature, severity, progression, and/or chronicity of IL-13– and/or Th2-mediated disorders. More research into the specific mechanisms by which adenosine influences the

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Figure 8 ADA enzyme therapy lowers lung adenosine levels and pulmonary inflammation in IL13–overexpressing mice. (A) HPLC was used to quantify lung adenosine levels in 3-month-old control (wild-type) and IL-13–overexpressing mice, and 3-month-old IL-13–overexpressing mice after 1 month of ADA enzyme therapy. Data are presented as mean nanomoles of adenosine per milligram protein SEM, n ¼ 4. (B) Total cells were counted in bronchial alveolar lavage fluid collected from the lungs of 3-month-old control and IL-13–overexpressing mice, and 3-monthold IL-13–overexpressing mice after 1 month of ADA enzyme therapy. Data are adapted from Blackburn et al. (2003) and are presented as total cells SEM, n ¼ 4. Tg, transgenic.

production of IL-13 is needed to further validate the role of this amplification loop in the exacerbation of chronic lung disease (Fig. 9). 5.6. Model for Adenosine Amplification of Lung Disease Most cells in the body generate adenosine and possess adenosine receptors. A challenge in understanding this ubiquitous signaling system is to determine the factors that govern its regulation and dictate whether it serves to maintain

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Figure 9 Model for adenosine-mediated amplification of lung inflammation and damage. A large variety of insults, including allergy, infection, injury, and environmental insult, can lead to lung inflammation. Inflammation in the lung can then set up a cascade of events that lead to airway changes including mucus metaplasia, fibrosis, other aspects of airway remodeling, and airway hyperresponsiveness. It is these attributes that ultimately lead to the loss of lung function. The hypoxia and cellular stress associated with lung inflammation and damage lead to the generation of high concentrations of adenosine through pathways that likely involve enhanced ATP release and ecto-50 -nucleotidase (CD73) activity. Elevations in adenosine may be enhanced by the local downregulation of ADA in the lung. Elevations in adenosine may be associated with heightened or aberrant adenosine receptor signaling. Whereas adenosine signaling may be serving some antiinflammatory roles in the lung, most of the accumulated data suggest that increased adenosine signaling may serve to amplify existing lung inflammation and airway damage by directly influencing proinflammatory signaling molecules and cell types.

homeostasis, protect tissues from injury, or trigger the promotion of inflammation or tissue damage. Experiments in ADA-deficient mice demonstrate that chronically elevated adenosine can lead to lung inflammation and damage. Furthermore, lowering adenosine levels by ADA enzyme therapy can prevent or reverse certain aspects of lung inflammation and damage. These findings suggest that increases in adenosine levels are detrimental in ADA deficiency; however, studies are needed to determine to what degree adenosine is elevated in patients with COPD and other chronic lung diseases and whether or not these elevations activate antiinflammatory or proinflammatory pathways. It is

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possible that adenosine may serve both pro- and antiinflammatory roles that are dictated by the levels of adenosine found in the lung (Cronstein and Hasko, 2004; Fig. 9). Lung inflammation and tissue damage likely results in a hypoxic environment conducive to the generation of adenosine. At lower levels of adenosine accumulation, engagement of high-affinity receptors, such as the A1 and A2A receptor, might play important roles in controlling the degree of inflammation or repair processes in the lung. As the damage becomes more severe and local adenosine levels increase, low-affinity receptors, such as the A2B adenosine receptor, might serve to access signaling pathways that lead to the exacerbation of the lung inflammation and damage. In this manner, chronic elevations in lung adenosine levels may serve important roles in promoting the chronic nature of lung diseases such as asthma and COPD (Fig. 9). In this sense, the regulation of mediators such as IL-13 by adenosine may be important. Examination of the specific adenosine receptors involved in these processes must be elucidated to help define the mechanisms involved. Doing so will help identify targets for the development of potential adenosine-based therapeutics for the treatment of chronic lung disease. 6. Additional Physiological Consequences of Elevated Adenosine ADA deficiency in humans is most often associated with SCID. Indeed, it is the immunodeficiency and consequences thereof that lead to the demise of ADA-deficient humans who are not diagnosed and treated. However, nonimmune phenotypes have been noted, including neurological (Hirschhorn et al., 1980), renal (Ratech et al., 1985), hepatic (Bollinger et al., 1996), and bony (Cederbaum et al., 1976) abnormalities and pulmonary insufficiencies (Stephan et al., 1993). Studies in ADA-deficient mice suggest that these abnormalities may not be secondary to the immunodeficiency seen but may be a direct consequence of the metabolic disturbances associated with ADA deficiency. As discussed earlier, the pulmonary insufficiencies seen in ADAdeficient mice are mediated by the accumulations of adenosine in the lungs of these animals, and they have served as useful models for examining the role of adenosine signaling in lung inflammation and damage (Blackburn, 2003). Whether the pulmonary insufficiencies in ADA-deficient humans are associated with aberrant adenosine signaling is not known; however, this feature has not been examined closely. It has been noted that ADA-deficient patients may have a higher incidence of asthma and eosinophilia, and elevations in IgE have been noted (Hirschhorn, 1999; Kawamura et al., 1998; Levy et al., 1988). More research is needed to determine whether these are primary consequences of purine metabolic disturbances in the lungs of ADA-deficient individuals or whether this represents a species- and model-specific outcome.

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ADA-deficient mice have served as useful biological screens for adenosine signaling phenotypes that are associated with ADA deficiency. Ongoing research will clarify the pathways involved and their relevance to human disease. To date, ADA-deficient mice have been used to describe the contribution of adenosine signaling in two physiological processes that have not previously been appreciated in ADA deficiency: defects in alveolar development in the lung (Banerjee et al., 2004) and the reduction in brain matter and secondary ventriculomegaly (Turner et al., 2003). Alveogenesis and microvascular maturation are the final stages in lung development in mammals. Alveogenesis in the mouse begins on postnatal day 5, when the process of secondary septation plays a pivotal role in the expansion of the alveolar sacs and microvascular maturation (Ten HaveOpbroek, 1991). ADA-deficient mice exhibit abnormalities in alveogenesis in association with elevated lung adenosine levels (Banerjee et al., 2004). Largescale gene expression analysis of ADA-deficient lungs, using microarrays, revealed novel relationships between gene expression patterns and elevated lung adenosine during the stages of alveolar maturation. Genes regulating apoptosis, proliferation, and vascular development are altered, and decreased cell proliferation in association with increased alveolar type II cell apoptosis is shown to contribute to abnormal secondary septation in these mice. ADA enzyme therapy allowed for normal patterns of apoptosis, proliferation, and alveolar development in association with prevention of adenosine elevations. These findings were correlated with the presence of adenosine receptors in the developing lung, suggesting the involvement of receptor signaling. These studies provide evidence that elevated lung adenosine can lead to abnormal alveogenesis by disrupting patterns of cell proliferation and apoptosis. The presence of alveolar defects has not been documented in ADA-deficient humans; however, such defects may contribute to the idiopathic pulmonary insufficiencies that are occasionally seen. ADA-deficient mice have also proved useful in the analysis of neurological defects associated with elevations in brain adenosine levels. Periventricular leukomalacia is a neurological disorder characterized by a reduction in brain matter and secondary ventriculomegaly and is a major cause of developmental delay and cerebral palsy in premature infants (Melhem et al., 2000; Volpe, 2001). In animal models, features of periventricular leukomalacia can be induced by hypoxia and by activation of A1 adenosine receptors (Latini and Pedata, 2001; Turner et al., 2002). This implies that elevations in brain adenosine levels after hypoxia can mediate the development of periventricular leukomalacia through A1 adenosine receptor engagement early in life. However, no direct evidence of endogenous adenosine elevations had been

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demonstrated to support this. Studies utilized ADA-deficient mice to test the hypothesis that endogenous elevations of adenosine could directly cause ventriculomegaly. Analysis of brain pathology in ADA-deficient mice on postnatal day 14 demonstrated that there is a decrease in brain matter, and secondary ventriculomegaly is evident in these mice (Turner et al., 2003). Furthermore, this phenotype is associated with marked elevations in brain adenosine levels. These findings add to the emerging hypothesis that adenosine, via signaling through the A1 adenosine receptor, can mediate ventriculomegaly during early postnatal development. These studies suggest that the pharmacological blockade of the A1 adenosine receptor may have clinical utility in the treatment of adenosine-induced brain injury. Neurological abnormalities have been documented in ADA-deficient individuals (Hirschhorn et al., 1980); however, they are relatively uncommon, and the diagnosis of ventriculomegaly or cerebral palsy has not been made. Therefore, the pronounced neurological defects seen in ADA-deficient mice may be unique to this model because of species differences or may be due to the high levels of adenosine allowed to develop in these animals. Most ADAdeficient individuals are diagnosed early in life and are managed on treatment protocols such as ADA enzyme replacement therapy. Therefore, it is less likely that phenotypes such as ventriculomegaly are allowed to develop in ADAdeficient patients. The same may be true for other phenotypes noticed in ADA-deficient mice such as bony and renal abnormalities (Blackburn et al., 1998). These abnormalities have been documented in some of the earliest identified ADA-deficient patients. However, their prevalence appears to be decreasing, which may be indicative of improved diagnosis and treatment. The mechanisms underlying the bony and renal defects seen in ADA-deficient mice are still being investigated. Efforts to understand the association of these defects with the metabolic disturbances seen will help identify the role of 20 -deoxyadenosine cytotoxicity and/or adenosine signaling in these organ systems. 7. Concluding Remarks Human genetic disorders can serve as naturally occurring genetic screens that reveal unexpected genotype–phenotype relationships. In this way, rare genetic disorders can provide significant insight and medical impact that extend well beyond the number of individuals affected with the specific genetic disorder. Such is the case of ADA deficiency (Fig. 10). The relationship between ADA activity and lymphocyte development prompted the use of ADA inhibitors as immune suppressants and antileukemic agents (Gan et al., 1987; O’Dwyer

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Figure 10 Information acquired from the study of ADA deficiency. Observations in ADAdeficient humans, ex vivo systems, and ADA-deficient mice have provided useful information about the mechanisms underlying the immunodeficiency seen in this disorder, as well as novel information about pathways that impact a broad range of physiological systems. The immunodeficiency seen in association with ADA deficiency is likely due to the accumulation of 20 -deoxyadenosine in primary and secondary immune organs, which, through increases in cellular dATP pools, leads to apoptosis. Efforts to treat the immunodeficiency have led to the advancement of ADA enzyme replacement therapy and gene therapy protocols. In addition, examination of the metabolic basis of the immune deficiency has led to the development of novel chemotherapeutic approaches for the treatment of certain leukemias. Work stemming predominantly from observations in ADA-deficient mice has led to novel hypotheses concerning the role of adenosine signaling in chronic lung disease, development, and neurological function. Further examinations of ADAdeficient mice will yield important information about the role of chronic adenosine elevations in these and other disorders.

et al., 1988). In addition, efforts to understand the metabolic consequences of ADA deficiency led to the development and use of nucleoside analogs and other antimetabolites as pharmacologic agents to treat leukemias and autoimmune disease (Beutler and Carson, 1993). These latter developments came from the realization that 20 -deoxyadenosine is cytotoxic to developing T cells, which is likely the major underlying feature that accounts for the immunodeficiency seen in ADA-deficient individuals. Thus, efforts to understand ADA deficiency have provided major inroads to the treatment of deadly diseases, particularly cancer.

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Studies conducted in ADA-deficient mice have provided insight into the role of adenosine signaling in development, physiology, and disease (Blackburn, 2003). With the realization that mammalian cells contain four types of adenosine receptors came the interest in deciphering the role of these receptors in mammalian cell signaling. Some investigators have approached these issues pharmacologically, using receptor-specific agonists and antagonists (Day et al., 2004; Harada et al., 2000). More recently, investigators have used gene-targeting strategies to create mice lacking specific adenosine receptors (Chen et al., 1999; Salvatore et al., 2000; Sun et al., 2001). The generation of ADA-deficient mice (Blackburn et al., 1998) has provided a complementary approach in which the biological consequences of chronically elevating the ligand adenosine can be observed in vivo. In doing so, chronic elevations in lung adenosine have been shown to be important in the activation of pathways associated with the exacerbation of chronic lung disease (Blackburn, 2003). Combining pharmacological approaches and genetic approaches to assess the function of the various adenosine receptors on different aspects of lung disease in ADA-deficient mice has begun to identify novel pathways that may develop into therapeutic targets for the treatment of chronic lung diseases such as asthma and COPD. In addition to pulmonary phenotypes, developmental (Banerjee et al., 2004) and neurological (Turner et al., 2003) abnormalities in ADA-deficient mice have been attributed to abnormal adenosine signaling. Thus, ADA-deficient mice have proved useful for the identification of cellular events and physiological processes that are sensitive to elevations in endogenous adenosine. ADA deficiency has been the testing ground for the development of novel therapies with considerable potential for many areas of medicine. For example, ADA deficiency was among the first of the immunodeficiencies for which bone marrow transplantation, enzyme replacement therapy, and gene therapy were attempted (Hershfield and Mitchell, 2001). Bone marrow transplantation and enzyme therapy have proved successful in treating ADA deficiency and other genetic disorders (Buckley et al., 1997). Gene therapy attempts were useful in initial assessments of the safety of this procedure and provided evidence of the feasibility of long-term genetic modification of target cells, using ex vivo approaches (Blaese et al., 1995; Bordignon et al., 1995; Kohn et al., 1998). Inadequately low levels of expression remain a problem in most cases, as does the complication of assessing the benefit of ADA gene therapy with persistent PEG–ADA treatment. However, these studies did lay the groundwork for the more successful use of gene therapy to treat X-linked SCID (Cavazzana-Calvo et al., 2000). In this regard, ADA-deficient mice will serve as useful tools for advancing aspects of ADA gene therapy that will benefit the broader development of this novel medical therapy.

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Acknowledgments We thank Dr. Claudia Andreu-Vieyra and Rebecca Corrigan for helpful comments on this manuscript. This work was supported by National Institutes of Health Grants AI43572 and HL70953 (to M.R.B.) and DK46207 and HD34130 (to R.E.K.). In addition, M.R.B. is supported by a Junior Investigator Grant from the Sandler Program for Asthma Research.

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Mechanism and Control of V(D)J Recombination versus Class Switch Recombination: Similarities and Differences Darryll D. Dudley,1 Jayanta Chaudhuri, Craig H. Bassing, and Frederick W. Alt Howard Hughes Medical Institute, The Children’s Hospital Boston, CBR Institute for Biomedical Research, and Harvard Medical School, Boston, Massachusetts 02115 Abstract .................................................................................................. Overview: V(D)J and Class Switch Recombination ............................................... Antigen Receptor Gene Rearrangement............................ ................................ Regulation of V(D)J Recombination ................................ ................................ Class Switch Recombination Employs Distinct Mechanisms for V(D)J Recombination ............................................................................. 5. CSR-Related Diseases ................................................................................. 6. Concluding Remarks ................................................................................... References ............................................................................................... 1. 2. 3. 4.

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Abstract V(D)J recombination is the process by which the variable region exons encoding the antigen recognition sites of receptors expressed on B and T lymphocytes are generated during early development via somatic assembly of component gene segments. In response to antigen, somatic hypermutation (SHM) and class switch recombination (CSR) induce further modifications of immunoglobulin genes in B cells. CSR changes the IgH constant region for an alternate set that confers distinct antibody effector functions. SHM introduces mutations, at a high rate, into variable region exons, ultimately allowing affinity maturation. All of these genomic alteration processes require tight regulatory control mechanisms, both to ensure development of a normal immune system and to prevent potentially oncogenic processes, such as translocations, caused by errors in the recombination/mutation processes. In this regard, transcription of substrate sequences plays a significant role in target specificity, and transcription is mechanistically coupled to CSR and SHM. However, there are many mechanistic differences in these reactions. V(D)J recombination proceeds via precise DNA cleavage initiated by the RAG proteins at short conserved signal sequences, whereas CSR and SHM are initiated over large target regions via activationinduced cytidine deaminase (AID)–mediated DNA deamination of transcribed 1 Present address: Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104.

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target DNA. Yet, new evidence suggests that AID cofactors may help provide an additional layer of specificity for both SHM and CSR. Whereas repair of RAG-induced double-strand breaks (DSBs) involves the general nonhomologous end-joining DNA repair pathway, and CSR also depends on at least some of these factors, CSR requires induction of certain general DSB response factors, whereas V(D)J recombination does not. In this review, we compare and contrast V(D)J recombination and CSR, with particular emphasis on the role of the initiating enzymes and DNA repair proteins in these processes. 1. Overview: V(D)J and Class Switch Recombination The lymphoid arm of the vertebrate immune system has evolved to respond and protect against a diverse set of antigens constantly encountered by the host. Lymphocytes generate a nearly limitless diversity of antigen receptors via processes that direct somatic rearrangements and mutations into the germline DNA sequences of antigen receptor genes. Variable region exons of antigen receptors expressed on B and T lymphocytes are generated via somatic assembly of component variable (V), diversity (D), and joining (J) gene segments in a process called V(D)J recombination. As the usage of particular gene segments for a given locus is to a certain extent stochastic, this combinatorial joining process generates a highly diverse set of antigen receptors from a limited number of germline gene segments. B cells are capable of undergoing two additional forms of genetic alteration that enhance the ability of an antigen-specific B cell to recognize and respond to its cognate antigen. Somatic hypermutation (SHM) introduces a high rate of mutations into the germline DNA sequences of assembled immunoglobulin heavy (IgH) and light (IgL) chain variable region exons and allows the selection of B cells with receptors that have increased affinity for a given antigen. IgH class switch recombination (CSR) adjoins a rearranged variable region exon initially associated with the Igm constant region (Cm) exons to one of several downstream sets of CH exons (referred to as CH genes) through the deletion of intervening germline DNA sequences. This allows expression of an antibody with the same antigen-binding specificity but with altered CH effector function. Initiation of the V(D)J recombination reaction requires the products of recombination activating genes 1 and 2 (RAGs) (Oettinger et al., 1990; Schatz et al., 1989), which are expressed only in developing lymphocytes (Chun et al., 1991; Mombaerts et al., 1992). RAGs were identified by their ability to confer recombinational activity to a fibroblast cell line harboring a drug-selectable recombination substrate (Oettinger et al., 1990; Schatz et al., 1989). Deficiency in either RAG-1 or RAG-2 leads to a complete block in lymphocyte development at progenitor stages, the first stages at which V(D)J

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recombination normally takes place (Mombaerts et al., 1992; Shinkai et al., 1992). RAGs introduce a DNA double-strand break (DSB) precisely between a variable region gene-coding segment and an associated recombination signal (RS) sequence (reviewed in Fugmann et al., 2000a; Jung and Alt, 2004). Each RS is made up of conserved heptamer and nonamer sequences and an intervening spacer sequence that is either 12 or 23 bp in length. RAGs will mediate recombination only between antigen receptor gene segments that have RS spacer sequences of 12 and 23 bp, referred to as the 12/23 rule. RAG-induced DNA breaks are repaired by ubiquitously expressed nonhomologous endjoining (NHEJ) proteins, forming precise signal end joints (SJs) and imprecise coding end joints (CJs) (reviewed in Bassing et al., 2002b; Jung and Alt, 2004). Lymphoid-specific expression of RAGs limits V(D)J recombination to B and T lymphocytes (reviewed in Nagaoka et al., 2000). However, to ensure that T cell receptor (TCR) genes are rearranged to completion only in T cells and that immunoglobulin genes are rearranged to completion only in B cells, the regulation of V(D)J recombination also involves the lineage-specific accessibility of gene segments (Yancopoulos and Alt, 1985). Such regulated accessibility of antigen receptor gene segments directs developmental stage-specific rearrangement. In developing B cells IgH genes are assembled before IgL genes, whereas in developing ab T cells TCRb genes are assembled before TCRa genes (reviewed in Willerford et al., 1996). Regulated accessibility also likely contributes to the ordered rearrangement of IgH and TCRb genes, wherein D-to-J rearrangements proceed to completion before the onset of V-to-DJ rearrangements (Alt et al., 1984; Born et al., 1985; Sleckman et al., 2000). Recombinational accessibility correlates with transcriptional activity of a given antigen receptor locus, as eliminating transcriptional enhancers often ablates rearrangement of associated gene segments (reviewed in Bassing et al., 2002b; Sleckman et al., 1996). CSR and SHM, unlike V(D)J recombination, are dependent on activationinduced cytidine deaminase (AID), a protein expressed only in activated germinal center B cells (Muramatsu et al., 2000). Conversely, CSR and SHM do not require the presence of RAGs, as B cells derived by site-specific targeting of rearranged IgH and IgL transgenes into the corresponding endogenous loci of RAG-deficient mice undergo normal levels of CSR (Lansford et al., 1998) and SHM (Zheng et al., 1998). AID was identified via subtractive cloning of a cell line capable of switching from IgM to IgA on appropriate cellular stimulation (Muramatsu et al., 1999). The absence of AID results in the loss of CSR and SHM in humans and mice and eliminates gene conversion in chickens, a process related to SHM that allows gene diversification in some animals (Arakawa et al., 2002; Muramatsu et al., 2000; Revy et al., 2000). In addition, expression of AID in nonlymphoid cell lines induces CSR and

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SHM of transfected substrates, implying that AID is the only lymphoidspecific factor necessary to effect these processes (Okazaki et al., 2002; Yoshikawa et al., 2002). Evidence demonstrates that AID deaminates cytidines of single-stranded DNA (ssDNA), thereby introducing DNA lesions that effect CSR and SHM (Bransteitter et al., 2003; Chaudhuri et al., 2003; Petersen-Mahrt et al., 2002; Pham et al., 2003; Sohail et al., 2003; Yu et al., 2004). Multiple DNA repair pathways including base excision repair (BER), mismatch repair (MMR), and NHEJ appear to be required for the processing and resolution of the AID-initiated DNA lesions during SHM and CSR (Chaudhuri and Alt, 2004; Petersen-Mahrt et al., 2002). The NHEJ factors Ku and DNA-PKcs appear to be required for normal levels of CSR (Casellas et al., 1998; Manis et al., 1998a, 2002a) and may be involved in the resolution of DNA lesions, including DNA DSB intermediates induced by AID (Bross et al., 2000; Chen et al., 2001; Papavasiliou and Schatz, 2000; Wuerffel et al., 1997). In contrast to the site-specific RSs that target V(D)J recombination, CSR is targeted to large regions (1–12 kb) of repetitive DNA sequences, known as switch (S) regions, located upstream of all CH genes except Cd (which is regulated at the level of alternate RNA splicing) (Davis et al., 1980; Honjo and Kataoka, 1978; Kataoka et al., 1980). Likewise, SHM mutates nonconserved sequences of rearranged VHDJH and VLJL exons (reviewed in Harris et al., 1999; Jacobs and Bross, 2001). CSR requires the transcription of S region target sequences, as disruption of specific S region transcriptional units eliminates CSR to the corresponding isotype (reviewed in Manis et al., 2002b), whereas transcription has not been shown to be directly (i.e., mechanistically) involved in V(D)J recombination. In this regard, the transcriptional orientation of an S region is important, as inverted S regions are impaired in their ability to mediate CSR in vivo (Shinkura et al., 2003), in accord with a direct role of transcription in the process of CSR, as opposed to V(D)J recombination, which clearly involves a different mechanism. Thus, although enhanced Vk germline transcription in vivo enhances Vk rearrangement (Casellas et al., 2002), germline promoter location, rather than transcription through gene segments, may target gene segment accessibility via chromatin remodeling in a polymerase-independent manner (Sikes et al., 1998). The identification of RAG-1 and -2 was instrumental in elucidating the V(D)J recombination mechanism, which is now understood in some detail (Fugmann et al., 2000a). Likewise, the identification of AID has led to rapid advances in our understanding of SHM and CSR mechanisms (reviewed in Honjo et al., 2002; Kenter, 2003; Manis et al., 2002b). This review compares and contrasts the targeting, initiation, and resolution of V(D)J recombination and CSR.

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2. Antigen Receptor Gene Rearrangement 2.1. Genomic Organization of Murine Antigen Receptor Loci The antigen receptor expressed on the surface of a B cell normally consists of four polypeptides that are made up of two identical IgH chains and two identical IgL chains, with IgL chains being derived from the rearrangement of either Igk or Igl genes (reviewed in Gorman and Alt, 1998). T cells express surface receptors made up of either ab or gd heterodimers (reviewed in Kisielow and von Boehmer, 1995). The assembly of the variable region exons of Igk and Igl in developing B cells, as well as the assembly of the variable region exons of TCRa and TCRg in developing T cells, involves the rearrangement of V and J gene segments (reviewed in Bassing et al., 2002b). In contrast, IgH, TCRb, and TCRd variable region exons are assembled from component V, D, and J gene segments, thus increasing the level of diversification of rearranged products (reviewed in Bassing et al., 2002b). The variable region exons of all antigen receptors are then linked to constant region exons via RNA splicing and subsequently expressed at the cell surface (Fig. 1). The murine IgH locus consists of some several hundred different V gene segments distributed throughout an approximate 1-Mb region beginning about

Figure 1 Schematic diagram of the murine IgH locus before and after V(D)J recombination. The VH, DH, and JH gene segments are depicted as rectangles. The 12-bp RS sequences are shown as open triangles, and the 23-bp RS sequences as solid triangles. The m constant region exons are shown as shaded rectangles, and the switch m region as an oval. The position of the iEm enhancer is indicated by a shaded diamond. The positions of the VH and I exon promoters are shown as solid circles. Distances between the various elements are not drawn to scale.

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Figure 2 Schematic diagram of the murine B cell receptor loci. The V, D, and J gene segments are depicted as rectangles. The 12-bp RS sequences are shown as open triangles, and the 23-bp RS sequences as solid triangles. Only functional constant region exons are shown, represented by squares. The positions of various enhancer elements are indicated by circles. The estimated number of antigen receptor gene segments for the VH and Vk loci is indicated above each locus. Distances between the various elements are not drawn to scale. Adapted from Hesslein and Schatz (2001).

100 kb upstream of Cm on chromosome 12 (reviewed in Honjo and Matsuda, 1995) (Fig. 2). Four J gene segments are positioned in a cluster about 7.5 kb upstream of Cm, and 13 known D gene segments are dispersed between the VH and JH gene segments (reviewed in Hesslein and Schatz, 2001). The VH gene segments are flanked at their 30 ends with RSs containing 23-bp sequences (23-bp RS), as are the JH gene segments at their 50 ends (reviewed in Hesslein and Schatz, 2001). The DH gene segments, on the other hand, are flanked on both sides by RSs with 12-bp spacer sequences (12-bp RS) (reviewed in Hesslein and Schatz, 2001). Thus the 12/23 rule prohibits direct VH-to-JH joining and ensures the usage of DH gene segments during normal V(D)J rearrangement, augmenting junctional diversification. The Igk locus spans approximately 3 Mb of chromosome 6 and contains about 140 Vk gene segments that can rearrange to 1 of 4 functional Jk gene segments positioned just upstream of a single Ck gene (reviewed in Gorman and Alt, 1998; Schable et al., 1999) (Fig. 2). There is also one nonfunctional Jk gene segment (reviewed in Hesslein and Schatz, 2001). Unlike the IgH and Igl loci, Vk gene segments are found in both transcriptional orientations and thus allow for rearrangement by both deletion and inversion of intervening sequences (reviewed in Gorman and Alt, 1998). Vk gene segments are flanked by 12-bp RSs, and Jk segments by 23-bp RSs (reviewed in Gorman and Alt, 1998). The Igl locus in most mouse strains spans about 200 kb on chromosome 16 and has only three functional Vl gene segments, each with a flanking 23-bp RS

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(reviewed in Gorman and Alt, 1998; Selsing and Daitch, 1995; Fig. 2). There are three functional and one nonfunctional Cl genes, each of which is associated with an upstream Jl gene segment flanked by a 12-bp RS (reviewed in Gorman and Alt, 1998; Selsing and Daitch, 1995). Two of the Vl gene segments are located upstream of all four Jl–Cl units whereas Vl1 is positioned upstream of only the two 30 -most Jl–Cl units and is therefore restricted in potential rearrangements (reviewed in Gorman and Alt, 1998; Selsing and Daitch, 1995). The TCRb locus contains two Cb genes, each associated with a single Db and six functional Jb gene segments positioned upstream (reviewed in Glusman et al., 2001) (Fig. 3). The entire locus spans nearly 700 kb of mouse chromosome 6 (reviewed in Glusman et al., 2001; Fig. 3). The Jb gene segments are associated with 12-bp RSs, whereas the Db segments are flanked on the 50 side by 12-bp RSs and on the 30 side by 23-bp RSs (reviewed in Hesslein and Schatz, 2001). There are about 34 Vb gene segments flanked by 23-bp RSs located upstream of the DJb clusters, 14 of which appear to be nonfunctional pseudogenes (reviewed in Hesslein and Schatz, 2001). There is also one Vb segment, Vb14, found 30 of Cb2 that rearranges by inversion (reviewed in Hesslein and Schatz, 2001). The gene segments and associated RSs of the TCRb locus are organized in such a way that according to the 12/23 rule, direct Vb-to-Jb rearrangement should be allowed, yet such rearrangements do not normally occur (Bassing et al., 2000a; Davis and Bjorkman, 1988; Ferrier et al., 1990). Additional constraints, referred to as beyond 12/23 restriction, ensure that Db

Figure 3 Schematic diagram of the murine T-cell receptor loci. The V, D, and J gene segments are depicted as rectangles. The 12-bp RS sequences are shown as open triangles, and the 23-bp RS sequences as solid triangles. Only functional constant region exons are shown, represented by solid squares. The positions of various enhancer elements are indicated by circles. The estimated number of antigen receptor gene segments for each locus is indicated above each locus. Distances between the various elements are not drawn to scale. Adapted from Hesslein and Schatz (2001).

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gene segments are utilized during Vb(D)Jb rearrangement of the TCRb locus and limit direct Vb-to-Jb joining (Bassing et al., 2000a; Jung et al., 2003; Sleckman et al., 2000). Both TCRa and TCRd gene segments are spread throughout a region spanning more than 1.3 Mb of mouse chromosome 14 (reviewed in Glusman et al., 2001; Fig. 3). The single Cd, two Dd, and two Jd gene segments are positioned between the 30 -most Va and 50 -most Ja segments, and thus are deleted after Va-to-Ja rearrangement (reviewed in Hesslein and Schatz, 2001). There are more than 85 Va and 12 Vd gene segments, each adjoined by a 30 23-bp RS, some of which can function as either Va or Vd gene segments, located upstream of the Dd segments (reviewed in Hesslein and Schatz, 2001). There is also one Vd positioned 30 of Cd that has a promoter in the opposite transcriptional orientation and undergoes inversional recombination (reviewed in Hesslein and Schatz, 2001). Like the Db gene segments, the Dd gene segments have 50 12-bp RSs and 30 23-bp RSs (reviewed in Hesslein and Schatz, 2001). Furthermore, Jd gene segments have 50 12-bp RSs that according to the 12/23 rule might allow for direct Vd-to-Jd joining, although, as with the TCRb locus, this does not normally occur. At least 60 Ja gene segments are found upstream of a single Ca gene, each associated with a 50 12-bp RS (reviewed in Hesslein and Schatz, 2001). The TCRg locus is distributed across a region spanning approximately 200 kb of mouse chromosome 13 (reviewed in Glusman et al., 2001; Fig. 3). There are seven Vg gene segments and one Vg pseudogene segment interspersed among three functional Jg–Cg units and one nonfunctional Jg–Cg unit (reviewed in Hesslein and Schatz, 2001). All gene segments are positioned in the same transcriptional orientation, with Vg segments flanked by 23-bp RSs and Jg gene segments flanked by 12-bp RSs (reviewed in Hesslein and Schatz, 2001). 2.2. Initiation of V(D)J Recombination 2.2.1. Recombinant-Activating Genes 1 and 2 RAGs were identified by transfecting cDNAs into a fibroblast cell line carrying the stable integration of a V(D)J recombination substrate that can confer drug resistance on successful completion of an RS-directed rearrangement (Schatz and Baltimore, 1988). RAG-1 and RAG-2 are each encoded within a single coding exon, and the RAG genes are located within 20 kb of one another in the opposite transcriptional orientation (Oettinger et al., 1990). The close proximity of the two RAG genes, the lack of introns in their coding sequences, and

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their inverted orientation led to the hypothesis that the RAGs were once part of a transposable element that integrated into the vertebrate genome (Agrawal et al., 1998; Lewis and Wu, 1997; Spanopoulou et al., 1996; Thompson, 1995; van Gent et al., 1996a). In support of this theory, RAGs have been shown to carry out transposition of target sequences in vitro (Agrawal et al., 1998; Hiom et al., 1998) and have been implicated in mediating translocations that occur in vivo (Messier et al., 2003; Zhu et al., 2002). Null mutations in RAGs cause a severe combined immune deficiency (SCID) in humans (Schwarz et al., 1996) and mice (Mombaerts et al., 1992; Shinkai et al., 1992) caused by a complete block in B-and T-cell development. The block in lymphocyte development occurs at the B and T progenitor stages (Mombaerts et al., 1992; Shinkai et al., 1992), the stages at which B cells normally rearrange IgH genes and T cells rearrange TCRb, g, and d genes (reviewed in Fehling et al., 1999; Willerford et al., 1996). Furthermore, mutations that lead to partial RAG activity in humans cause Omenn syndrome (Villa et al., 1998, 1999; Wada et al., 2000), an SCID disorder characterized by hepatosplenomegaly, lymphadenopathy, eosinophilia, elevated IgE, lack of circulating B cells, and a variable number of oligoclonal T cells (reviewed in Notarangelo et al., 1999; Villa et al., 2001). Aberrant RAG activity has been implicated in translocations between immunoglobulin or TCR and oncogenes such as c-Myc, Bcl-2, and Bcl-6 among human T and B lineage lymphomas (reviewed by Mills et al., 2003; Roth, 2003). Some such translocations may involve interchromosomal V(D)J recombination involving cryptic RSs in the oncogene loci; whereas others may involve aberrant joining of RAG-initiated DSBs at antigen receptor loci to general DSBs on other chromosomes. Clear evidence for the latter process has come from studies of mouse pro-B lymphomas that arise in an NHEJ- and p53-deficient background (Difilippantonio et al., 2002; Guidos et al., 1996; Zhu et al., 2002). In addition, work has suggested that RAGs initiate translocations by introducing ssDNA nicks, which can be converted to DSBs, at cryptic RS or other non-B form DNA structures at various chromosomal locations, such as around the major breakpoint cluster region of human Bcl-2, and, thereby, initiate translocations (Lee et al., 2004; Raghavan and Lieber, 2004; Raghavan et al., 2004). 2.2.2. RAGs Recognize Site-Specific Target Sequences RAGs recognize and bind to site-specific RSs positioned adjacent to all antigen receptor gene-coding segments (reviewed in Tonegawa, 1983). Each RS consists of a conserved 7-bp sequence (heptamer; consensus, 50 -CACAGTG), a conserved 9-bp sequence (nonamer; consensus, 50 -ACAAAAACC), and an intervening, relatively nonconserved 12  1 or 23  1 bp spacer sequence

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(Early et al., 1980; Hesse et al., 1989; Max et al., 1979; Sakano et al., 1979). Although overall highly conserved, there is variation between heptamer and nonamer sequences of individual RSs, with those most closely resembling the consensus sequences being the most efficiently rearranged (reviewed in Lewis, 1994a). Moreover, not all of the positions within the conserved heptamer and nonamer sequences appear to be important for RAG-mediated cleavage. Whereas changes in the first three nucleotide positions of the heptamer or in the sixth or seventh positions of the nonamer greatly reduce RAG-mediated cleavage of plasmid substrates, changes at other positions are better tolerated (Hesse et al., 1989). The spacer sequences also play an essential role in V(D)J recombination, as RAG-mediated cleavage will occur only when an RS with a 12-bp spacer sequence is paired in complex with an RS with a 23-bp spacer sequence, a constraint referred to as the 12/23 rule (Eastman et al., 1996; Sakano et al., 1981; van Gent et al., 1996b). The 12/23 rule appears to be enforced at the level of binding and assembly of RAGs to paired RSs (Hiom and Gellert, 1998; Mundy et al., 2002) as well as the subsequent cleavage step (West and Lieber, 1998; Yu and Lieber, 2000). Although much less conservation exists in RS spacer sequences compared with heptamer and nonamer sequences, these sequences have also been shown to influence RAG-mediated cleavage and RS usage (Jung et al., 2003; Nadel et al., 1998). As described above, like gene segments (e.g., all V segments) for any given antigen receptor locus are each associated with RSs with the same length spacer sequences, and thus the 12/23 rule prevents nonproductive V-to-V or J-to-J joining. The configuration of the heavy chain locus ensures that D gene segments flanked with 12-bp RSs will be utilized in all successful VHDJH rearrangements, as VH and JH gene segments all have 23-bp RSs (Fig. 2). In contrast, the configuration of the TCRb locus, with 23-bp RSs flanking the Vbs and 12-bp RSs flanking the Jbs, should allow direct Vb-to-Jb rearrangement according to the 12/23 rule, yet this rarely occurs in vivo (Bassing et al., 2000; Sleckman et al., 2000; Wu et al., 2003) (Fig. 3). Even when Db1 was deleted on both alleles in mice, Vb-to-Jb1 rearrangements rarely took place and subsequent ab T-cell development was severely impaired (Bassing et al., 2000). Several studies have shown that this so-called beyond 12/23 restriction is enforced at the level of specific RSs (Bassing et al., 2000; Jung et al., 2003; Tillman et al., 2003). Indeed, when a Vb 23-bp RS was replaced by the 30 Db1 23-bp RS, the ‘‘beyond 12/23 restriction’’ was broken, and direct Vb-to-Jb rearrangement was detected (Wu et al., 2003). The strength and efficiency with which the 30 Db1 23-bp RS mediates rearrangement imply that this RS might contribute to ordered rearrangement in which Db1-to-Jb rearrangement takes place before the onset of Vb-to-DJb rearrangement.

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In a coupled cleavage reaction involving both 12- and 23-bp RSs, RAGs introduce DNA DSBs between the heptamers and flanking coding sequences, followed by subsequent ligation of the two blunt RS ends and two modified coding ends. Recombination that takes place between RSs found in the opposite chromosomal orientation will therefore result in the deletion of intervening DNA sequences in the form of covalently sealed DNA circles (Fujimoto and Yamagishi, 1987; Okazaki et al., 1987; Sakano et al., 1979). Subsequent rounds of cell division result in the permanent loss of these sequences from the genome (Kabat, 1972; Sakano et al., 1979; Tonegawa et al., 1977). On the other hand, recombination between RSs that are in the same chromosomal orientation leads to an inversion of intervening DNA sequences and retention of these sequences in the genome (Alt and Baltimore, 1982; Lewis et al., 1982; Malissen et al., 1986; Weichhold et al., 1990; Zachau, 1993). As the presence of an accessible RS is all that is necessary to render a piece of DNA susceptible to RAG-mediated cleavage, plasmid substrates have been engineered that retain either the RS or coding ends, allowing detailed analysis of each type of DNA junction (Hesse et al., 1987; Lewis et al., 1985). 2.2.3. Assembly of Precleavage Complex In vitro, RAGs are found to cooperatively associate with 12- and 23-bp RSs and their flanking coding gene segments to form a synaptic complex (Bailin et al., 1999; Hiom and Gellert, 1997; Leu and Schatz, 1995). Contacts between RAG-1 and nonamer sequences are essential for RS binding, whereas interactions with the heptamer or coding sequences appear to help provide specificity to the RAG-binding complex and to promote efficient DNA cleavage (Difilippantonio et al., 1996; Roman and Baltimore, 1996). RAG-1 binding to nonamer sequences involves the region between residues 376 and 477 of RAG-1, with a GGRPR motif (amino acids 389–393 of murine RAG-1) that is also found in members of the bacterial DNA invertase family forming the main site of interaction (Difilippantonio et al., 1996; Spanopoulou et al., 1996). Independently, RAG-1 binds only weakly to heptamer sequences; however, the presence of RAG-2 has been shown to help stabilize this interaction (Aidinis et al., 2000; Akamatsu and Oettinger, 1998; Fugmann and Schatz, 2001; Spanopoulou et al., 1996; Swanson and Desiderio, 1999). The region of RAG-1 that makes contact with the heptamer has been mapped to residues 528–760 and also appears to contain the main site of RAG-2 interaction (Arbuckle et al., 2001; Peak et al., 2003). Although the RAG-2 protein does not bind DNA independently, RAG-2 does make contact with the RS heptamer sequence when in a complex with RAG-1 (Difilippantonio et al., 1996; Spanopoulou et al., 1996; Swanson and Desiderio, 1999).

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Synaptic complex assembly begins in vitro with the binding of RAGs to a single 12-bp RS referred to as a single complex (SC), followed by integration of the companion 23-bp RS target DNA into a paired complex (PC) (Jones and Gellert, 2002; Mundy et al., 2002; Swanson, 2002b). The DNA-bending proteins HMG1 and HMG2 facilitate the integration of the 23-bp RS and assembly of the SC (Rodgers et al., 1999; Swanson, 2002a) and appear to promote RAG-mediated cleavage (Swanson, 2002a; van Gent et al., 1997). The coordinated assembly of the PC and subsequent coupled cleavage requires the presence of Mg2þ divalent cation, whereas in vitro the presence of Mn2þ allows cleavage to take place on single RS-containing substrates (van Gent et al., 1996b). By replacing Mn2þ or Mg2þ divalent cations with Ca2þ in vitro, DNA cleavage by RAGs is blocked and the SC consisting of RAGs bound to a single RS can be isolated as an intermediate of the reaction (Hiom and Gellert, 1997). This made it possible to detect two distinct complexes that form on a single RS, single complex 1 (SC1) and single complex 2 (SC2) (Mundy et al., 2002; Swanson, 2002b). The number of RAG-1 subunits in the SC1, SC2, and PC appears to be the same, although whether there are two (Swanson, 2002b) or more (Mundy et al., 2002) molecules of RAG-1 per complex is still not clear. Studies have consistently found that the slower migrating SC1 contains two subunits of RAG-2, whereas only a single subunit of RAG-2 exists in SC2 (Mundy et al., 2002; Swanson, 2002b). However, crystallization of the complex may ultimately be required to unequivocally ascertain the stoichiometry. 2.2.4. Biochemistry of the Cleavage Reaction After assembly of the PC, RAGs introduce a single-strand nick in the DNA between the border of the RS heptamer and the gene-coding segment in a coupled cleavage reaction that in vivo requires the presence of both a 12-bp RS and a 23-bp RS (McBlane et al., 1995; van Gent et al., 1996b). This creates a 30 -OH on one DNA strand of the gene-coding segment and a 50 -phosphate group on the corresponding RS-containing DNA strand (Fig. 4). The 30 -OH then acts as a nucleophile in attacking the opposite DNA strand in a transesterification reaction, forming a covalently sealed hairpin coding end and a blunt, 50 -phosphorylated RS end (McBlane et al., 1995; Roth et al., 1992) (Fig. 4). After cleavage, the DNA ends are held together in a postcleavage complex that includes the RAGs and all four DNA ends (Agrawal and Schatz, 1997; Hiom and Gellert, 1998; Jones and Gellert, 2001; Qiu et al., 2001; Tsai et al., 2002; Yarnell Schultz et al., 2001). Mutational studies have identified active catalytic residues in RAG-1 that when mutated result in defects in DNA nicking and hairpin formation, although these residues do not appear to be required for assembly of the PC (Fugmann et al., 2000b; Kim et al., 1999; Landree et al., 1999). The three

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Figure 4 Biochemistry of V(D)J recombination. Standard V(D)J recombination results in the formation of precise signal joints and modified coding joints. Products of aberrant V(D)J recombination include hybrid joints, open and shut joints, and transposition events. The rectangles represent V, D, or J gene segments and the solid and open triangles represent 12- and 23-bp RSs, respectively. RAG cleavage and subsequent processing and joining via the NHEJ pathway leads to the standard V(D)J recombination products shown on the left. Hybrid joints can form when the 30 -OH of an RAG-liberated RS end attacks the hairpin-coding end of the partner gene segment in the coupled reaction, as shown in the center. RAG-mediated transposition of a liberated 30 -OH into an intact piece of double-stranded DNA is depicted on the right.

identified acidic residues (D600, D708, and E782) are all contained within the active core RAG-1 protein and likely constitute a DDE motif similar to that found in many integrase/transposase family proteins (reviewed in Haren et al., 1999). The DDE triad is thought to function in coordinating two divalent metal ions (Mg2þ) that facilitate the trans-esterification reaction, one acting as a general base and the other as a general acid (reviewed in Haren et al., 1999). The presence of the DDE motif is consistent with the theory that RAGs started out as components of a transposable element that integrated into the vertebrate genome (Agrawal et al., 1998; Spanopoulou et al., 1996; Thompson, 1995; van Gent et al., 1996a). Full-length RAG proteins are relatively insoluble, and therefore elucidation of the biochemistry behind RAG-mediated V(D)J recombination has largely made use of highly truncated ‘‘core’’ RAG proteins (Kirch et al., 1996;

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McBlane et al., 1995; Sadofsky et al., 1993, 1994; Sawchuk et al., 1997). Although truncated core RAG proteins are capable of mediating complete V(D)J recombination in vitro, the core RAGs carry out the reaction at reduced efficiency both in vitro and in vivo (Akamatsu et al., 2003; Dudley et al., 2003; Kirch et al., 1998; Liang et al., 2002). In the absence of the C-terminal portion of RAG-2, V-to-DJ rearrangements appear more severely affected than D-to-J rearrangements, suggesting that the noncore region of RAG-2 may play a specific role during ordered rearrangement of IgH and TCR genes (Kirch et al., 1998; Roman et al., 1997). In addition to normal CJs and SJs (see below), RAGs can mediate open and shut joints, hybrid joints (HJs), and transpositions both in vitro and in vivo (Fig. 4) (Agrawal et al., 1998; Lewis et al., 1988; Messier et al., 2003; Morzycka-Wroblewska et al., 1988; Sekiguchi et al., 2001). An HJ is defined as the joining of the liberated RS end from one coding segment to the partner hairpin-coding end participating in the recombination reaction (Fig. 4) (Lewis et al., 1988). A transposition event is similar to a hybrid joint, with insertion of the liberated RS end into a double-stranded DNA target sequence instead of joining with an RAG-generated coding end (Fig. 4) (Agrawal et al., 1998; Hiom et al., 1998). The truncated core RAGs mediate an increased rate of HJs in NHEJ-deficient cells compared with full-length RAGs, suggesting that the noncore regions normally function to suppress such aberrant joining events (Sekiguchi et al., 2001). Furthermore, there is an increase in the frequency of transposition events in core RAG-2–expressing cells compared with controls (Elkin et al., 2003; Tsai and Schatz, 2003), which form by a similar mechanism as that of hybrid joints. Taken together, these studies imply that the noncore regions of RAGs may have evolved to ensure that RAG-liberated DNA ends are properly joined, thus preventing transposition and other deleterious or ineffective recombination reactions (Agrawal and Schatz, 1997; Elkin et al., 2003; Hiom et al., 1998; Messier et al., 2003; Sekiguchi et al., 2001; Steen et al., 1999; Tsai and Schatz, 2003). 2.2.5. Postcleavage Complex After cleavage, the RAGs remain associated with the four DNA ends in a postcleavage complex, possibly playing a role in the protection of DNA ends from degradation, the juxtaposition of ends before rejoining, or recruitment and activation of end-joining factors for both CJ and SJ formation (Fig. 5) (Agrawal and Schatz, 1997; Hiom and Gellert, 1998; Jones and Gellert, 2001; Qiu et al., 2001; Tsai et al., 2002; Yarnell Schultz et al., 2001). Stability of the postcleavage complex may also function to inhibit DSB-induced cell cycle arrest and apoptosis, as well as to prevent potentially deleterious transposition events (Jones and Gellert, 2001; Perkins et al., 2002). However, studies have

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Figure 5 Joining of RAG-mediated DNA double-strand breaks. (A) RAG-1 and RAG-2 cleavage occurs between RS and coding segments. (B) Ku70 and Ku80 bind to the broken DNA ends. (C) DNA-PKcs and Artemis facilitate the opening and processing (opening) of covalently sealed hairpin coding ends. (D) TdT adds random nucleotides to opened coding ends. XRCC4 and Lig4 seal the blunt signal ends and processed coding ends to produce precise signal joints and modified coding joints. In addition, DNA-PKcs functions independently of Artemis to form normal signal joints.

shown that signal ends must be deproteinized before rejoining by NHEJ factors in vitro (Leu et al., 1997; Ramsden et al., 1997). In this regard, it was demonstrated that the N terminus of RAG-1 has E3 ubiquitin ligase activity (Yurchenko et al., 2003), suggesting a function for RAG-1 in steps beyond recognition and DNA cleavage. For instance, once the appropriate end-joining proteins have been recruited or have performed their function, RAG-1– mediated ubiquitination could tag RAG-2 or NHEJ proteins within the complex for proteasomal degradation, thus promoting disassembly of the complex and ligation of the DNA ends. 2.2.6. Coding and Signal Joint Formation RAG-mediated cleavage generates hairpin-coding ends that must be opened and processed before rejoining, whereas the RS ends do not require any additional processing and are religated by NHEJ proteins to form precise

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SJs (reviewed in Fugmann et al., 2000a; Fig. 5). Although studies have demonstrated that the RAGs themselves can mediate hairpin opening in vitro (Besmer et al., 1998; Ma et al., 2002; Shockett and Schatz, 1999), evidence has shown that the NHEJ protein Artemis, in association with DNA-PKcs, likely performs this role in vivo (Ma et al., 2002; Rooney et al., 2002, 2003) (Fig. 5). Hairpin-coding ends are opened via the introduction of a DNA nick, usually within four or five nucleotides 30 of the apex of the hairpin (reviewed in Fugmann et al., 2000a; Lieber, 1991; Nadel et al., 1995). Once the hairpins are opened, the 30 overhangs can be filled in via DNA polymerases, thus generating short stretches of palindromic sequences at the junctions of CJs, referred to as P nucleotides (Lafaille et al., 1989; Lewis, 1994b; reviewed in Lewis, 1994a; Lieber, 1991). Alternatively, nucleases can chew back the 30 overhangs, resulting in a loss of germline nucleotides at the junction of CJs (reviewed in Fugmann et al., 2000a; Lieber, 1991; Nadel et al., 1995). To further diversify junctions, the lymphoid-specific protein terminal deoxynucleotidyltransferase (TdT) adds random, nontemplated nucleotides to 30 coding ends and introduces so-called N-nucleotide additions, further increasing the diversity of antigen receptor variable regions (Alt and Baltimore, 1982). Moreover, a splice variant of TdT appears to function to remove nucleotides from coding junctions (Thai et al., 2002). Although TdT is not required for either V(D)J recombination or lymphocyte development, it does affect overall repertoire diversification (Gilfillan et al., 1993; Komori et al., 1993). Finally, DNA polymerase m (polm)–deficient mice have a significant reduction in the length of Vk-to-Jk junctions, suggesting polm plays a role in maintaining CDR3 length of Igk chains (Bertocci et al., 2003). It is still unclear whether polm regulates the processing of coding ends by protecting them from exonucleolytic attack or by filling in 30 overhangs (Bertocci et al., 2003). Notably, polm shares homology with TdT. 2.3. Joining of RAG-Mediated DNA Double-Strand Breaks DNA DSBs can be induced by a variety of agents including ionizing radiation (IR), oxidative stress incurred during normal cellular metabolism, and RAGs during V(D)J recombination. Mammalian cells have evolved two different pathways to repair such potentially catastrophic lesions. Homologous recombination (HR) is a high-fidelity process that repairs breaks, using a homologous chromosome as a DNA template (reviewed in Hoeijmakers, 2001). NHEJ repairs broken DNA ends in the absence of long stretches of homology, allowing both the loss and addition of nucleotides at the repair junction (reviewed in Khanna and Jackson, 2001). HR takes place predominantly in S and G2 phases of the cell cycle, when homologous templates are both

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available and in close proximity (reviewed in Hoeijmakers, 2001). Conversely, NHEJ appears to be the preferred repair pathway during the G1 phase of the cell cycle, corresponding to the stage at which RAGs are both expressed and active for recombination (reviewed in Jackson, 2002; Lin and Desiderio, 1995). However, it is clear that NHEJ can function outside of the G1 phase and complement the repair activities of HR (Couedel et al., 2004; Mills et al., 2004). Studies involving the transfection of recombination substrates into IRsensitive cell lines implicated several members of ubiquitously expressed NHEJ proteins as having direct roles in V(D)J recombination (reviewed in Bassing et al., 2002b; Taccioli et al., 1993). Members of the NHEJ repair pathway known to be involved in V(D)J recombination include Ku70, Ku80, DNA-PKcs, XRCC4, ligase 4 (Lig4), and Artemis (reviewed in Mills et al., 2003; Rooney et al., 2004). Cells isolated from patients that are unable to complete RAG-initiated V(D)J recombination of transiently transfected plasmid substrates and exhibit IR sensitivity have implicated a seventh potential member of the NHEJ group, as genetic analyses have ruled out defects in Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, or Lig4 (Dai et al., 2003). 2.3.1. Ku70 and Ku80 Ku70 and Ku80 form a heterodimer (Ku) that directly associates with DNA DSBs as well as telomeric regions of chromosomes (reviewed in Critchlow and Jackson, 1998). Ku-deficient cell lines are IR sensitive and defective in both CJ and SJ formation of transiently transfected recombination substrates, demonstrating that these proteins are essential for normal V(D)J recombination (Gu et al., 1997; Nussenzweig et al., 1996; Taccioli et al., 1993, 1994; Zhu et al., 1996). Potential functions for Ku during NHEJ include (1) the protection of DNA ends generated during V(D)J recombination from unwanted processing or degradation (Boulton and Jackson, 1996; de Vries et al., 1989; Getts and Stamato, 1994), (2) the juxtaposition of DNA ends produced by RAG cleavage before religation (Boulton and Jackson, 1996; Cary et al., 1997), and (3) the recruitment or activation of DNA repair or DNA damage-sensing proteins (Gottlieb and Jackson, 1993; Lieber et al., 1997; Ramsden and Gellert, 1998; West et al., 1998). 2.3.2. DNA-PKcs and Artemis DNA-PKcs is a serine/threonine kinase and member of the phosphatidylinositol-3-kinase (PI-3 kinase) family that includes the DNA damage response proteins ataxia telangiectasia mutated (ATM) and ataxia telangiectasia related (ATR) (reviewed in Smith and Jackson, 1999). DNA-PKcs–deficient cell lines display varying degrees of IR sensitivity (Gao et al., 1998; Lees-Miller et al.,

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1995; Taccioli et al., 1998). Furthermore, cell lines derived from SCID mice, which harbor a mutation in DNA-PKcs (Blunt et al., 1995), are severely impaired in their ability to form CJs, although SJ formation is relatively unaffected in these cells (Blackwell et al., 1989; Lieber et al., 1988; Malynn et al., 1988). DNA-PKcs–deficient embryonic stem (ES) cells fail to make CJs but make fully normal SJs (Gao et al., 1998); however, mouse embryonic fibroblasts from DNA-PKcs–deficient mice, which are also fully deficient for CJ formation, are also somewhat impaired in SJ formation, with many RS joins in such cells harboring abnormal deletions (Bogue et al., 1998; Errami et al., 1998; Fukumura et al., 1998, 2000; Gao et al., 1998; Priestley et al., 1998). Therefore, although not fully required, DNA-PKcs has some unknown role in SJ formation, and this role is independent of Artemis (see below) and may be substituted by other factors (e.g., in ES cells). Finally, hairpin-coding ends were shown to accumulate in lymphocytes derived from DNA-PKcs–deficient mice (Roth et al., 1992), thus implicating a function for DNA-PKcs in the processing of these V(D)J intermediates. More recently, DNA-PKcs has been shown to phosphorylate Artemis, another member of the NHEJ repair pathway required for the formation of CJs but not SJs (Ma et al., 2002; Nicolas et al., 1998; Rooney et al., 2002). The phosphorylated form of Artemis has an endonuclease activity that in vitro is capable of opening DNA hairpins produced by RAGs (Ma et al., 2002). Sequences of CJ junctions generated from both DNA-PKcs– and Artemisdeficient cells show an increased rate of P-nucleotide additions (Rooney et al., 2003) consistent with aberrant opening of the hairpin ends (Kienker et al., 1991; Lewis, 1994b; Rooney et al., 2002; Schuler et al., 1991). In contrast to DNA-PKcs deficiency, SJ formation is normal (both in quantity and quality) in all types of Artemis-deficient cells examined (Noordzij et al., 2003; Rooney et al., 2003), again supporting a non-Artemis–mediated role for DNA-PKcs in V(D)J recombination and NHEJ. 2.3.3. XRCC4 and DNA Ligase 4 The role of XRCC4 in V(D)J was discovered by expression cloning via complementation of an IR-sensitive, V(D)J recombination-defective hamster cell line with a human cDNA library, which was shown to completely complement all IR sensitivity and V(D)J recombination defects of this line (Li et al., 1995). XRCC4 was then shown to associate with DNA Lig4 in vitro (Critchlow et al., 1997; Grawunder et al., 1997). XRCC4- and Lig4-deficient cells were both shown to exhibit IR sensitivity and an inability to generate either SJs or CJs (Frank et al., 1998; Gao et al., 1998). Thus DNA Lig4 in association with XRCC4 rejoins the four broken DNA ends generated by RAG-mediated cleavage and likely has a similar role in NHEJ in general.

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3. Regulation of V(D)J Recombination 3.1. RAG-1 and RAG-2 Expression 3.1.1. Lymphoid-Specific Expression of RAGs The expression of RAG-1 and RAG-2 is predominantly limited to developing lymphocytes, although low levels of RNA transcripts have been detected in the murine central nervous system and in peripheral lymphoid tissues (reviewed in Nagaoka et al., 2000). In fact, transcription of the RAG-1 locus is detected in the earliest lymphocyte progenitors isolated thus far from the bone marrow of mice (Igarashi et al., 2002). However, the only known defect in RAG-deficient mice is a complete block in lymphocyte development, and therefore the RAGs do not appear to play a role in the development or function of the central nervous system (Mombaerts et al., 1992; Shinkai et al., 1992). The RAG-2 promoter is lymphoid specific and differentially regulated in B and T cells, whereas the basal RAG-1 promoter in both mice and humans does not impart lymphoid specificity (Lauring and Schlissel, 1999; Monroe et al., 1999a). A more distal element 50 of RAG-2 directs the coordinate and lymphoid-specific expression of fluorescently tagged RAG-1 and RAG-2 from a bacterial artificial chromosome (BAC) transgene integrated into mice (Yu et al., 1999a). Moreover, RAG transcription appears to be regulated by different cis elements in B and T lymphocytes (Hsu et al., 2003), with both a silencer and antisilencer important for proper tissue- and stage-specific expression (Yannoutsos et al., 2004). Several studies have detected RAG expression in mature B and T cells, leading to the hypothesis that RAGs could function to maintain self-tolerance via secondary rearrangements of autoreactive receptors (Han et al., 1996; Hikida et al., 1996; Papavasiliou et al., 1997). However, targeted replacement of the RAG-2 gene with sequences encoding a RAG-2:GFP (green fluorescent protein) fusion protein demonstrated that the low level of RAGs detected in the periphery likely came from immature lymphocytes that had not completely shut off RAG expression and yet migrated to the peripheral lymphoid tissues (Kuwata et al., 1999; Monroe et al., 1999b; Yu et al., 1999b). Moreover, RAG2:GFP expression was not detected when GFP-negative lymphocytes isolated from the periphery of RAG-2:GFP mice were adoptively transferred into RAG-1–deficient host animals after immunization (Yu et al., 1999b), thus indicating that RAG-2 was not reexpressed in mature lymphocytes. 3.1.2. Allelic Exclusion and Feedback Regulation Coincident with the onset of RAG expression, IgH rearrangements begin in B220þCD43þckitþCD19þ progenitor B cells (pro-B), and TCRb, TCRg, and TCRd rearrangements take place in CD4CD8 double-negative (DN) T cells

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(reviewed in Willerford et al., 1996). Because of the inherently imprecise nature of CJs, only one in three rearrangements will be in-frame and capable of expressing a functional protein. Theoretically, lymphocytes could make a different receptor chain for each allele and express multiple receptors, each of a different specificity. However, almost all B cells express the functional products of only one IgH allele and one IgL allele, and in mature ab T cells only one TCRb allele is functionally rearranged and expressed, a process referred to as allelic exclusion (reviewed in Gorman and Alt, 1998; Kisielow and von Boehmer, 1995; Melchers et al., 1999). Thus for the IgH, Igk, Igl, and TCRb loci, only those cells in which the first V(D)J rearrangement is nonproductive go on to rearrange their second allele, preventing the assembly of multiple antigen receptors in a single cell (Alt et al., 1984; Yancopoulos and Alt, 1985). Both stochastic and regulated models have been proposed to explain allelic exclusion, but the absolute mechanism, which may involve different mechanistic aspects for different loci, remains enigmatic; although it seems clear that there must be some form of feedback regulation to prevent opening of the second allele (reviewed by Mostoslavsky et al., 2004). In this context, epigenetic factors, such as asynchronous replication, monoallelic demethylation, and variegated, monoallelic transcriptional activation, which render only a single allele capable of V-to-(D)J rearrangement initially (Liang et al., 2004; Mostoslavsky et al., 1998), may contribute to initiation of allelic exclusion before the feedback signals that maintain allelic exclusion in the face of continued expression of RAG. The product of a functionally rearranged IgH gene, mIgH, and expression of a TCRb chain initiate signals that enforce feedback regulation at nonrearranged IgH and TCRb alleles, respectively, thus preventing further rearrangements (reviewed in Gorman and Alt, 1998; Kisielow and von Boehmer, 1995; Melchers et al., 1999). In this regard, mIgH associates with the surrogate light chain proteins, Vpre-B and l5, to form the pre-B cell receptor (pre-BCR) (Melchers et al., 1993). A productive TCRb chain then associates with the preTa protein to form the pre-T cell receptor (pre-TCR) (Saint-Ruf et al., 1994). Expression of the pre-BCR and pre-TCR provides the necessary signals to mediate feedback regulation, cellular expansion, and differentiation to the B220þCD43lo/ckitCD19þ pre-B and CD4þCD8þ double-positive (DP) T-cell stages, respectively (reviewed in Kisielow and von Boehmer, 1995; Rolink et al., 2001; von Boehmer et al., 1999). Thus, introduction of rearranged IgH (Spanopoulou et al., 1994; Young et al., 1994) or TCRb (Shinkai et al., 1993) transgene into an RAG-deficient background promotes development to the pre-B or DP T-cell stages, respectively. RAG expression is terminated during the phase of cellular proliferation that occurs during the transition from pro-B to pre-B in developing B cells, and

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from DN to DP in developing T cells (Grawunder et al., 1995). To facilitate this, RAG-2 is specifically tagged for degradation by cell cycle–dependent phosphorylation and ubiquitination (Lin and Desiderio, 1993; Mizuta et al., 2002). After the cellular proliferation signaled by expression of the pre-BCR or pre-TCR, RAGs are once again expressed, thus allowing rearrangement of IgL and TCRa genes in pre-B and DP T cells, respectively (reviewed in Nagaoka et al., 2000). During this second wave of RAG expression, further rearrangement of the IgH and TCRb loci does not occur, and therefore these loci have somehow been rendered inaccessible to the RAGs (reviewed in Krangel, 2003). Individual B cells produce immunoglobulin containing either Igk or Igl light chains, but not both, referred to as IgL isotype exclusion (reviewed in Mostoslavsky et al., 2004). In pre-B cells, the Igk locus is the first to rearrange, with subsequent rearrangement of Igl genes usually occurring only in cells that have failed to generate a productive Igk gene rearrangement (reviewed in Gorman and Alt, 1998). The mechanism of sequential Igk versus Igl rearrangement and whether it is regulated or stochastic also remain unsolved problems (reviewed in Mostoslavsky et al., 2004). Once a productive IgL chain is formed, it pairs with the previously rearranged IgH chain to create a mature BCR in the form of IgM that is expressed at the surface of the developing B cell. Surface expression of IgM provides further feedback regulation and allelic exclusion, as well as signaling for the differentiation to the immature B cell stage and beyond (reviewed in Rolink et al., 2001; Willerford et al., 1996). IgL rearrangements sometimes result in Igk and Igl protein products that fail to associate with mIgH, and thus are functionally nonproductive (Alt et al., 1980). Similarly, productive TCRa rearrangement in DP T cells allows for expression of a functional TCRa/b complex that induces progression through positive and negative selection steps that lead to the development of mature CD4þ or CD8þ single-positive (SP) T cells (reviewed in Kisielow and von Boehmer, 1995; Willerford et al., 1996). Unlike other antigen receptor gene loci, the TCRa, g, and d loci do not undergo feedback regulation or allelic exclusion at the level of gene rearrangement (Casanova et al., 1991; Davodeau et al., 1993; Malissen et al., 1988, 1992; Padovan et al., 1993; Sleckman et al., 1998). Preferential pairing of one TCRa chain to the expressed TCRb chain essentially prevents the expression of more than one antigen-specific receptor at the surface of ab T cells carrying two productive TCRa rearrangements (reviewed in Fehling and von Boehmer, 1997; Malissen et al., 1992). If a newly generated B cell expresses a productive but self-reactive receptor, signaling via the Ig receptor appears to prolong, or possibly reactivate, RAG expression and allow further rearrangement of IgL chains (reviewed in

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Nemazee and Weigert, 2000; Nussenzweig, 1998). This may lead to the replacement of a self-reactive receptor with a non-self-reactive one in a process termed receptor editing (Gay et al., 1993; Radic et al., 1993; Tiegs et al., 1993). There also is some evidence that suggests potential editing of TCRb genes in peripheral T cells (reviewed by Mostoslavsky and Alt, 2004). In contrast, both alleles of the TCRa locus appear to rearrange concomitantly in T cells and continue to rearrange throughout the DP T cell stage until positive selection on self-MHC has successfully occurred (Borgulya et al., 1992; Malissen et al., 1988; Petrie et al., 1995). 3.1.3. Effect of Deregulated RAG Expression Tight regulatory control over RAG expression is important for normal lymphocyte development. Overall numbers of B and T cells are dramatically reduced in transgenic mice expressing RAGs continually during all stages of lymphocyte development (Barreto et al., 2001; Wayne et al., 1994a). The reduction in T-cell numbers reflects a selective reduction in ab T cells, with gd T cell development appearing relatively normal (Barreto et al., 2001). As ab T cells undergo a burst of proliferation after productive TCRb rearrangement and expression of the pre-TCR, the impairment would be consistent with p53-induced cell cycle arrest or apoptosis caused by an abundance of RAG-generated DNA DSBs. Surprisingly, allelic exclusion is maintained even in mice continually expressing RAGs throughout lymphocyte development, indicating that the regulation of antigen receptor gene accessibility is an extremely efficient process (Barreto et al., 2001; Wayne et al., 1994b). Finally, transgenic mice that ubiquitously express RAGs die prematurely and are significantly smaller than control littermates, although it is unclear why (Barreto et al., 2001). 3.2. Regulated Accessibility of Antigen Receptor Gene Segments 3.2.1. Regulated Accessibility and V(D)J Recombination IgH and TCRb rearrangements take place in an ordered fashion, with DH-toJH (Alt et al., 1984) and Db-to-Jb (Born et al., 1985) rearrangements proceeding to completion on both IgH and TCRb alleles, respectively, before the onset of subsequent V-to-DJ rearrangements. Lymphoid-restricted RAG expression limits V(D)J recombination to developing B and T lymphocytes but cannot account for the ordered or stage-specific rearrangement of immunoglobulin and TCR loci (Yancopoulos and Alt, 1985). Regulated gene accessibility imparts ordered rearrangement of antigen receptor genes such that IgH genes assemble before IgL genes in developing B cells, and TCRb genes assemble before TCRa genes in developing ab T cells (reviewed by Krangel,

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2003; Mostoslavsky et al., 2003; Yancopoulos and Alt, 1986). Moreover, during IgH and TCRb gene assembly, D-to-J rearrangements precede V-to-DJ rearrangements and take place on both alleles before the onset of V-to-DJ rearrangements (Alt et al., 1984; Born et al., 1985). In this regard, when nuclei isolated from RAG-deficient lymphocytes are incubated with RAGs in vitro, RS cleavage reflects the lineage and stage specificity of the cell from which the nuclei were isolated (Stanhope-Baker et al., 1996). Moreover, the efficiency of RAG-mediated cleavage of RS-containing extrachromosomal substrates was found to be substantially reduced when the substrate was incorporated into a nucleosome compared with the same substrate in the form of naked DNA (Golding et al., 1999; Kwon et al., 1998). Thus higher order chromatin structure plays an integral role in the regulated accessibility of antigen receptor gene rearrangement (reviewed in Bassing et al., 2002b; Hesslein and Schatz, 2001; Krangel, 2003). 3.2.2. Transcription and V(D)J Recombination Recombinational activity of integrated immunoglobulin or TCR transgenes is often largely dependent on the presence and function of associated transcriptional enhancer elements (reviewed in Ferrier et al., 1990; Krangel, 2003; Raulet et al., 1985; Sleckman et al., 1996). As the site of integration and number of integrated copies can influence the expression of transgenes, gene-targeted ablation of regulatory cis elements at endogenous loci was used to assess more directly their function in recombination. There are transcriptional enhancer and promoter elements associated with all antigen receptor loci (reviewed in Hempel et al., 1998; Hesslein and Schatz, 2001). Early observations that V(D)J recombination generally correlated with the appearance of germline transcripts at various antigen receptor loci implicated regulatory cis elements as playing a role in recombinational accessibility (Alessandrini and Desiderio, 1991; Fondell and Marcu, 1992; Goldman et al., 1993; Schlissel and Baltimore, 1989; Yancopoulos and Alt, 1985). In the IgH locus, gene-targeted deletion of the intronic IgH enhancer (iEm) substantially reduced VH-to-DJH but not DH-to-JH rearrangement in developing B lymphocytes (Chen et al., 1993; Serwe and Sablitzky, 1993). In the Igk locus, elimination of both the intronic Igk (iEk) and 30 (30 Ek) enhancers completely abolished Vk-to-Jk rearrangement (Inlay et al., 2002) and separate deletion of one or the other led to a substantial reduction in Vk-to-Jk rearrangement (Gorman et al., 1996; Xu et al., 1996). In T cells, deletion of the TCRb enhancer (Eb) substantially reduced the level of TCR DJb and VbDJb transcripts, although some Vb-associated germline transcripts were still present (Bories et al., 1996; Bouvier et al., 1996; Mathieu et al., 2000). A corresponding decrease in levels of DJb and VbDJb

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rearrangements in Eb/ lymphocytes was also found, and overall ab T-cell development was severely impaired (Bories et al., 1996; Bouvier et al., 1996). Gene-targeted ablation of the TCRa enhancer (Ea) resulted in a severe reduction in germline Ja transcripts and TCRa rearrangement in developing T cells (Sleckman et al., 1997). On the other hand, deletion of Ea did not substantially alter the level of TCRd rearrangements in these same cells, even though TCRd gene segments are distributed among TCRa gene segments in this locus (Sleckman et al., 1997). In contrast, mice carrying a deletion of the TCRd enhancer (Ed) had substantial impairment in VdDJd rearrangements but were normal for TCRa/b development (Monroe et al., 1999c). Whereas Ed was required for TCRd transcripts in DN thymocytes, TCRd transcripts were unaffected in the few gd T cells that developed in the absence of Ed (Monroe et al., 1999c). Thus Ed differentially regulates early but not late gd T-cell processes. Antigen receptor gene segment–associated germline promoters have also been shown to affect V(D)J recombination. Deletion of the germline Db1 promoter substantially reduced germline Db1 transcripts and Db-to-Jb1 rearrangement levels but did not affect transcription or rearrangement involving Db2/Jb2 gene segments (Whitehurst et al., 1999). Similarly, deletion of the T early a (TEA) germline promoter upstream of the 50 -most Ja gene segments eliminated germline transcripts associated with upstream Ja gene segments and reduced Va-to-Ja rearrangements corresponding to these same Ja segments (Villey et al., 1996). However, germline Ja transcripts initiating downstream of TEA were detectable in thymocytes lacking TEA, and overall levels of TCRa rearrangements were normal (Villey et al., 1996). Regarding lineage specificity, targeted replacement of the TCRb enhancer with iEm promoted transcription and rearrangement of the TCRb locus at levels substantially higher than in developing T cells with Eb deleted (Bories et al., 1996; Bouvier et al., 1996), demonstrating that iEm can function outside of the IgH locus to promote accessibility of heterologous sequences. VbDJb rearrangements did not occur at appreciable levels in B lineage cells carrying iEm in place of Eb, although germline JbCb transcripts were readily detectable in these same cells (Bories et al., 1996). However, when a larger region encompassing Cb2 and Eb was replaced with iEm, significant levels of Db-to-Jb rearrangements were detected in splenic B cells (Eyquem et al., 2002), suggesting that elements within the larger region have the ability to suppress TCRb accessibility in B lineage cells. When Eb was replaced with the TCRa enhancer (Ea), there was a significant reduction in Db germline transcripts in CD25þCD44CD4CD8 (DN3) thymocytes, the stage at which rearrangements of TCRb gene segments normally take place. However, levels of germline Db transcripts were normal in CD4þCD8þ

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thymocytes carrying the Eb-to-Ea replacement, the stage during which TCRa is normally active. Thus Ea affected TCRb transcription in a manner corresponding to its normal functional stage. However, levels of Vb germline transcripts in DN3 thymocytes appeared normal, and overall levels of DJb and VbDJb rearrangements were only modestly reduced, demonstrating that Ea could function to promote VbDJb recombination differently than it would normally at the TCRa locus. Finally, TCRa rearrangements were dramatically reduced when Ea was replaced with either the TCRd enhancer (Ed) or iEm (Bassing et al., 2003b), demonstrating that Ea must carry elements important for the regulated rearrangement of TCRa genes that cannot be replaced by other enhancer sequences. Thus, promoter/enhancer interactions clearly influence transcription as well as RAG-mediated recombination at specific sites within antigen receptor loci (reviewed in Bassing et al., 2000; Krangel, 2003). 3.2.3. Chromatin Modifications Chromatin is made up of complexes of protein and DNA that allow the packaging of approximately 1- to 10-cm lengths of unwound chromosomal DNA into a nucleus with a diameter of only 3–10 mm (Alberts et al., 1983). The structure of chromatin begins with 146 bp of DNA wrapped around a complex of histone proteins that form a nucleosome. Each nucleosome consists of eight histone molecules, two copies each of histone family proteins H3, H4, H2A, and H2B (Alberts et al., 1983). The histone protein H1 then links nucleosomes into the higher ordered structure of 30-nm fibers, which are condensed even further during interphase of the cell cycle (reviewed in Belmont et al., 1999). The regulation and control of higher order chromatin is an integral component of transcriptional activation and repression of eukaryotic genes (reviewed in Udvardy, 1999), as well as DNA replication (reviewed in Gerbi and Bielinsky, 2002). The epigenetic regulation of chromatin accessibility is associated with histone acetylation, phosphorylation, methylation, and ubiquitination (reviewed in Berger, 2002), as well as DNA methylation (reviewed in Richards and Elgin, 2002). Histone modifications have been associated with actively recombining extrachromsomal substrates and endogenous antigen receptor loci (reviewed in Krangel, 2003; Oettinger, 2004). Treatment with histone deacetylase inhibitors has been shown to induce RS accessibility and V(D)J recombination within the Igk, TCRg, and TCRb loci of cells otherwise inaccessible because of higher order chromatin (Agata et al., 2001; Mathieu et al., 2000; McBlane and Boyes, 2000). In addition, deletions of cis-regulatory elements necessary for endogenous V(D)J recombination have been linked to reduced levels of histone acetylation of antigen receptor locus–associated sequences (Agata et al.,

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2001; Mathieu et al., 2000). Furthermore, chromatin near the Db1Jb1 region of Eb-deficient thymocytes, which have reduced levels of TCRb rearrangements (see above), contained alterations in histone acetylation, methylation, and phosphorylation (Spicuglia et al., 2002). These data are consistent with the hypothesis that such combinatorial interactions and modifications lead to an epigenetic regulatory system or a so-called ‘‘histone code’’ that in some manner may promote recombinational accessibility at antigen receptor loci (reviewed in Bassing et al., 2002b; Jenuwein and Allis, 2001; Oettinger, 2004; Strahl and Allis, 2000). Methylation of specific lysine residues on histone H3 has also been shown to correlate with recombinational activity at the IgH and TCRb loci (Morshead et al., 2003; Ng et al., 2003; Spicuglia et al., 2002; Su et al., 2003). However, targeted recruitment of a histone methyltransferase to chromosomal recombination substrates blocks transcription and recombination of nearby segments (Osipovich et al., 2004), illuminating the complexity of such regulation. DNA hypomethylation at CpG dinucleotides has been shown to correlate with transcription in general (Razin and Riggs, 1980), as well as with specific antigen receptor gene segments (Kelley et al., 1988; Mather and Perry, 1981, 1983). Regarding recombinational accessibility, methylation at a single CpG site within the 30 RS of Db1 did not allow cleavage by RAGs (Whitehurst et al., 2000). In addition, Vk-to-Jk rearrangements appear limited to hypomethylated alleles, and thus DNA methylation may also play a role in allelic exclusion (Mostoslavsky et al., 1998). However, as not all actively recombining antigen receptor loci display hypomethylated status (Villey et al., 1997), DNA methylation does not always result in elevated recombinational accessibility (Cherry et al., 2000). Therefore, the overall state of recombinationally accessible antigen receptor gene segments likely involves a variety of interacting modifications involving chromatin and DNA (reviewed in Krangel, 2003). 3.2.4. H2AX Chromatin modifications along antigen receptor loci are also important for monitoring the chromosomal V(D)J recombination reaction to ensure the normal NHEJ-mediated repair of RAG-generated DSBs. There are three subfamilies of histone H2A, of which H2AX comprises 10–15% of total H2A protein in most mammalian cells (Mannironi et al., 1989). In response to IRinduced DNA DSBs, H2AX is phosphorylated on Ser-139, thus producing g-H2AX. g-H2AX is found in discrete foci at the site of DSBs, and these foci occur at a frequency comparable to the number of induced DSBs (Rogakou et al., 1999). Several DNA repair factors including Rad50, Rad51, and Nijmegen breakage syndrome protein (NBS1) have been shown to colocalize with g-H2AX after the induction of DSBs (Chen et al., 2000; Paull et al., 2000).

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Both DNA-PKcs and ATM are able to phosphorylate H2AX in vitro and/or in vivo (Burma et al., 2001; Rogakou et al., 1998; Stiff et al., 2004). g-H2AX and NBS1 have been shown to undergo RAG-dependent colocalization with the TCRa locus in normal thymocytes (Chen et al., 2000), and H2AX-deficient mice have a slight reduction in overall numbers of B and T lymphocytes (Bassing et al., 2003a; Celeste et al., 2002). Nevertheless, the formation of V(D)J-associated CJs and SJs was found to be normal in H2AXdeficient mice (Bassing et al., 2003a; Celeste et al., 2002) and from recombination substrates transiently transfected into H2AX-deficient ES cells (Bassing et al., 2002a). Clearly, H2AX is not essential for the repair of RAG-induced DNA DSBs. However, approximately 4% of nontransformed ab T cells from H2AX-deficient mice contain potential TCRa/d locus translocations (Celeste et al., 2002) and H2AX-deficient mice may exhibit an increased predisposition to thymic lymphomas with potential TCRa/d locus translocations (Bassing et al., 2003a). Thus, H2AX likely serves a critical role in the suppression of aberrant V(D)J recombination, possibly through the proposed ‘‘anchoring’’ function of H2AX in forming a nucleation site for a number of DNA–protein– protein–DNA interactions that might serve to stabilize synaptic complexes of chromosomal RAG-cleaved antigen receptor loci (Bassing and Alt, 2004). 4. Class Switch Recombination Employs Distinct Mechanisms for V(D)J Recombination 4.1. Overview of Class Switch Recombination and Somatic Hypermutation The consequence of successful V(D)J recombination of IgH and IgL chains in developing B cells is the surface expression of IgM and/or IgD. Activation by antigen in the context of certain cytokine stimuli can induce the process of CSR, whereby the V(D)JH exon initially associated with Cm exons is adjoined to one of several groups of downstream CH exons (e.g., Cg, Ce, and Ca, referred to as CH genes) (Fig. 6). Recombination takes place between repetitive sequences, termed S regions, which lie just upstream of the various CH genes (reviewed in Chaudhuri and Alt, 2004). The exchange in CH genes alters the isotype of expressed antibody from IgM to either IgG, IgE, or IgA, along with associated changes in effector function, while maintaining antigenbinding specificity (reviewed in Manis et al., 2002b). DNA sequences located between the recombining S regions can be detected in the form of circularized DNA that has been excised from the genome of the effected B cell (Iwasato et al., 1990). The liberation of circular DNA in CSR is consistent with the participation of DSB intermediates, analogous to excised SJs in V(D)J

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Figure 6 Schematic diagram of the murine IgH locus before and after class switch recombination between Sm and Se. The VH gene segments are depicted as shaded rectangles, the DH segments as solid rectangles, and the JH segments as open rectangles. The Sm regions exons are shown as striped ovals and constant region exons as solid squares. The position of the iEm and 30 RR enhancers are indicated by diamonds. The positions of the VH and I exon promoters are shown as solid circles. Distances between the various elements are not drawn to scale.

recombination that require synapsis and repair over appreciable chromosomal distances. CSR is a mature B-cell–specific process and, unlike V(D)J recombination, does not occur in T lineage cells. In addition to CSR, activation of mature B cells in the context of a germinal center reaction can introduce mutations at a high rate (103 to 104 per base pair per generation) into assembled IgH and IgL variable region exons via a process called SHM. Selection of B cells in which mutated V regions create an antigen receptor of higher affinity than the original results in ‘‘affinity maturation’’ and the generation of a more effective immune response. CSR and SHM rely on the activity of the aicda gene, which encodes activation-induced deaminase (AID), which in turn deaminates cytidine residues on DNA and, thus, forms dU/dG mismatched DNA base pairs (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Petersen-Mahrt et al., 2002; Pham et al., 2003; Sohail et al., 2003; Yu et al., 2004). CSR and SHM likely proceed via subsequent excision of the mismatched dU by the base excision repair protein uracil DNA glycosylase (UNG). UNG creates an abasic site, and differential repair of this lesion apparently leads to either SHM or CSR (Di Noia and Neuberger, 2002; Petersen-Mahrt et al., 2002; Rada et al., 2002b). The mismatch repair (MMR) proteins Msh2/Msh6 can also bind and process the

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dU:dG mismatch and contribute to the CSR and SHM. In this regard, UNG and MMR deficiencies can impair CSR and alter the spectrum of mutations sustained by V genes during SHM. Intriguingly, however, some UNG mutants that have lost the uracil glycosylase activity retain the ability to mediate CSR in mice, leading to the suggestion that UNG may participate in an as yet unidentified manner in CSR that extends beyond its known enzymatic activity (Begum et al., 2004a). Transcription of target S region or variable region target sequences is essential for both CSR (Bottaro et al., 1994; Zhang et al., 1993) and SHM (Bachl et al., 2001; Betz et al., 1994; Fukita et al., 1998; Peters and Storb, 1996). Each CH gene is organized into a transcriptional unit differentially regulated by cytokine-specific transcription factors, thus providing the necessary specificity for directing isotype-specific switching (Fig. 6) (reviewed in Manis et al., 2002b; Stavnezer, 2000). AID deaminates cytidines of ssDNA in vitro (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Pham et al., 2003) but requires transcription of double-stranded dsDNA to generate an appropriate ssDNA substrate (Chaudhuri et al., 2003; Ramiro et al., 2003). Thus, transcription provides an appropriate target substrate for AID to act on (Chaudhuri et al., 2003; Ramiro et al., 2003). The remainder of this review focuses on CSR, although contrasts will be made between CSR and SHM where appropriate. 4.2. Organization of Heavy Chain Constant Region Genes In mice there are eight CH genes located on chromosome 12 and positioned downstream of the antigen receptor gene segments in a region spanning approximately 200 kb (Fig. 6). Cm and Cd are the most JH-proximal group of CH genes, and Cm is the first to associate with a functionally rearranged V(D)JH exon, and therefore IgM is the first isotype expressed on the surface of a B cell. Later in development, expression of IgD occurs via alternative RNA splicing of the V(D)JH exon to the Cd exons. Thus, IgM and IgD are often simultaneously expressed on the surface of the same B cell. Expression of all other immunoglobulin isotypes requires CSR between Sm and downstream S region sequences (e.g., Sg, Se, or Sa), with subsequent loss of the intervening Cm and Cd genes (reviewed in Chaudhuri and Alt, 2004). All CH genes, except Cd, are integrated into transcriptional units consisting of an intervening (I) exon (Lutzker and Alt, 1988), S region, CH exons, and downstream polyadenylation signal sequences corresponding to the membrane and secreted versions of immunoglobulin. Finally, a region downstream of Ca containing enhancer elements, referred to as the 30 regulatory region (30 RR), is important

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for the production of germline transcripts (GTs) and CSR to the various CH genes except Cg1 (reviewed in Manis et al., 2002b). 4.3. Regulation of Class Switch Recombination 4.3.1. B-Cell Activation and Class Switch Recombination V(D)J recombination and early B-cell development take place in the bone marrow. On successful expression of surface immunoglobulin in the form of IgM, immature B cells migrate from the bone marrow to peripheral lymphoid tissues located in the spleen, lymph nodes, and gut-associated lymphoid tissue. Here, at discrete anatomic sites referred to as germinal centers, and often in association with T cells, B cells encounter antigen and undergo antigen-driven clonal expansion that can lead to CSR and/or SHM; although CSR (Macpherson et al., 2001) and SHM (William et al., 2002) can also take place outside of germinal centers as well. CSR is induced in vivo by both T-dependent (TD) and T-independent (TI) antigens. B-cell activation by TD antigens requires interaction of CD40 ligand expressed on activated T cells and CD40 on the surface of B cells. T-independent antigens can activate B cells in the absence of direct T- and B-cell interactions. Type 1 TI antigens, such as lipopolysaccharide (LPS), can act as polyclonal B-cell activators at high concentrations and are able to activate B cells in the complete absence of T cells. Type 2 TI antigens, on the other hand, which usually consist of highly repetitious molecules, do not require direct B- and T-cell interactions to induce B-cell activation or CSR, although B-cell activation and CSR occur inefficiently in the absence of T-cell–derived cytokines. TD antigen stimulation can be mimicked in vitro by culturing B cells in the presence of anti-CD40 along with specific cytokines, and TI activation can be mimicked by treatment with LPS plus or minus the addition of specific cytokines (reviewed in Manis et al., 2002b; Stavnezer, 2000). In concert with antigen-dependent activation, cytokine-induced signaling provides specificity to CSR (reviewed in Manis et al., 2002b; Stavnezer, 2000). For instance, LPS induces isotype switching to IgG2b and IgG3, whereas LPS plus interleukin 4 (IL-4) induces isotype switching to IgG1 and IgE (reviewed in Manis et al., 2002b; Stavnezer, 2000). 4.3.2. Germline CH Transcripts Isotype switching to a particular CH gene is preceded by transcription of the corresponding germline sequences (reviewed by Chaudhuri and Alt, 2004; Manis et al., 2002b; Stavnezer, 2000). Transcripts initiate upstream of I exons found 50 of each CH gene and terminate at polyadenylation sites located

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downstream of CH genes (Lutzker and Alt, 1988). These transcripts undergo RNA splicing between the I and CH exons to form processed GTs in which the intervening S region sequences are deleted (reviewed in Chaudhuri and Alt, 2004). Transcription is regulated by I region promoter sequences that are activated by CD40-, LPS-, and cytokine-mediated signals (Fig. 6) (reviewed in Stavnezer, 2000). Deletion of I region promoters and subsequent loss of recombination involving the associated CH gene demonstrated the dependence on S region transcription for CSR (Jung et al., 1993; Lorenz et al., 1995; Zhang et al., 1993). Furthermore, targeted replacement of I promoters with heterologous, constitutively active promoters rescues S region transcription and directs isotype switching to the corresponding CH genes (Bottaro et al., 1994; Lorenz et al., 1995; Seidl et al., 1998). GTs do not encode functional proteins, but several studies have implicated RNA splicing and processing as potentially playing a role in the CSR process, as targeted mutations in specific splice sites strongly inhibited CSR to the corresponding CH gene (Hein et al., 1998; Lorenz et al., 1995). However, it now seems clear that the major role of GTs is to provide the appropriate ssDNA substrate for AID (see Sections 4.4 and 4.5, below) (reviewed in Chaudhuri and Alt, 2004). In the IgH locus, VH-to-DJH rearrangements are impaired in the absence of the intronic enhancer (iEm) that lies in the intronic region between the JH gene segments and Cm just uptream of Sm; thus it appears that iEm serves an essential role in promoting VH-to-DJH rearrangement in developing B cells (Sakai et al., 1999a; Serwe and Sablitzky, 1993). In this regard, CSR is also reduced in the absence of iEm, which serves a promoter function for Im-Cm–containing GTs (Bottaro et al., 1998; Gu et al., 1993; Sakai et al., 1999a; Su and Kadesch, 1990). Conceivably, CSR that occurs in the absence of iEm may be mediated by transcription of Im sequences initiated by heterologous promoters such as those associated with VH or DH gene segments (Gu et al., 1993; Kuzin et al., 2000). Notably, removal of iEm along with a large region of potential upstream promoters resulted in reduced Sm recombination, but left substantial recombination within downstream S regions, which manifest as internal S region deletions (Gu et al., 1993). Thus, iEm does not appear to function as an essential enhancer element for promoting CSR to downstream CH genes, which may be more dependent on the activity of the 30 RR (see below). The production of GTs is influenced by enhancer-like elements located in the region found approximately 15 kb downstream of Ca. The 30 RR is an 40kb region composed of four elements corresponding to hypersensitivity sites (50 -HS3a-HS1,2-HS3b-HS4-30 ), which in various combinations have been shown to possess locus control region (LCR)–like activity (reviewed in

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Madisen and Groudine, 1994; Manis et al., 2002b). Targeted replacement of HS1,2 with a pgk-driven neomycin resistance gene cassette (pgk neor) disrupts the production of GTs and CSR to all CH genes except IgG1 and IgA, some of which are located as far as 120 kb upstream (Cogne et al., 1994). This suggests that either I promoter–driven transcription of CH genes is dependent on this site, or that pgk neor blocked the activity of an unknown downstream element (Cogne et al., 1994; Manis et al., 1998b). In support of the latter possibility, production of GTs and CSR was rescued when the neor cassette was deleted from the locus, leaving a loxP site in place of HS1,2 (Manis et al., 1998b). Similar studies demonstrated that HS3a is also dispensable for CSR (Manis et al., 1998b). Furthermore, targeted insertion of the pgk neor cassette at several locations throughout the IgH locus inhibited the production of GTs and CSR of CH genes located upstream of the pgk neor insertion, but not downstream (Seidl et al., 1999). Thus the pgk neor cassette appears to preferentially compete with CH promoters for an enhancer activity found in the region downstream of HS1,2. Clean deletion of HS3b and HS4 together has the identical effect as the targeted replacement of HS1,2 or HS3a with the pgk neor cassette, namely a significant reduction in the production of all immunoglobulin isotypes except IgM and IgG1, with a modest reduction in IgA (Pinaud et al., 2001). Finally, insertion of the pgk neor cassette downstream of HS4 did not reduce CSR or the production of GTs, which, together with the other data, strongly implies that the enhancer activity is contained within the HS3b or HS4 sequences, and possibly both (Manis et al., 2003). 4.3.3. Class Switch Recombination and Somatic Hypermutation Are Region-Specific Events CSR is targeted to S regions, found upstream of each CH gene, that consist of 1–12 kb of repetitive sequences (Fig. 6) (Kataoka et al., 1980). Each unique S region is made up of tandem repeat units varying in length between 5 bp (Sm) and 80 bp (Sa) (reviewed in Honjo et al., 2002) with some varying degrees of overall homology (reviewed in Stavnezer, 1996). In contrast to the sitespecific cleavage mediated by RAGs at RSs in V(D)J recombination, CSR between two S regions can occur throughout, and even outside, the core repeat sequences (Dunnick et al., 1993; Lee et al., 1998; Luby et al., 2001). Sequencing the break points of CSR junctions failed to identify a consensus target sequence either in relation to the overall S regions or in the context of the short pentameric repeats, confirming that CSR is a region-specific rather than site-specific event (Dunnick et al., 1993; Lee et al., 1998). The lack of consensus sequences at CSR junctions suggests that rather than depending on sequence-specific recognition, the CSR ‘‘recombinase’’ might instead be targeted via the formation of S region–specific higher order

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structures. The S regions of most vertebrates are rich in G and C nucleotides, with a bias for purine nucleotides on the nontemplate strand of DNA (Shinkura et al., 2003). Such properties have been shown to promote the formation of RNA–DNA hybrids (Mizuta et al., 2003; Reaban and Griffin, 1990; Reaban et al., 1994; Tian and Alt, 2000) that could lead to the generation of structures such as R loops (Tian and Alt, 2000; Shinkura et al., 2003; Yu, 2003) and G quartets (Dempsey et al., 1999; Sen and Gilbert, 1988). In contrast, the S region sequences in Xenopus contain approximately 60% A and T nucleotides, consistent with the overall nucleotide composition of the Xenopus genome (Mubmann et al., 1997). However, Xenopus S regions are highly repetitive and contain palindromic sequences similar to those that target SHM, which conceivably could effect CSR in the absence of potential higher order structures. (Mubmann et al., 1997; Zarrin et al., 2004). Deletion of the core tandem repeat sequences of Sm significantly reduced CSR in mice (Luby et al., 2001). The remaining level of CSR detected in these mice might be due to the retention of a considerable amount of G-rich and short palindromic sequences left upstream of Cm (Luby et al., 2001), although complete deletion of all Sm tandem repeats further reduces, but does not eliminate, CSR (Khamlichi et al., 2004). Thus, transcriptional activation from the iEm enhancer may by itself be enough to induce low levels of recombination (Khamlichi et al., 2004). On the other hand, complete deletion of Sg1 sequences in mice essentially blocks CSR to the deleted allele (Shinkura et al., 2003). Finally, the lack of an associated S region apparently prevents the usage of the cCg gene in humans, as it is located in the correct transcriptional orientation and lacks mutations in its coding sequence that result in frameshift or stop codons or would otherwise prevent functional expression (Bensmana et al., 1988). Therefore, the presence of an S region appears critical for normal CSR. Potential functions of mammalian S regions are discussed in more detail below. SHM is also region specific, as mutations begin in the region just downstream of an IgH or IgL V promoter, are found throughout the variable region exons, and are detected as far as 2 kb downstream of V promoters within the intronic region between J and C exons (reviewed in Harris et al., 1999). However, most of the introduced mutations occur within the assembled variable regions that form the antigen-binding portion of an antibody molecule or in nearby flanking sequences (reviewed in Harris et al., 1999; Papavasiliou and Schatz, 2002b). Mutations are frequently associated with RGYW sequence motifs (where R is A or G, Y is C or T, and W is A or T). The most common changes are point mutations, with transitions being slightly favored over transversions, although small deletions and duplications are also detected. Targeting to RGYW sequences likely reflects specific recruitment and

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specificity of the recombinase machinery (Chaudhuri et al., 2004; see below), whereas mutations concentrated in sequences making up antigenbinding regions are partly the result of the selection for high-affinity antigen receptors in response to antigen during affinity maturation (Griffiths et al., 1984). 4.3.4. Induced Mutations in and Around S Region Sequences Sequencing of CSR junctions reveals frequent DNA alterations in the form of single-nucleotide mutations and small deletions (Dunnick et al., 1993; Lee et al., 1998). These mutations are AID dependent (Petersen et al., 2001) and can be found both 50 and 30 of the Sm region in wild-type B cells activated for, but that have not undergone, CSR (Dudley et al., 2002; Nagaoka et al., 2002; Petersen et al., 2001). CSR is therefore frequently associated with mutations that resemble those induced by SHM, likely reflecting the common AID deamination event in the initiation of both CSR and SHM. Thus, the resolution of a common DNA lesion generated by AID by different downstream repair pathways could result in either DNA recombination or mutation (reviewed in Chaudhuri and Alt, 2004). 4.4. Activation-Induced Cytidine Deaminase 4.4.1. Discovery and Isolation of Activation-Induced Cytidine Deaminase The discovery of AID and the subsequent demonstration of its essential role in CSR and SHM have led to rapid advances toward the elucidation of the mechanisms that effect CSR and SHM (reviewed in Kenter, 2003; Reynaud et al., 2003). AID was isolated via a subtractive cloning screen from a murine B-cell line (CH12) that on activation switches from IgM to IgA (Muramatsu et al., 1999). Expression of AID is limited to developing germinal center B cells (Muramatsu et al., 1999) and can be induced in vitro by culturing splenic B-cells in the presence of activating stimuli known to induce CSR (Muramatsu et al., 1999). AID deficiency completely abrogates CSR and SHM in both humans (Revy et al., 2000) and mice (Muramatsu et al., 2000), and expression of AID in nonlymphoid cell lines induces at least limited CSR (Okazaki et al., 2002) and SHM (Martin et al., 2002b; Yoshikawa et al., 2002). Furthermore, overexpression of AID in bacteria can lead to mutations in several transcribed genes (Petersen-Mahrt et al., 2002b; Ramiro et al., 2003). Thus, analogous to RAGs with respect to V(D)J recombination, AID is both necessary and sufficient to effect CSR and SHM in the context of proteins expressed in nonlymphoid cells. It is to be noted, however, that the rate of CSR and the

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spectrum of mutations observed in nonlymphoid cells on artificial substrates do not faithfully recapitulate that observed at endogenous loci in B cells, suggesting that B-cell–specific factors and/or AID modifications (see below) contribute to these processes. 4.4.2. Activation-Induced Cytidine Deaminase Expression High-level expression of AID via stable integration of an AID transgene can induce mutations within actively transcribed nonimmunoglobulin genes, including the AID transgene itself, both in lymphoid and nonlymphoid cell lines (Martin and Scharff, 2002b; Yoshikawa et al., 2002). Constitutive and ubiquitous expression of AID in mice via a transgene leads to T-cell lymphomas and adenosarcomas (Okazaki et al., 2003) and deregulated AID expression has been detected in several types of human non-Hodgkin lymphomas (Greeve et al., 2003; Hardianti et al., 2004a,b). Moreover, mature human and mouse B lineage tumors often have translocations that fuse S regions with oncogene loci (reviewed by Mills et al., 2003). Work has shown, in mouse models, that such translocations are dependent on AID (Ramiro et al., 2004). Thus, tight regulatory control of AID expression is necessary to prevent generalized genomic mutations and genomic instability. AID is expressed in activated B lymphocytes in the context of a germinal center reaction, precisely in those cells that undergo SHM and CSR in vivo (Muramatsu et al., 1999). Expression of AID is modulated by inhibitors of differentiation (Id) proteins, as ectopic expression of Id2 or Id3 reduces AID expression in activated splenic B cells and inhibits CSR (Gonda et al., 2003; Sayegh et al., 2003). Id proteins are best known as antagonists of the E family of transcription factors (E proteins), a class of basic helix–loop–helix proteins that bind DNA at conserved E box sites as homo- and heterodimers (reviewed in Quong et al., 2002; Sun, 2004). Id proteins form heterodimers with E proteins that are unable to bind DNA, thus negatively regulating transcriptional activation by E proteins (Benezra et al., 1990a,b; Christy et al., 1991; Riechmann et al., 1994; Sun et al., 1991). Id proteins have also been shown to interact with members of Pax and Ets families of transcription factors, likewise inhibiting their DNA-binding functions (Roberts et al., 2001; Yates et al., 1999). E12, E47, and Pax5 are vital for B-cell development (Bain et al., 1994; Urbanek et al., 1994; Zhuang et al., 1994), and their expression is highly induced in mature B cells by CSR-inducing stimuli (Gonda et al., 2003; Quong et al., 1999). E47 and Pax5 have both been shown to bind regulatory elements upstream of AID in vivo (Gonda et al., 2003; Sayegh et al., 2003), and the Pax5 element was shown to be essential for AID gene expression (Gonda et al., 2003).

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4.4.3. Activation-Induced Cytidine Deaminase Deaminates dC Residues of Single-Stranded DNA Substrates: Interaction with Replication Protein A AID is a ssDNA cytidine deaminase (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Pham et al., 2003; Sohail et al., 2003) and such ssDNA substrates are probably revealed during transcription through the S regions (reviewed in Chaudhuri and Alt, 2004). Transcription of S regions, in their physiological orientation, generates ssDNA in the context of R loops in vitro (Shinkura et al., 2003; Tian and Alt, 2000; Yu et al., 2003) and in vivo (Yu et al., 2003) and such transcribed DNA can serve as targets of AID deamination in vitro (Chaudhuri and Alt, 2004; see below). In addition to R loops, other mechanisms may operate to target AID activity to S regions (see below). In this regard, variable region exons do not have repetitive sequences or unusual GC content that would lead to R loop formation and do not form R loops when transcribed in vitro (reviewed in Chaudhuri and Alt, 2004; Papavasiliou and Schatz, 2002b), yet they are targeted by AID during SHM. These observations suggested a specific cofactor to target AID during SHM and such a cofactor was identified (Chaudhuri et al., 2004) as replication protein A (RPA), a heterotrimeric ssDNA-binding protein involved in replication, recombination, and repair (reviewed in Wold, 1997). RPA stabilizes ssDNA (Wold, 1997) and can bind short stretches of ssDNA bubbles and recruit nucleotide excision and base excision repair proteins (reviewed by Binz et al., 2004; Matsunaga et al., 1996). AID forms a specific complex with RPA that facilitates AID-induced DNA deamination of transcribed RGYW-containing substrates (Chaudhuri et al., 2004). The efficiency of substrate binding and deamination by RPAAID complexes was dependent on the number of RGYW motifs, and deamination was observed at or around these sequences (Chaudhuri et al., 2004). Thus RPA likely functions to target AID to transcribed SHM hot spots found in V region exons of IgH genes. Significantly, the AIDRPA complex is B-cell specific, and this specificity appears regulated, at least in part, by the phosphorylation status of AID in B cells (Chaudhuri et al., 2004). Other findings support the notion that the AIDRPA complex may also be involved in CSR, particularly in the context of Xenopus S regions that lack R loop-forming ability but contain regions of RGYW motifs (Zarrin et al., 2004; see below). Also, it is possible that RPA that remains bound to the deaminated mammalian S region substrate, as proposed for SHM, also can actively recruit proteins that are downstream of deamination, such as UNG and MMR proteins, to the site of initial DNA lesions in CSR.

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4.4.4. Apobec1 and Other Deaminase Family Members AID shares significant sequence homology with Apobec-1 (34% amino acid identity), a known cytidine deaminase (Muramatsu et al., 1999), and has been shown to catalyze the deamination of free CTP nucleotides in vitro (Muramatsu et al., 1999; Papavasiliou and Schatz, 2002a). Apobec-1 functions as an RNA-editing enzyme, inducing a C-to-U conversion at position 6666 of the ApoB mRNA transcript, hence changing Gln-2153 into an in-frame stop codon (reviewed in Chan et al., 1997). The edited transcript encodes ApoB-48, a protein that, although colinear with the N-terminal 2152 residues of fulllength ApoB-100, has significantly altered biological function (reviewed in Chan, 1992). The shared homology with Apobec-1 led to the proposal that AID may edit an mRNA transcript of unknown function, thus generating a novel class switch recombinase and/or V region mutator (Muramatsu et al., 2000). This model, which contrasts with most data arguing for a DNA deamination activity for AID in CSR and SHM, was supported, albeit quite indirectly, by the finding that de novo protein synthesis is required for AID to induce CSR (Begum et al., 2004b; Doi et al., 2003). The cytidine deaminase ApoBec3G acts as an inhibitor of the human immunodeficiency virus type (HIV-1) retrovirus, not by mutating the genomic viral RNA or RNA transcripts, but by introducing dG-to-dA mutations into the newly synthesized viral DNA (reviewed in Neuberger et al., 2003). Furthermore, although RNA is the physiological substrate for Apobec-1, AID (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Ramiro et al., 2003) and other Apobec-1 family members (Harris et al., 2002; Petersen-Mahrt and Neuberger, 2003) can deaminate dC residues of nontranscribed ssDNA or transcribed dsDNA substrates in vitro. Furthermore, overexpression of Apobec-1 in bacteria, as well as other Apobec-1 family members including AID, can lead to dC-to-dG mutations in bacterial DNA (Harris et al., 2002; Petersen-Mahrt et al., 2002). These mutations were dependent on the catalytic function of the transfected deaminase vectors, as mutations in Znþ coordination motifs required for deaminase activity abolished this mutagenic effect (Harris et al., 2002). Thus Apobec-1 family members including AID, but not Apobec-1 itself, function via a DNA deamination process that is dependent on ssDNA, rather than the previously proposed RNA-editing model, further weakening the argument that AID acts via RNA editing. Notably, Apobec-1 is unable to induce CSR or SHM when overexpressed in B cells, as does AID (Eto et al., 2003; Fugmann et al., 2004), although Apobec-1 can function in vitro to deaminate DNA; it is conceivable that lack of such complementing activity may, in part, reflect inability to recruit cofactors such as RPA. Overall, current evidence suggests the possibility that

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AID may well have evolved from a family of DNA-editing proteins, with Apobec-1 being the outlying protein that may have evolved a new RNA-editing function. 4.5. Mechanism of Class Switch Recombination and Somatic Hypermutation 4.5.1. S Region Transcription and Activation-Induced Cytidine Deaminase Substrate Formation Mammalian S regions display a strong G-rich nontemplate strand bias (reviewed in Manis et al., 2002b). In this regard, inversion of the Sg1 region in vivo significantly reduces recombination of the corresponding allele (Shinkura et al., 2003). This strongly suggests that CSR, at least to Cg1, is influenced by the transcriptional orientation of S region sequences in vivo. Targeted replacement of Sg1 sequences with randomly generated purine- or pyrimidine-rich sequences supported these findings (Shinkura et al., 2003). Replacement of Sg1 with a 1-kb sequence that when transcribed produces a highly purine-rich transcript was able to target recombination to Cg1, albeit at a reduced level compared with the endogenous Sg1 sequences (Shinkura et al., 2003). The reduction in CSR efficiency is likely due in significant part to the difference in overall length of available target sequences (1 kb for the synthesized regions compared with 12 kb for the endogenous Sg1 region) (A. Zarrin, and F. Alt, unpublished data). When this 1-kb sequence was inverted, so that pyrimidine-rich instead of purine-rich transcripts are generated, recombination was reduced to levels comparable to that of an allele completely lacking Sg1 (Shinkura et al., 2003). In vitro, transcription of substrates with pyrimidine-rich sequences on the template strand produces purine-rich transcripts that form stable RNA–DNA heteroduplexes (R loops and collapsed R loops) with the DNA template strand and result in stretches of ssDNA that have been shown to exist in vitro (Fig. 7) (Mizuta et al., 2003; Reaban and Griffin, 1990; Reaban et al., 1994; Tian and Alt, 2000). Furthermore, R loop formation is orientation dependent, as substantial levels of R loops do not form when these same sequences are transcribed in the opposite transcriptional orientation (Shinkura et al., 2003; Tian and Alt, 2000). Thus R loop structures form under circumstances that also lead to CSR in vivo and promote stretches of ssDNA that could provide the necessary substrate for AID deamination (Fig. 7). Notably, such stable R loop structures were demonstrated to occur within endogenous S regions when they were transcribed in vivo, indicating that they may well serve the physiological function of providing an AID substrate that was generated from in vitro studies (Yu et al., 2003).

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Figure 7 Transcription of DNA results in the formation of secondary structures that provide the target substrate for AID. The indicated secondary structural changes induced into S regions (Sx) by transcription have all been proposed to play a role in CSR and/or SHM. R loops, which have been demonstrated to form in vivo, occur when RNA transcripts stably interact with the DNA template strand. AID deaminates cytidine residues preferentially on the coding strand, thus leading to DNA lesions that effect CSR and SHM. Adapted from Chaudhuri and Alt (2004).

Inversion of endogenous Sg1 sequences did not completely abolish recombination in vivo (Shinkura et al., 2003). This would imply that either RNA– DNA hybrid structures are still generated at a reduced level when Sg1 is transcribed in the nonphysiological orientation, or there are other means of providing the necessary substrates for AID and CSR. In addition, as Xenopus S regions are A-T rich instead of G-C rich, the transcription of Xenopus S region sequences would not be predicted to form R loops (Mubmann et al., 1997). Transcription of palindrome-containing sequences found in S regions is also prone to the formation of stem–loop structures (Kataoka et al., 1981; Mubmann et al., 1997; Tashiro et al., 2001). Unlike R loops, stem–loop structures should form on transcription of palindromic sequences regardless of transcriptional orientation. However, like R loops, stem loops can promote the formation of short stretches of ssDNA that could provide appropriate substrates for AID. Therefore, there may be several ways in which the unusual

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sequence content of S regions could lead to the generation of structures containing ssDNA that would be a substrate for AID activity. Finally, all S regions (from Xenopus to mammals) have a high concentration of the consensus SHM motif (most notably AGCT), and evidence suggests that this sequence could target AID in CSR in the context of an AIDRPA complex as it does in SHM (Zarrin et al., 2004). It has been proposed that S regions may have evolved from sequences containing large numbers of SHM motifs (e.g., AGCT) in lower organisms to the form found in mammals in which high levels of such motifs remain (to target AID in the context of RPA), but in mammalian S regions, ssDNA generation may have been further augmented via evolution of ability to form R loops (Zarrin et al., 2004). 4.5.2. AID-Induced Cytidine Deamination Cytidine deaminases catalyze the conversion of dC to dU residues via the hydrolytic removal of the amino group at the fourth position of the pyrimidine ring of cytidine (Betts et al., 1994). The deamination mechanism is likely to resemble that of adenosine deaminases as both involve a zinc atom in the active site (Betts et al., 1994; Harris et al., 2002). Deamination of dC residues by AID thereby induces dU/dG mismatches in DNA, the type of DNA lesions normally corrected by the base excision repair and MMR pathways (reviewed in Lindahl, 2000). 4.5.3. Base Excision Repair and Uracil DNA Glycosylase The base excision repair pathway has evolved to provide protection against structural alterations that can occur in DNA as a result of various endogenous alkylating agents and metabolic reactive oxygen species (reviewed in Lindahl, 2000). Such alterations lead to the formation of aberrant nucleotide residues that are frequently recognized by DNA glycosylases (reviewed in Krokan et al., 1997). These glycosylases recognize the aberrant nucleotide and remove it from the DNA backbone, leaving behind an abasic site (reviewed in Krokan et al., 1997). In the case of cytidine deamination, this function is performed by UNG. The abasic site is then cleaved by an apyrimidic (AP) endonuclease, followed by nucleotide replacement via the action of a polymerase, which is normally polymerase b (polb) (reviewed in Lindahl, 2000). The final repair step is ligation, likely involving DNA ligase III (reviewed in Lindahl, 2000). 4.5.4. DNA Deamination Model The DNA deamination mechanism for CSR and SHM was initially proposed on the basis of the observation that AID expression in bacteria caused mutations that somewhat resemble those induced by SHM (Petersen-Mahrt et al.,

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Figure 8 The DNA deamination model for CSR and SHM. AID deaminates cytidine residues in S regions, and in the variable region exons of IgH and IgL genes. Steps that lead to SHM are shown on the left and those that effect CSR on the right. UNG, uracil deglycosylase; AID, activation-induced cytidine deaminase. The minor pathway for CSR refers to CSR in the absence of UNG, likely involving mismatch repair (MMR). Adapted from Rada et al. (2002) and Di Noia and Neuberger (2002).

2002). In this model, replication or repair of dU-containing DNA strands leads to both CSR between S regions and SHM of immunoglobulin VH and VL exons (Fig. 8; Petersen-Mahrt et al., 2002). Mutations could be generated if the dU residue resulting from AID-induced cytidine deamination is not removed by base excision repair before DNA replication, wherein the dU is read as a dT, thus resulting in dC-to-dT and dG-to-dA transitions (Fig. 8; Petersen-Mahrt et al., 2002). Alternatively, if the abasic site produced by the function of UNG undergoes replication via a translesional polymerase (reviewed by Chaudhuri and Alt, 2004; Reynaud et al., 2003), subsequent repair of the abasic site would lead to both transitions and transversions (Fig. 8; Petersen-Mahrt et al., 2002). Moreover, if an error-prone polymerase were recruited to the abasic site during replication, then mutations could be introduced both in and around the initial deaminase-induced lesion, explaining mutations that arise at non-dC

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or non-dG residues in SHM (reviewed in Reynaud et al., 2003). The dU:dG mismatch could also be recognized and processed by components of the MMR machinery, ultimately leading to the generation of DNA breaks. Regarding CSR, if multiple cytidine residues are deaminated in close proximity and on opposite strands, then base excision repair could lead directly to staggered DNA breaks (Petersen-Mahrt et al., 2002). Processing of the ends of staggered DNA breaks could then result in the formation of DNA DSBs that are either blunt or have short overhangs (reviewed in Reynaud et al., 2003). The subsequent repair of DNA DSBs induced in two different S regions by the NHEJ pathway could thus result in CSR. In addition, there are models whereby MMR could also lead to DSBs after AID deamination (reviewed in Martin and Scharff, 2002a). In support of the DNA deamination model with respect to SHM, the pattern of hypermutations observed in the hypermutating chicken cell line DT40 changes from transversion-dominated mutations to transitions when UNG function is inhibited (Di Noia and Neuberger, 2002). Furthermore, there is a dramatic shift in the SHM pattern to transitions in immunoglobulin V genes of murine UNG-deficient B cells (Rada et al., 2002b). As transversions are primarily dependent on the removal of dU residues by UNG, these data provide further support for the DNA deamination model. With respect to class switching, CSR is substantially reduced in UNGdeficient mice and humans, in accordance with the predictions of the model (Rada et al., 2002b). In addition, as mentioned above, AID has been shown to be capable of direct deamination of DNA (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Petersen-Mahrt et al., 2002; Pham et al., 2003; Ramiro et al., 2003; Sohail et al., 2003; Yu et al., 2004). Finally, the observation that AID associates with transcribed S regions (Chaudhuri et al., 2004; Nambu et al., 2003) provides strong support for the DNA deamination model. As mentioned above, work has questioned the precise role of UNG in CSR and SHM, as certain UNG mutants that are catalytically inactive in U removal activity in vitro are still proficient in mediating CSR and SHM in vivo, suggesting that the role of UNG in CSR and SHM is beyond its DNA glycosylase activity (Begum et al., 2004a). These results were surprising given that human patients with similar mutations have profound defects in CSR (Imai et al., 2003a), leading to the speculation that there may be secondary mutations in these patients that contribute to the phenotype (Begum et al., 2004a) or that there is some undetected UNG activity in vivo in the mouse mutants; overall, these apparently conflicting findings await further experimentation for full resolution.

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4.5.5. Class Switch Recombination versus Somatic Hypermutation Specific Factors Hyper-IgM syndrome (HIGM) is caused by defects in CSR leading to reduced levels of IgG, IgA, and IgE (reviewed in Durandy, 2001). Until recently, the known causes of HIGM have been thought to affect both CSR and SHM (reviewed in Durandy, 2001). Some patients with HIGM have now been identified with a novel form of HIGM that results in impaired CSR but normal levels of SHM (Imai et al., 2003b). B cells isolated from the peripheral blood of HIGM4 patients were activated for CSR in vitro and shown to express substantial levels of AID and GTs, yet failed to secrete detectable levels of IgE or IgG (Imai et al., 2003b). Furthermore, using a ligation-mediated polymerase chain reaction (LM-PCR) assay, DNA DSBs corresponding to Sm region sequences were readily detectable in DNA isolated from activated HIGM4 and control B cells (Imai et al., 2003a). Thus HIGM4 may arise from defects in processes downstream of DNA deamination that are distinct between CSR from SHM. CSR and SHM share the requirements for AID-induced DNA deamination; however, AID mutants have now been identified that can differentially effect CSR or SHM. Mutations in the C terminus of AID retain SHM activity but are unable to promote CSR in AID/ B cells (Barreto et al., 2003; Ta et al., 2003). AID C-terminal mutants retained DNA deamination function (Barreto et al., 2003; Ta et al., 2003), and loss of CSR was not due to failure in nuclear transport (Barreto et al., 2003). Furthermore, several mutations in the N terminus of AID had nearly normal CSR activity but were unable to mediate SHM of a retroviral GFP expression construct (Shinkura et al., 2004). Although some of these C-terminal mutants had defects in nuclear import, their ability to effect CSR suggests inefficient nuclear transport is not the cause of defective SHM (Shinkura et al., 2004). AID mutations that uncouple the related but distinct processes of CSR and SHM suggest specific cofactors might exist that interact with these different domains of AID, with RPA being one such potential factor. In this regard, the C terminus of AID has been shown to contain a nuclear export sequence that facilitates nuclear export in a CRM1-dependent pathway (McBride et al., 2004). Although AID must be present in the nucleus to effect CSR and SHM, AID is predominantly found in the cytoplasm (Rada et al., 2002a). Thus AID cofactors such as RPA may play a role in the retention of AID in the nucleus as well as target specificity. 4.5.6. AID-Induced Double-Strand Breaks Past studies have documented DNA DSBs in S regions of cells stimulated for CSR (Chen et al., 2001; Wuerffel et al., 1997) and in variable regions of B cells stimulated for SHM (Bross et al., 2000; Papavasiliou and Schatz, 2000). These

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findings led to the proposal that DSBs may be intermediates in these processes. On identification of AID, early efforts focused on determining whether AID functioned in the induction or resolution of DNA DSBs (reviewed in Chua et al., 2002). The IgH locus has been shown to colocalize with NBS-1 and gH2AX foci, which are normally associated with DSBs, following CSR activation of wild-type but not AID-deficient B cells (Begum et al., 2004b; Petersen et al., 2001). Colocalization was interpreted to reflect the induction of DNA DSBs in S regions; and, therefore, these studies concluded that AID directly participates in the formation of DNA DSBs during CSR (Petersen et al., 2001). However, H2AX was also found to be associated with V genes (Woo et al., 2003), and, therefore, it is not clear whether the H2AX foci indeed represent breaks at S regions or those at V genes, particularly given that H2AX foci can extend for up to 1 Mb from a DSB (Rogakou et al., 1999). In support of DSB intermediates in CSR, LM-PCR assays have detected both AID- and UNG-dependent S region breaks (Catalan et al., 2003; Imai et al., 2003a). In several studies, DNA breaks in V gene segments were found at similar frequencies in both wild-type and AID-deficient B cells stimulated to undergo SHM (Bross et al., 2002; Papavasiliou and Schatz, 2002a). These results led to the suggestion that AID was not involved in the induction of DNA DSBs during SHM, and thus it was speculated that AID might instead be somehow involved in the repair of these DSBs (Papavasiliou and Schatz, 2002a). However, it remains unclear whether the DSBs observed in these studies were actually related to SHM; so the significance of the findings remains unclear (reviewed in Chua et al., 2002). Overall, it seems likely that CSR works via a DSB intermediate, whereas SHM does not; this interpretation has been reinforced by the requirement for factors involved in the DSB response (H2AX, DNA-PKcs, Ku, 53BP1, etc.) in CSR but not SHM (see below). 4.5.7. Internal S Region Deletions Are Analogous to Class Switch Recombination A high frequency of internal S region (intra-S) deletions is detected in the Sm region of normal B cells and B cell lines activated for CSR (Alt et al., 1982; Bottaro et al., 1998; Hummel et al., 1987; Winter et al., 1987). Intra-Sm deletions are largely AID dependent, can occur in the absence of an acceptor S region, and are accompanied by mutations in 30 flanking sequences analogous to those seen in CSR junctions (Dudley et al., 2002). Thus an intra-S region deletion probably reflects the normal CSR mechanism, but in which recombination has taken place within homologous sequences of a single S region rather than between two heterologous S regions. This could result

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via the failure to induce DNA lesions in downstream S regions or in the absence of S region synapsis. 4.5.8. S Region Mutations AID mutants with N-terminal alterations that result in selective loss of SHM activity, but retain the ability to mediate CSR, also generate mutations in Sm (Shinkura et al., 2004). Mutations in AID that selectively lose SHM activity could be the result of failure to target the AID mutant to V region sequences, for instance because of inability to interact with RPA (Chaudhuri et al., 2004). In this regard, induction of Sm mutations by SHM-defective AID mutants would simply reflect the differential targeting of AID and not SHM-versus CSR-specific functions of AID. However, loss of AID-targeting activity does not preclude an SHM-specific function of AID not directly related to a separate CSR-specific activity. Separate studies involving human or murine C-terminal mutants of AID that promote SHM and gene conversion but not CSR have given conflicting results regarding the induction of Sm mutations (Barreto et al., 2003; Shinkura et al., 2004). CSR-defective mutants of murine AID induced normal levels of Sm mutations, whereas most human CSR-defective AID mutants were unable to promote Sm lesions. Sm mutations induced by CSR-defective murine AID mutants (Barreto et al., 2003) could be due to the retention of low-level CSR activity as suggested by an analogous human C-terminal AID mutant, and thus be independent of SHM activity (Shinkura et al., 2004). This would imply that AID contains distinct functions for promoting SHM and CSR; or that the CSR-defective mutant forms of AID fail to target S regions. Alternatively, the CSR-defective AID mutants may properly target S regions and effect DNA lesions, as evidenced by the ability to induce mutations in Sm sequences but fail to complete actual CSR. In this scenario, CSR-defective mutants might be unable to interact with cofactors essential for DNA repair or to facilitate S region synapsis (reviewed by Chaudhuri and Alt, 2004). 4.6. Class Switch Recombination and S Region Synapsis Recombination between two different S regions takes place over large chromosomal distances (up to 175 kb), and these regions must be juxtaposed before being joined. Adjoining of S regions could be mediated via association with transcriptional promoters, enhancers, chromatin factors, DNA repair proteins, or AID-associated factors, or by interactions involving the S region sequences themselves.

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4.6.1. Promoter/Enhancer Interactions CSR is reduced in the absence of iEm, even though the level of steady-state transcription through Sm appeared unaffected (Bottaro et al., 1998). Promoter/ enhancer interactions between iEm and downstream I promoters could effect juxtaposition of Sm with the S regions of downstream CH genes. However, as Sm transcription detected in the absence of iEm could be driven by VH or DH promoters, normal levels of CSR may be dependent on the specific site of transcriptional initiation rather than overall levels of transcription (Bottaro et al., 1998). In this regard, replacement of iEm with a pgk promoter returns the rate of CSR to approximately normal levels (Bottaro et al., 1998). Similarly, although deletions of HS3b and HS4 in the 30 RR located downstream of the IgH locus result in the reduction of GTs, a role for these sites in the synapsis of S region sequences cannot be excluded (Pinaud et al., 2001). 4.6.2. H2AX Effective long-range synapsis of S regions likely relies on chromatin modifications and associated factors, as indicated by studies of H2AX deficiency (Reina-SanMartin et al., 2003). As noted above, AID-dependent H2AX foci are found at the IgH locus in conjunction with IgH CSR (Petersen et al., 2001). SHM is unaffected in H2AX-deficient mice, whereas CSR is substantially impaired (Reina-SanMartin et al., 2003). Intra-S region deletions were detected in H2AX-deficient B cells activated for CSR, demonstrating that accessibility of S regions to the CSR machinery and the basic joining mechanism required for CSR is not impaired by the absence of H2AX (Reina-San-Martin et al., 2003). The recruitment and assembly of repair factors at sites of DNA DSBs by g-H2AX has been proposed to facilitate the juxtaposition of broken DNA ends and subsequent repair by NHEJ proteins (Bassing and Alt, 2004). Thus gH2AX might similarly promote long-range S region synapsis for the efficient recombination between heterologous S regions. In this regard, H2AX-deficient mice, in the absence of the cell cycle checkpoint protein p53, have been shown to undergo translocations involving S region sequences, perhaps indicating that proper synapsis of S regions during CSR is important for genome stability as well as CSR (Bassing et al., 2003a). The finding that another protein, 53BP1, proposed to work in the H2AX anchoring mechanism, is also required for CSR (but not SHM) further supports this general model (Manis et al., 2004). 4.6.3. DNA-PKcs Pro-B cells that lack DNA-PKcs are defective for switching to the IgE isotype (Rolink et al., 1996). However, significant levels of CSR to all immunoglobulin isotypes were detected in a study involving SCID mice reconstituted with

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rearranged IgH and IgL transgenes, which carry a catalytic mutation in DNAPKcs that abrogates the kinase activity of DNA-PKcs (Bosma et al., 2002; Cook et al., 2003). In contrast, DNA-PKcs–deficient mice had a significant reduction in CSR to all isotypes except IgG1 (Manis et al., 2002). Expression of DNAPKcs, albeit catalytically inactive, can be detected in cells from SCID mice, leading to the intriguing possibility that serine/threonine kinase activity of DNA-PKcs is dispensable for CSR, whereas the presence of a noncatalytic DNA-PKcs can provide a necessary function for CSR (Bosma et al., 2002). In the context of this model, why there should still be CSR to IgG1 in DNAPKcs–deficient mice remains a mystery. The fact that DNA-PKcs–deficient B cells switch to IgG1 and not other isotypes implies that recombination between Sm and Sg1 may be mechanistically different than that of CSR between Sm and other S regions. Alternatively, a general reduction in CSR efficiency in the absence of DNA-PKcs could result in the preferential detection of IgG1 simply because it occurs the most efficiently because of its large size. Whatever the case, it is notable that DNA-PKcs is able to promote synapsis of broken DNA ends in vitro (DeFazio et al., 2002), consistent with such a function in CSR. In this regard, transformation/transcription domainassociated protein (TRRAP), a distantly related member of the PI-3 kinase family found in humans with homologs in both yeast and Caenorhabditis elegans, apparently lacks kinase activity and appears to instead function as a scaffolding protein during chromatin remodeling (McMahon et al., 1998). 4.6.4. Mismatch Repair Mlh1- and Pms2-deficient mice have a modest reduction in CSR activity, and sequences isolated from S junctions of Mlh1- and Pms2-deficient B cells have an increased rate of microhomologies compared with wild-type B cells (Ehrenstein et al., 2001; Schrader et al., 2002). Yeast homologs of PMS2 and MLH1 can bind two different DNA molecules simultaneously (Hall et al., 2001), leading to the proposal that PMS2 and MLH1 might facilitate S region synapsis during CSR (Schrader et al., 2003). 4.6.5. Other Factors LR-1 is a B-cell–specific heterodimeric protein composed of nucleolin and heterogeneous nuclear ribonucleoprotein D (hnRNP D), in which each subunit is capable of low-affinity binding to S region–specific duplex sequences, and with high affinity to sequences in the form of G quartets or G4 DNA (Dempsey et al., 1999; Williams and Maizels, 1991). Consequently, it has been proposed that LR-1 might bind and capture DNA from two different S regions and facilitate their synapsis, thus contributing to CSR (Dempsey et al., 1999).

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4.7. Class Switch Recombination and Double-Strand Break Repair The detection of closed circles of DNA composed of intervening sequences between two different S regions implied that intermediates of CSR occur in the form of DNA DSBs (reviewed in Iwasato et al., 1990; Kenter, 2003). Sequences from CSR junctions demonstrate little or no sequence homology between donor and acceptor S regions, and CSR junctions frequently contain short deletions or insertions of nucleotides, all of which are consistent with the NHEJ pathway of DNA DSB repair (Dunnick et al., 1993). Lending further support for DSBs as intermediates in CSR, deficiencies in assayed NHEJ proteins reduce CSR in mice (Casellas et al., 1998; Manis et al., 1998a, 2002a). Finally, deficiency for 53BP1, a DNA damage-sensing protein that becomes activated in response to DSBs and is found associated with H2AX, also leads to significantly reduced levels of CSR (Manis et al., 2004; Ward et al., 2004). 4.7.1. Ku Ku-deficient mice do not develop B or T cells; therefore rearranged IgH and IgL genes must be introduced into these animals to derive mature B cells (Casellas et al., 1998; Manis et al., 1998a). The only detectable IgH isotype in the serum of these mice is IgM, and splenic B cells isolated from these animals and stimulated in vitro to undergo specific CSR fail to secrete anything other than IgM (Casellas et al., 1998; Manis et al., 1998a). The presence of GTs from downstream CH genes and DSBs detected in Sg3 sequences suggested that the defect in CSR was not due to an inability to initiate the process (Casellas et al., 1998; Manis et al., 1998a). However, as Ku-deficient B cells are also defective in proliferation, the lack of CSR could be explained by decreased survival of activated B cells (Manis et al., 1998a). Potentially countering this argument, cells that have undergone several rounds of cell division still do not undergo CSR (Reina-San-Martin et al., 2003), although it is not clear whether these cells might represent those that have failed to be completely activated. 4.7.2. DNA-PKcs and Artemis DNA-PKcs–deficient mice have significantly reduced levels of serum isotypes (Manis et al., 2002a), whereas SCID mice that carry DNA-PKcs kinase inactive mutations undergo CSR at nearly normal levels (Bosma et al., 2002; Cook et al., 2003). In this regard, CSR occurs normally in the absence of Artemis (Rooney et al., submitted), which is activated on phosphorylation by DNA-PKcs (Ma et al., 2002), whereas Artemis is essential for opening the hairpin-coding ends

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generated during V(D)J recombination (Rooney et al., 2002). Therefore, DNA-PKcs may provide functions for the repair of DNA damage induced during CSR, such as stabilization of the repair complex, synapsis of target sequence (see above), or recruitment of other essential proteins to the site of DNA breaks, outside its role in Artemis activation that is required for V(D)J CJ formation. 4.7.3. Ataxia Telangiectasia Mutated Human patients with ataxia telangiectasia mutated (ATM) have normal levels of SHM in their V region sequences, although an overall reduction in serum immunoglobulin isotypes and an increase in homology at S region junctions suggest that ATM does influence CSR (Pan et al., 2002; Pan-Hammarstrom et al., 2003; Waldmann et al., 1983; reviewed in Regueiro et al., 2000). ATM is activated by DNA damage, thereby phosphorylating and activating cell cycle control proteins p53 and Chk2, and thus inducing cell cycle arrest in cells containing DSBs (reviewed in Khanna and Jackson, 2001; Shiloh, 2001). However, ATM likely functions beyond sensing DNA damage and cell cycle regulation, as indicated by its ability to phosphorylate the DNA repair protein NBS1 (Gatei et al., 2000; Lim et al., 2000; Wu et al., 2000; Zhao et al., 2000). In addition to the increase in homology at CSR junctions, there are fewer mutations and insertions in the sequences around CSR junctions of ATM-deficient B cells than are found in control B cells (Pan et al., 2002). Thus it appears that ATM may function during the repair phase of CSR, although secondary effects caused by defects in B- and T-cell development and survival could also contribute to the observed immunodeficiencies in patients with ATM. In this regard, ATM-deficient mice initially were not found to have clear-cut defects in the production of serum IgH isotypes (Barlow et al., 1996; Xu et al., 1996). However, more detailed analyses have now clearly shown a defect in CSR but normal internal Sm deletions similar to what is seen in H2AX deficiency, which supports a role for the DNA DSB response in this process and potentially synapsis (Reina-San-Martin et al., 2004; see below). 4.7.4. 53BP1 The role of NHEJ proteins and the likely generation of DNA DSBs during CSR imply the need to sense and respond to such DNA lesions. 53BP1 was found to interact with the DNA damage response and cell cycle checkpoint protein p53 (Xia et al., 2001). 53BP1 was rapidly phoshphorylated in response to IR (Anderson et al., 2001) and was found in foci that are thought to represent sites of DNA damage (Anderson et al., 2001; Schultz et al., 2000). Furthermore, 53BP1 colocalized with g-H2AX in nuclear foci that appear after

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DSB induction (Rappold et al., 2001; Rogakou et al., 1999). 53BP1-deficient B cells were dramatically impaired for CSR; although the production of germline transcripts and induction of AID expression were normal (Manis et al., 2004; Ward et al., 2004). In contrast, both V(D)J recombination and SHM occurred normally in 53BP1-deficient mice (Manis et al., 2004; Ward et al., 2004). Thus CSR is highly dependent on DNA damage-sensing proteins downstream of AID induction and, thus, likely to influence the DNA repair/ S region joining phase. Finally, rare Sm-Sg1 switch junctions amplified from 53BP1-deficient B cells are qualitatively similar to wild-type junctions, demonstrating that 53BP1 does not mechanistically affect CSR (Manis et al., 2004). In the context of these observations, it has been suggested that 53BP1 may work with H2AX for S region synapsis via an anchoring mechanism (Bassing and Alt, 2004; Manis et al., 2004). 4.7.5. H2AX Phosphorylation of H2AX on Ser-139 occurs within minutes after treatments that introduce DNA DSBs in yeast and mammalian cells (Downs et al., 2000; Rogakou et al., 1998). g-H2AX appears in discrete nuclear foci that correlate in frequency and nuclear location with induced DSBs (Rogakou et al., 1999). The rapid appearance of g-H2AX foci after the induction of DSBs precedes that of DNA repair proteins, suggesting that g-H2AX may be involved in the recruitment of specific repair factors such as BRCA1, MRE11, RAD50, and NBS1 to sites of DNA damage (Paull et al., 2000). Whereas H2AX is required for efficient CSR and AID-dependent foci formation at the IgH locus (see above), it is not required for the process of intra-S region deletions and has been suggested to be therefore involved in long-range synapsis (Reina-San-Martin et al., 2003), which might occur via an anchoring mechanism as outlined above (Bassing and Alt, 2004; Manis et al., 2004). 4.7.6. NBS1 NBS1 is a DNA repair protein associated with the hMre11/hRad50/NBS1 complex that forms nuclear foci in response to DSB-inducing DNA damage and is a target of ATM-mediated phosphorylation (Carney et al., 1998; Maser et al., 1997; Nelms et al., 1998; Wu et al., 2000; Zhao et al., 2000). In yeast, scmre11 and scrad50 mutants have defects in NHEJ and have been linked genetically to the same NHEJ pathway as yku70 and lig4, and Mre11, also implicated in microhomology-mediated DNA break repair (reviewed in Critchlow and Jackson, 1998; Paull and Gellert, 2000). Furthermore, NBS1 has been detected at nuclear foci that colocalize with the IgH loci in B cells activated to undergo CSR, and this colocalization was dependent on the

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presence of AID (Petersen et al., 2001). However, the levels of most serum isotypes in patients with Nijmegan breakage syndrome (NBS) are not substantially reduced (reviewed in Shiloh, 1997). B cells of patients with NBS do have an increased frequency of S region junctions with ‘‘imperfect’’ microhomology (four or more nucleotides, with only one mismatch) compared with controls, albeit from a limited number of samples (Pan et al., 2002). Thus NBS1 may play a role in the DNA repair phase of CSR or, given its association with g-H2AX, it could be involved in S region synapsis. 4.7.7. Mismatch Repair During DNA replication, MMR proteins recognize improperly paired nucleotide base pairs and mediate the removal and reinsertion of the correct nucleotide based on the DNA template strand (Buermeyer et al., 1999). Several studies have found an overall decrease in the rate of CSR in the absence of certain MMR proteins (Ehrenstein and Neuberger, 1999; Schrader et al., 1999, 2002). CSR junctions were found to occur more frequently in consensus GAGCT and GGGGT sequences, reminiscent of ‘‘hot spot’’ targeting of SHM in the absence of Msh2 (Ehrenstein and Neuberger, 1999; Phung et al., 1998). Moreover, CSR junctions isolated from B cells of Msh2-deficent mice were found to have slightly decreased lengths of microhomology (Schrader et al., 2002). This would be consistent with the DNA deamination model of CSR, as MMR proteins can extend the region of mutations beyond the original dU residue induced by the function of AID and UNG (Petersen-Mahrt et al., 2002). These results are consistent with Msh2 playing a more important role in end processing, specifically the removal of 30 nonhomologous overhangs outside potential regions of microhomology (Schrader et al., 2002). In contrast, there was an increase in microhomology length detected in the CSR junctions of Mlh1- and Pms2-deficient B cells (Schrader et al., 2002). The increase in microhomology at CSR junctions of Pms2- or Mlh1-deficient B cells might reflect a role for stabilizing CSR intermediates or for S region synapsis, thus requiring increased sequence homology in their absence for adequate base pair interactions (Schrader et al., 2002, 2003). 5. CSR-Related Diseases 5.1. Hyper-IgM Syndrome Types 1 and 3 Hyper-IgM (HIGM) syndromes are immunodeficiencies caused by genetic defects that result in abrogation or impairment in CSR (reviewed in Durandy and Honjo, 2001). The first described was X-linked hyper-IgM, or

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HIGM type 1 (HIGM1), caused by mutations in the gene encoding the CD40 ligand (CD40L), a membrane glycoprotein expressed on activated T cells (Allen et al., 1993; Aruffo et al., 1993; DiSanto et al., 1993). CD40L interacts with CD40, a member of the tumor necrosis factor receptor family, that is constitutively expressed on B cells and is variably expressed on T cells, monocytes, basophils, dendritic cells, and endothelial cells (reviewed in Grammer and Lipsky, 2000). CD40L binds to CD40 and induces B cell proliferation (Nishioka and Lipsky, 1994; Tohma and Lipsky, 1991), AID induction (Muramatsu et al., 1999) and the production of some immunoglobulin GTs (Fujita et al., 1995; Jumper et al., 1994; Warren and Berton, 1995). Removal of either CD40 or CD40L through the use of anti-CD40L antibodies (Foy et al., 1993, 1994) blocks germinal center formation, SHM, and CSR in response to T-dependent antigens. Genetic defects in CD40 lead to an autosomal recessive form of hyper-IgM, HIGM3, similar to that caused by the absence of CD40L (Ferrari et al., 2001). Thus HIGM1 and HIGM3 are caused by the ablation of upstream signaling pathways leading to CSR and SHM activation. 5.2. Hyper-IgM Syndrome Type 2 Autosomal recessive hyper-IgM syndrome type 2 is caused by mutations abrogating the expression or function of AID (Revy et al., 2000). Patients lacking AID have enlarged lymph nodes with correspondingly expanded germinal centers (Revy et al., 2000), a characteristic also seen in AID-deficient mice (Muramatsu et al., 2000). These oversized germinal centers likely reflect the presence of activated B cells that are unable to effect CSR or SHM, and thus accumulate in B-cell follicles of the peripheral lymph tissue. 5.3. Hyper-IgM Syndrome Type 4 Patients with HIGM4 are substantially impaired for CSR, whereas SHM can be detected in VH regions at levels comparable to that of controls (Imai et al., 2003b). Defects in AID, UNG, or in the expression of GTs were eliminated as possible causes of HIGM4. Evidence for the existence of a factor differentially involved in CSR versus SHM is in keeping with the DNA deamination model, in which CSR is effected via DSB intermediates, whereas SHM can be induced in the absence of DSBs (Petersen-Mahrt et al., 2002). Thus HIGM4 is likely caused by defect(s) in factors associated with the targeting of AID to S regions that affect the synapsis of S regions and/or that are involved in an aspect of DNA DSB repair (reviewed in Manis and Alt, 2003).

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5.4. X-Linked Hypohydrotic Ectodermal Dysplasia Another human disease, X-linked hypohydrotic ectodermal dysplasia (XHMED), is characterized by hyper-IgM immunodeficiency caused by missense mutations in the gene encoding NF-kB essential modulator (NEMO) (Doffinger et al., 2001; Jain et al., 2001). NEMO interacts with NF-kB kinases IKK1 (IkB kinase 1) and IKK2 and is essential for NF-kB activation (Yamaoka et al., 1998). Engagement of CD40 on the surface of B cells with T-cell– expressed CD40L leads to the induction of NF-kB family transcription factors (Berberich et al., 1994; Francis et al., 1995; Lalmanach-Girard et al., 1993). NF-kB family transcription factors mediate the production of Ig1-Cg1 (Lin and Stavnezer, 1996; Lin et al., 1998) and Ie-Ce (Iciek et al., 1997) GTs. Not all immunoglobulin GTs are dependent on NF-kB; thus mutations affecting NF-kB signaling would be predicted to abrogate CSR to some but not all CH genes. However, patients with XHM-ED have undetectable levels of all serum IgGs, and B cells activated in vitro with anti-CD40 fail to effect CSR (Jain et al., 2001). Thus NF-kB signaling in B cells, as with upstream CD40and CD40L-mediated signaling, is likely involved in overall activation of CSR, perhaps as an activator of AID, although an affect on AID expression in these patients has yet to be reported. The developmental aspects of XHM-ED syndrome can be attributed to defective NF-kB signaling through tumor necrosis factor (TNF) family receptors expressed on embryonic and fetal ectoderm-derived tissues (Doffinger et al., 2001). Thus genetic mutations that affect CD40, CD40L, or CD40-mediated downstream signaling molecules all lead to immunodeficiencies with hyper-IgM characteristics. 6. Concluding Remarks V(D)J recombination and CSR (and the related process of SHM) lead to the direct alteration of DNA sequences and content in cells of the vertebrate immune system. V(D)J recombination occurs both in developing B and T lineage cells; whereas CSR and SHM occur only in mature B lineage cells. The potential for deleterious or catastrophic consequences during the manipulation of a cell’s genetic material is obvious; and aberrant V(D)J recombination and CSR, and perhaps SHM, have all been implicated in translocations and other genetic alterations that underlie T lineage [V(D)J recombination] and B lineage [V(D)J recombination, CSR, and SHM] lymphomas. Therefore, all three of these potentially dangerous genomic alteration processes require tight regulatory control mechanisms. In this context, the proteins that initiate these genetic alterations, namely RAGs for V(D)J recombination and AID for CSR

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and SHM, are expressed in tissue- and lineage-specific fashion and are subject to strict control via posttranslational regulatory processes. Likewise, there is control of the substrate DNA, RS sequences for V(D)J recombination, and S regions for CSR, such that its availability to the initiating enzymes is largely limited to the appropriate cell types and sequences. Among the major outstanding questions is the issue of precisely what aspects of target RS or S region DNA in chromatin make them good substrates for RAG or AID in the appropriate cell types, lineages, and/or activation stages. One fundamental difference between CSR and V(D)J recombination is in the nature of the target sequences recognized by the ‘‘recombinase.’’ RAGmediated cleavage at the junction of antigen receptor gene-coding segments is site specific and dependent on short, well-defined cis-acting RSs. In contrast, AID deamination of cytidine residues, which appears to initiate CSR (and SHM), is targeted to large S regions that lie upstream of CH genes in the IgH locus, with recombination occurring throughout the 1- to 12-kb repetitive sequences. Thus, CSR is region specific rather than site specific. Moreover, AID does not appear to recognize specific target sequences with the same degree of specificity as RAGs, which, in general, recognize specific RS sequences. Instead, AID has been thought to rely on transcription-dependent DNA structures such as R loops that are formed when sequences with certain base compositions are actively transcribed. Yet, S regions are composed of tandem repeat units with frequent repeats of specific motifs favored by SHM. Thus, in this context, there may still be specific sequences, such as the SHM consensus, that are preferentially targeted by AID in conjunction with its RPA partner to provide a further degree of specificity in CSR. Although we now have some idea about how AID is targeted, there is still much to be learned about how AID targeting is so specific for S regions and variable regions and why there is not more wide-scale deamination leading to a higher level of mutation and translocations involving other genes in activated B cells. In both V(D)J recombination and CSR, the initiating lesion by RAG and AID ultimately appears to lead to a DSB and, subsequently, to employ DSB repair pathways, most likely NHEJ pathways, for the resolution of the DNA breaks. Clearly, the classic NHEJ pathway seals both coding and signal joints in the context of V(D)J recombination. Some evidence suggests this pathway is also responsible for ligating CSR junctions, although more evidence on this point is needed. A significant difference in the joining phase of V(D)J recombination and CSR lies in their relative reliance on the DNA DSB response. Thus, V(D)J recombination occurs relatively unimpaired in the absence of DSB response factors such as H2AX and 53BP1. However, the absence of these factors dramatically impairs CSR. One possible explanation is that the factors are somehow involved in the long-range synapsis of S regions in

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the context of CSR via a general anchoring mechanism proposed to hold DSBs together in chromatin before their joining via NHEJ. In contrast, RAG-generated DSBs appear to be held together in a postcleavage synaptic complex by the RAGs themselves, which then recruit the NHEJ factors to complete the reaction. In both V(D)J recombination and CSR, however, we still know little about the actual process of synapsis and how the involved proteins contribute to it. Acknowledgments We thank Drs. JoAnn Sekiguchi and John Manis for helpful advice and suggestions. We thank Drs. Sean Rooney and Ali Zarrin for communicating unpublished data. C.H.B. is a Lymphoma Research Foundation Fellow. F. W. A. is an investigator with the Howard Hughes Medical Institute. This work was supported by an NIH grant to F.W.A. (NIH 5PO1A131541-14).

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Isoforms of Terminal Deoxynucleotidyltransferase: Developmental Aspects and Function To-Ha Thai1 and John F. Kearney Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35204

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Abstract ................................................................................................. V(D)J Recombination and Mediating Factors.................................................... Junctional Diversity................................................................................... Origin of TdT.......................................................................................... Transcriptional Regulation of the TdT Gene (Dntt) ............................................ TdT and Its Splice Variants ......................................................................... TdT Splice Variants and Junctional Diversity .................................................... The TdT Protein ...................................................................................... TdT-Interacting Proteins............................................................................. TdT Splice Variants and Repertoire Development .............................................. Biochemical Properties and Substrate Specificity of TdT ...................................... Expression of Human TdT in Human Leukemias............................................... Possible Aberrant Activity of Human TdT in Leukemias ...................................... Conclusions ............................................................................................ References .............................................................................................

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Abstract The immune system develops in a series of programmed developmental stages. Although recombination-activating gene (RAG) and nonhomologous end-joining (NHEJ) proteins are indispensable in the generation of immunoglobulins and T-cell receptors (TCRs), most CDR3 diversity is contributed by nontemplated addition of nucleotides catalyzed by the nuclear enzyme terminal deoxynucleotidyltransferase (TdT) and most nucleotide deletion is performed by exonucleases at V(D)J joins. Increasing TdT expression continuing into adult life results in N region addition and diversification of the T and B cell repertoires. In several species including mice and humans, there are multiple isoforms of TdT resulting from alternative mRNA splicing. The short form (TdTS) produces N additions during TCR and B-cell receptor (BCR) gene rearrangements. Other long isoforms, TdTL1 and TdTL2, have 30 ! 50 exonuclease activity. The two forms of TdT therefore have distinct and opposite functions in lymphocyte development. The enzymatic activities of the splice variants of TdT play an essential role in the diversification of lymphocyte repertoires by modifying the composition and length of the gene segments involved in the production of antibodies and T-cell receptors. 1 Present address: CBR Institute for Biomedical Research, 138 Warren Alpert Building, Boston, Massachusetts 02115.

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1. V(D)J Recombination and Mediating Factors Diversity in the antigen-binding or variable region of immunoglobulins and T-cell receptors (TCRs) results from combinatorial rearrangements of the variable, diversity, and joining gene segments by a process known as V(D)J recombination (Hozumi and Tonegawa, 1976). This process normally takes place at specific stages in developing B and T lymphocytes (Ghia et al., 1996; Haynes and Heinly, 1995; Melchers et al., 1995). V(D)J recombination is a site-specific reaction that is initiated by lymphoidspecific recombination-activating gene (RAG)-1 and RAG-2 proteins that synapse with recombination signal sequences (RSSs) flanking all immunoglobulin- and TCR-coding gene segments (Bassing et al., 2002; Fugmann et al., 2000; Grawunder and Harfst, 2001; Lewis, 1994; Oettinger, 1999; Schatz, 1997) (Fig. 1). RSSs are composed of a conserved palindromic heptamer and an AT-rich nonamer separated by nonconserved 12- or 23-bp spacers. V(D)J recombination occurs predominantly between two coding gene segments flanked by RSSs that contain 12- and 23-bp spacers, respectively. This phenomenon is known as the 12/23-bp rule. After synapsis of coding gene segments, DNA double-stranded breaks (DSBs) or single-stranded (ss) nicks are generated precisely by RAG proteins at the border of RSSs flanking each gene segment. This is followed by a trans-esterification reaction, catalyzed by RAG proteins, in which the 30 -OH of the coding strand attacks the opposite DNA strand (or strand transfer) to form closed hairpin coding ends and blunt 50 -phosphorylated RSS ends. Hairpins are then resolved by the endonuclease Artemis (Ma et al., 2002) or other endonucleases generating predominantly coding ends with 30 or 50 extensions. Subsequently, signal ends or RSS ends are usually precisely joined, whereas coding ends will undergo modifications such as nucleotide deletion and addition. The joining phase of the V(D)J reaction is completed by ubiquitously expressed nonhomologous end-joining (NHEJ) and DSB repair proteins, including Ku70, Ku86, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), XRCC4, and ligase IV. To date, immunoglobulins and TCRs of all vertebrate taxa examined, including mammals, avians, reptiles, amphibians, teleosts, and cartilaginous fish, are generated through V(D)J recombination (Hawke et al., 1996; Kerfourn et al., 1996; Partula et al., 1996; Tjoelker et al., 1990; Turchin and Hsu, 1996). 2. Junctional Diversity Although RAG and NHEJ proteins are crucial in the generation of immunoglobulins and TCRs, the majority of junctional diversity is contributed by nontemplated addition (N addition) and deletion of nucleotides at V(D)J joins

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(Fig. 1). V(D)J joins of terminal deoxynucleotidyltransferase (TdT)-deficient mice are devoid of N nucleotides, strongly demonstrating that TdT is responsible for N addition (Gilfillan et al., 1993; Komori et al., 1993). The identity of the exonucleases involved in nucleotide deletion has proved to be more elusive. TdT is a nuclear enzyme found predominantly in developing B and T cells in the bone marrow and the thymus, respectively. However, this strict definition may not hold true, because TdT transcripts can also be detected in bone marrow cells with the phenotypes of myeloid progenitors (see below). TdT is expressed in fetal human B- and T-cell precursors, and in fetal mice it is barely detectable. In many vertebrates, the TdT gene (Dntt) is conserved where V(D)J recombination occurs (Hawke et al., 1996; Kerfourn et al., 1996; Partula et al., 1996; Tjoelker et al., 1990; Turchin and Hsu, 1996). 3. Origin of TdT TdT belongs to the DNA polymerase (Pol) family X. The amino acid sequence of DNA polymerase family X members contains the conserved motif GGFRRGKLQGHDVDFLI, for which a function has not yet been determined. However, the underlined D residue is shown to be involved in nucleotide binding (see below). It has been suggested that TdT and Pol b, implicated in DNA base excision repair (Pelletier et al., 1994; Sawaya et al., 1994; Sugo et al., 2000), share a more recent common ancestral gene. A close examination of the Pol X family tree reveals that TdT and the newly identified mammalian Pol m (Aoufouchi et al., 2000; Dominguez et al., 2000) are derived from a more recent common ancestor. In addition, an ancient divergence occurs among TdT, Pol m, and Pol b, a close relative of Pol l (Aoufouchi et al., 2000). Pol m has 41% amino acid identity to TdT and is preferentially expressed in tonsillar germinal center B cells. These results may indicate that TdT transcripts reportedly detected in tonsillar B cells might be those of Pol m. If that is the case, TdT is not reexpressed in mature B cells, as has been suggested (Girschick et al., 2001). Pol m has intrinsic TdT activity; however, unlike TdT, Pol m polymerizing activity is enhanced by a template strand independently of cations, and nucleotide insertion is rather random. These data suggest that the switch to template independence evolved more recently and that the common ancestor of TdT and Pol m is probably template dependent. Advances in genomics have allowed us to tentatively identify the putative ancestral gene in the Ciona intestinalis genome from which TdT and Pol m are derived (Fig. 2). TdT, Pol m, Pol b, and Pol l appear to be descendents of the DNA-dependent DNA Pol X family belonging to the archaebacteria group.

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Figure 2 Identification of the Dntt putative ancestral gene. The rooted near-neighbor-joining phylogenetic tree was generated from aligned sequences retrieved from the NCBI, Takifugu, and Ciona databases. Note the sequence annotated as Takifugu TdT falls within the Pol m cluster, suggesting that this sequence might represent TdT, not Pol m.

4. Transcriptional Regulation of the TdT Gene (Dntt) Human, bovine, mouse, and rat TdT genes consist of 13 exons, most of which locate in the 30 half of the respective gene (Thai et al., 2002; Fig. 3). TdT gene expression is normally restricted to B- and T-cell precursors. Undoubtedly, mechanisms employed to regulate a gene that is inactive in mature lymphoid cells would be different from those of a gene such as Ig m that is constitutively active. Indeed, mapping studies reveal that the TdT promoter is unusual in many aspects (Ernst et al., 1996, 1999; Hahm et al., 1994). The minimal promoter spans a region from positions 111 to þ58, and three control elements are found within this region. The TdT promoter lacks a TATA box normally located 25–30 bp upstream of the transcriptional start site, and Figure 1 V(D)J recombination. Initiation of V(D)J recombination: RAG-1 and RAG-2 bind and create a single-strand nick at the border of the recombination signal sequence (RSS, triangles) and coding sequence. Nicking: The RAG complex binds stably to the pair of RSS, forming a synaptic complex that is necessary for the cleavage reaction to occur. Cleavage and hairpin formation: The synaptic complex converts the nick into double-strand breaks through a trans-esterification reaction, generating coding end hairpins and blunt signal ends, held together in a postcleavage complex by RAG-1/2. Hairpin resolution: The endonuclease Artemis, complexed with and phosphorylated by DNA-PKcs, resolves the hairpins; the signal joint is formed by precise, head–head ligation of RSS, using the NHEJ machinery. Coding end modification and joining: P addition to, nucleotide deletion of (by TdTL and other, yet to be identified exonucleases) and N addition to (by TdTS) coding ends take place before end joining by the NHEJ machinery.

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Figure 3 Genomic organization of human, bovine, mouse, and rat Dntt. The proposed genomic organization of human, bovine, mouse, and rat TdT genes was based on NCBI and Celera genomic databases and data from others (Thai and Kearney, 2004); newly identified exons II, VII, and XII are represented by slashed boxes, respectively.

instead contains an initiator (Inr) element (positions 3 to þ5) that overlaps the transcriptional start site. The TFIID complex can recognize the TATA box, as well as the Inr element. Mutations of nucleotides at a site where a TATA box might normally reside, at the 30 position, do not affect TdT promoter function. Likewise, substitution of a genuine TATA box at the 30 position only slightly enhances promoter activity relative to the wild-type promoter; however, promoter activity remains lymphocyte specific. In contrast, substitutions of two nucleotides within the Inr region completely disrupt promoter activity in lymphoid and nonlymphoid cell lines, despite the presence of a TATA box. These results suggest that, although weak, the Inr is the core promoter element that functions in concert with other regulators to activate TdT transcription in a non-tissue-restricted manner. However, despite the high degree of conservation between the human and the mouse promoter region, the Inr element is not totally conserved in the human promoter. The human promoter contains several inserts; of interest are the 27-bp insert near the site where the mouse Inr should be and the 9-bp insert close to the D0 element. The DNA sequence of the 27-bp insert resembles that of the Inr consensus, suggesting that spacing constraints between the start site and upstream elements are quite loose. The activity of the Inr is enhanced by downstream basal elements (DBEs) located downstream of the transcriptional start site (positions þ33 to þ58). Mutations in this region abrogate promoter activity in both lymphoid and nonlymphoid cell lines, but factors controlling DBE function have not been determined. TdT tissue-specific expression is regulated by a third region

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called the D0 element and located 60 bp upstream of the start site (positions 70 to 48). Biochemical and genetic studies identify two classes of transcription factors that bind to this region. The first factor, Elf-1, belongs to the Ets class of proteins. Trans-activation and expression studies show that Elf-1 activates TdT transcription in immature T and B cells by binding to the second region within the D0 element. Mutations that reduce Elf-1 binding but not other Ets proteins within this region result in the selective loss of promoter activity in lymphocytes, despite the presence of an inserted TATA box. It is puzzling why Elf-1 would confer stage-specific expression to the TdT gene, because Elf-1 has been implicated in the inducible activation of other genes in mature T and B cells. In addition, promoter methylation has also been implicated in TdT tissue-specific expression (Nourrit et al., 1999); however, this study needs to be confirmed. The second factor, Lyf-1, belongs to the Ikaros family of transcription regulators that intimately associate with pericentromeric heterochromatin and are implicated in heritable gene inactivation (Trinh et al., 2001). Studies demonstrate that Ikaros dimers bind to two regions within the D0 element and can compete with Elf-1 activator for binding. Mutations that disrupt Ikaros binding also abrogate TdT downregulation on lymphocyte differentiation, suggesting that binding of Ikaros dimers within the D0 region represses TdT transcription. Ikaros-dependent repression of TdT transcription is accompanied by chromatin remodeling, as shown by nuclease sensitivity. Both Ikaros binding and chromatin alterations precede pericentromeric repositioning of the TdT gene. Together, these results lead to the development of the current working model. On lymphocyte differentiation, Ikaros dimers compete with Elf-1 for occupancy at the D0 element, thus displacing Elf-1. This event appears to be reversible and is accompanied by chromatin remodeling. Binding of Ikaros dimers to the D0 element facilitates their association with multimeric Ikaros complexes positioned at pericentromeric foci, thereby recruiting the TdT gene to these foci to be inactivated. 5. TdT and Its Splice Variants Three alternative TdT splice variants are found in human, cattle, mouse, and rat (Doyen et al., 1993; Koiwai et al., 1986; Takahara et al., 1994) [Thai and Kearney, 2004; and National Center for Biotechnology Informal (NCBI) rat genomic data base]. In humans, mice, and cattle, the inclusion of exons XII and VII in the mature transcripts gives rise to TdTL1 and TdTL2, respectively. The exclusion of both exons VII and XII results in the generation of TdTS. In rat, the inclusion of exon II produces TdTL2, whereas TdTL1 is derived from mature transcripts containing exon XII. Rat TdTS (rTdTS) is generated through the constitutive splicing of both exons II and XII (Fig. 4). Although the presence of these alternatively spliced exons is conserved

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Figure 4 The proposed alternative splicing patterns of human, bovine, mouse, and rat Dntt. Red and blue dashed lines represent alternative splicing; green dashed lines indicate constitutive splicing during normal lymphocyte development.

evolutionarily, their nucleotide and thus deduced amino acid sequences are not (Fig. 5A and B). These observations suggest that the L1 and L2 inserts may serve as structural rather than protein interaction domains, which allow TdTL1 and TdTL2 to assume conformations different than those of TdTS. The change of conformation, in turn, may confer different DNA-modifying activities. In mice, during normal B-cell development in the bone marrow, mTdTS and mTdTL1 transcripts as well as proteins are detected in both cycling and noncycling pro-B cells where heavy (H) chain gene rearrangements occur. In contrast, in cycling and noncycling pre-B cells where light (L) chain gene rearrangements take place, mTdTL1 transcripts and proteins are primarily seen (Thai et al., 2002). Surprisingly, neither mTdTL2 transcripts nor proteins are found in either population during normal bone marrow B-cell development; however, the mTdTL2 isoform is readily detected in the transformed myeloid cell line HTX-1. In the bone marrow, the Mac1þGr1þB220þThy1þ and Mac1þGr1þB220Thy subpopulations express predominantly mTdTS whereas mTdTL1 expression is restricted primarily to the Mac1þGr1þB220Thy1þ population; mTdTL2 is not detected in any of these populations (T.-H. Thai, unpublished data). These observations suggest that Dntt transcription occurs in myeloid and T and B precursors in the bone marrow before commitment. In the thymus, all populations of T-cell progenitors, excluding the DN1 population, (DN2, DN3, ISP, and DP) express mTdTS and mTdTL1 both at the transcript and protein levels. Again, mTdTL2 is not detected during normal T-cell development in the thymus (T.-H. Thai, unpublished data).

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Unlike TdT expression patterns in the mouse, during normal human B-cell development, hTdTS and hTdTL2, not hTdTL1, are predominantly expressed in pro-B (CD34þCD19þ sIgM) and pre-B (CD34CD19þsIgM) cells. As expected, none of the isoforms are seen in the mature (CD34CD19þsIgMþ) population. However, this ordered pattern of expression is altered in transformed cell lines because, along with the other isoforms, hTdTL1 is detected in human cell line 697, representative of the pre-B-cell stage, where it is not normally detected (Thai, unpublished data). To date the expression of hTdT isoforms during normal human thymocyte development has not been formally assessed. In contrast to adult bone marrow pro-B cells, hTdTL1 is not detected in fetal thymocytes at any age. However, hTdTL1 is detected in transformed T cell lines. Both hTdTS and hTdTL2 are expressed in all stages of thymocyte development [DN (CD4CD8), DP (CD4þ CD8þ), and CD4þ and CD8þ thymocytes], as well as in transformed cell lines. The level of hTdTL2 transcripts is consistently higher than that of hTdTS. On fetal day 91, all three populations studied: DN, DP, and CD4þ and CD8þ thymocytes express less hTdTS than hTdTL2; and this level of differential expression persists until fetal day 111, albeit slightly less pronounced. Moreover, TdT proteins are readily detected in the respective thymocyte subpopulations by fluorescence-activated cell scrting (FACS) analyses, using TdT-specific monoclonal antibodies (mAbs) (Thai, 2004). In contrast to human fetal thymocytes, both hTdTS and hTdTL2 are expressed at similar levels in all stages of adult thymocyte development (Thai, 2004). These data support previous studies demonstrating that the degree of N addition in TCR-b DJ junctions of human thymocytes increases with age, but the extent of nucleotide nibbling remains constant (George and Schroeder, 1992). The persistent expression of hTdTS and hTdTL2 in all stages of thymocyte development also explains the presence of N addition and nucleotide deletion in human TCR-b, a, g, and d chain genes (Breit and Van Dongen, 1994; Dave et al., 1993; Genevee et al., 1994; Quiros Roldan et al., 1995; Yoshikai et al., 1986). Therefore, in normal human B and T cells, exon XII of human Dntt is always excluded, whereas exon VII can be included (generating hTdTL2) or excluded (generating hTdTS). In contrast, in normal mouse B and T cell progenitors, exon XII of mouse Dntt can be included (generating mTdTL1) or excluded (generating mTdTS), whereas exon VII is always excluded. In the case of cattle and rats, exons XII and VII can be included (producing bTdTL1 and bTdTL2, respectively) or excluded (producing bTdTS) (Takahara et al., 1994, and NCBI rat genomic database, respectively), because transcripts of all three splice variants can be detected in these two species (Fig. 4). Detailed studies on expression patterns of bovine and rat TdT splice variants during normal B- and T-cell development should be carried out to confirm these observations. Thus,

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alternative splicing, which appears to be species specific, regulates human, bovine, mouse, and rat TdT isoform expression. 6. TdT Splice Variants and Junctional Diversity It is evident that mammalian TdT splice variants are alternatively expressed during normal B- and T-cell development. In addition, we and others have shown that mTdTS and hTdTS clearly catalyze N addition in V(D)J joins (Benedict et al., 2000; Bentolila et al., 1995; Thai, 2005; Thai et al., 2002). Therefore, it is logical to ask whether the long isoforms possess enzymatic activity, and if so, do they contribute to junctional diversity? We have shown both biochemically and genetically that mTdTL1, hTdTL1, and hTdTL2 possess 30 ! 50 exonuclease activity. In a standard recombination assay, all three long isoforms catalyze the removal of nucleotides from artificial coding ends but not signal ends (Thai, 2005; Thai et al., 2002). Moreover, the concomitant expression of TdTS (transferase) and TdTL1 and/or TdTL2 (exonuclease) appears to modulate the activity of each other during V(D)J recombination. The activity of mTdTL2 has not been extensively studied, because its identity has just been determined in our laboratory. However, preliminary data suggest that it also possesses 30 ! 50 exonuclease activity. Thus, in mouse and human, both TdT long isoforms catalyze nucleotide deletion; however, there exists a strong evolutionary constraint on alternative splicing to express only one exonuclease (TdTL1 in mouse and TdTL2 in human) during V(D)J recombination in normal B and T progenitors. One possible explanation for the differential expression patterns of mammalian TdT splice variants is that the coexpression of all three human TdT isoforms greatly reduces the recombination frequency in the standard recombination assay (Thai, 2005). It is noteworthy to point out that coding joins retrieved from cells that express only the long isoform (TdTL1 or TdTL2) contain no P nucleotides, suggesting that TdTL1/2 may be responsible for P nucleotide removal. Superficial examination of CDR3 joins in bulk peripheral splenic B cells and bone marrow revealed little evidence of a decrease in exonucleolytic activity at V, D, or J coding ends in TdT-deficient mice (Gilfillan et al., 1993; Komori et al., Figure 5 Genomic and deduced amino acid sequence analyses of TdT splice variants. (A) The human, bovine, mouse, and rat TdT genomic sequences were retrieved from NCBI and Celera genome databases, and alignment was done to identify L1 and L2 inserts. (B) The deduced amino acid sequences of L1 and L2 inserts from all four species were aligned, L1 and L2 inserts are in red and blue, respectively; dashed lines indicate missing residues. In contrast, rat L2 (green) is encoded by exon II, not exon VII, as in human, bovine, and mouse. Moreover, rat exon IV is spliced into exon V, resulting in the deletion of 11 residues (purple dashed line) from exon V.

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1993). These observations have been construed as evidence that TdTL isoforms are redundant or are not responsible for N deletion during the joining process. However, careful examination of CDR3 joins in FACS-purified B cell progenitors in the C and C0 fractions revealed that only about 0–7% of wild-type CDR3 V–D and D–J joins show homology-directed recombination involving one to five nucleotides, whereas about 27–70% of CDR3 joins in TdT-deficient mice show homology-directed recombination involving one to five nucleotides (I. Ivalyo and H. Schroeder, personal communications). Therefore when C and C0 B cells are analyzed before the effects of BCR-mediated selection pressures, these homologies are preserved, most likely because of the lack of exonucleolytic activity in the absence of TdTL. Although this is indirect evidence, these observations strongly support a role for TdTL in the removal of these homologous nucleotides. Undoubtedly there may exist other exonucleases capable of catalyzing nucleotide deletion from coding ends. However, because of the expression of TdTL isoforms, which is confined primarily to developing B and T cells and their presence along with TdTS in cells undergoing V(D)J recombination, we propose that mammalian TdT long isoform exonuclease activity contributes to the generation of diversity in V(D)J joins (Fig. 1). 7. The TdT Protein TdT contains several structural and functional domains (Fig. 6). These domains appear to be conserved among species; therefore, only the human terminal deoxynucleotidyltransferase (hTdT) structure is described. Like most nuclear proteins, hTdT contains a conserved nuclear localization signal, PRKKRPR. Three conserved exonuclease (Exo) motifs and three putative

Figure 6 Domain structure of human, bovine, mouse, and rat TdT isoforms. (A) Domain structure of (top to bottom) human, bovine, and mouse TdT isoforms. (B) Domain structure of rat TdTL2; note the location of the L2 insert. Rat TdTS (rTdTS) and TdTL1 (rTdTL1) are similar in structure to those of the other three species.

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Figure 7 Identification of exonuclease motifs in human, bovine, mouse, and rat TdT. Amino acid sequence alignment of mouse, rat, bovine, and human TdTL isoforms was done to identify exonuclease core motifs; conserved residues are colored green. The third exonuclease motif is underlined to show its upstream position proximal to the L1 insert (red).

cAMP-dependent phosphorylation consensus sequences are found where threonine can be phosphorylated. cAMP site 1 locates just upstream of the Exo I motif, whereas site 2 abuts the N-terminal end of the Exo II motif, and all three motifs can be found in the amino acid sequences of human, mouse, bovine, and rat TdT splice variants (Fig. 7; residues in green have been shown to be important for exonuclease activity). Exo motifs have been shown to confer Exo activity to mTdTL1 (Thai et al., 2002). Although human and bovine TdT can be phosphorylated by a cAMP-dependent kinase in vitro, their activity does not appear to be modulated (Chang and Bollum, 1982). On the other hand, phosphorylation has been shown to mediate RAG-2 cell cycle– dependent degradation (Li et al., 1996); perhaps TdT is similarly marked for degradation during lymphocyte differentiation. Alternatively, phosphorylation may change the conformation, thereby revealing cryptic DNA-modifying activities in TdT, as seen in Artemis (Ma et al., 2002). Whether the proximity of two Exo motifs to two cAMP sites mutually affects their functions has yet to be determined. The DNA-binding domain is located just upstream of the third cAMP site and the L2 insert. This domain consists of two peptides (DTEGIPCLGSK and GIIEEIIEDGESSEVK) that have been shown to covalently cross-link to DNA under optimal photolabeling conditions (Farrar et al., 1991). The nucleotide-binding domain is located at the carboxyl end of the L2 insert and overlaps the Pol X consensus sequence (GGFRRGKKMGHDVDFLI), where the D residue is found to be crucial for nucleotide binding (Yang et al., 1994). The Exo III motif lies just upstream of the human, bovine, mouse, and rat TdT L1 insert, which is located at the N-terminal end of the putative tyrosine phosphorylation site. It has not yet been determined whether this site is actually phosphorylated and whether

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phosphorylation modulates TdT activity, but the site appears to be conserved in all TdT proteins identified to date. After DNA damage induced by radiation, carcinogens, or oxidative free radicals, cell cycle checkpoints are initiated to allow complete repair of damaged DNA, thus curtailing the propagation of permanently altered genetic materials. Cell cycle checkpoint and DNA repair proteins, including the tumor suppressor BRCA1 protein, the p53-binding protein (53BP1), the yeast cell cycle checkpoint protein RAD9, the DNA repair protein XRCC1, DNA ligases III and IV, and many others, all contain the globular BRCA1 Cterminal region domain, termed BRCT (Callebaut and Mornon, 1997; Huyton et al., 2000). BRCT modules have been shown to be involved in both BRCT– BRCT and BRCT–non-BRCT-mediated protein interactions and in associations with DNA strand breaks. Among all Pols in Pol X, only TdT and its close relatives Pol m and Pol l contain the BRCT domain located in the N terminus. The BRCT domain overlaps with Exo I and cAMP sites. A contribution of the BRCT domain to TdT function has not yet been determined. Studies have suggested that the hTdT BRCT domain interacts with Pso4, a human factor involves in DNA repair and recombination (Mahajan and Mitchell, 2003). 8. TdT-Interacting Proteins Because TdT is involved in the diversification of immunoglobulins and TCRs during V(D)J recombination, it follows that TdT must interact with other factors involved in V(D)J recombination. Indeed, many groups have devoted their resources to the search for such TdT-interacting proteins. It has been shown in Ku86-deficient mice that the majority of coding joins are devoid of N regions and that a high proportion of these joins have lost no nucleotides from either end relative to littermate and C.B-17 SCID controls (Bogue et al., 1997; Purugganan et al., 2001). These observations suggest that Ku proteins interact with TdTS and TdTL1 or TdTL2 (depending on the species) and then recruit them to coding joins. Subsequently, TdTwas shown to interact with Ku70, Ku86, and the Ku70– Ku86 heterodimer complex (Mahajan et al., 1999). This interaction appears to be DNA independent. On treatment of cells with a topoisomerase II inhibitor etoposide, TdT and Ku form discrete foci in the nucleus and, with increasing time, form intracellular complexes dispersed throughout the nucleus. Furthermore, removal of the first 131 amino acids from the TdT N-terminal domain, which contains the BRCT module, selectively and greatly reduces TdT association with Ku70 and the Ku70–Ku86 heterodimer; however, its interaction with Ku86 alone remains intact. TdT has also been shown to interact with DNA-PKcs, another crucial factor required for V(D)J recombination (Mickelsen et al., 1999). These results, although intriguing, fall short in

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explaining mechanisms and functional consequences of these associations and have not been confirmed. Through a series of yeast two-hybrid experiments, TdT was shown to interact with proliferating-cell nuclear antigen (PCNA) and a novel nuclear protein (TdIF1, terminal deoxynucleotidyltransferase-interacting factor 1) homologous to transcription factor p65, which belongs to the nuclear receptor superfamily (Ibe et al., 2001; Yamashita et al., 2001). The BRCT domain does not appear to mediate association with PCNA or TdIF1. Moreover, association with TdIF1 enhances and interaction with PCNA reduces TdT activity. PCNA and TdIF1 do not appear to play any roles in V(D)J recombination; therefore, the physiological consequences of these associations on cells are not apparent. However, these data suggest different mechanisms of TdT regulation and function independent of V(D)J recombination. 9. TdT Splice Variants and Repertoire Development The most common and direct approach to studying the function of a gene is to create gene-targeted mutant mice and then observe the loss of function and/ or change of phenotype. Indeed, TdT-deficient mice were created (Gilfillan et al., 1993; Komori et al., 1993). To date, the TdT deficiency mutation does not cause any deleterious diseases or overall phenotypic abnormalities in mice, suggesting that TdT is not required for normal development. However, close examination of the immune system reveals several phenotypic abnormalities (Gilfillan et al., 1995b). Studies with TdT-deficient mice incontrovertibly show that the majority (70–80%) of N regions in V(D)J joins are catalyzed by TdTS in vivo. In adult TdT-deficient mice, homology-directed recombination and canonical joins are enhanced to the levels seen in normal fetal immunoglobulins and TCRs (Fig. 8A and B). CD3hi single-positive selection is more efficient in the thymuses of TdT-deficient mice, and both CD4þ and CD8þ lineages are equally affected by the null mutation. The enhanced positive selection caused by the TdT deficiency mutation is not influenced by the size of thymuses or the genetic background (i.e., strain and MHC haplotype) of animals used. Moreover, the increased proportion of CD3hi singlepositive cells in TdT-deficient mice appears to result from more thymocytes successfully making the transition from the double-positive to the singlepositive stage rather than to a more rapid transition between these stages. The more efficient positive selection observed in TdT-deficient mice may be due to the shortened complementarity-determining region 3 (CDR3) loop of the ab TCR resulting from N-less joining. Although structural evidence is lacking, this may also account for the cross-reactive neonatal-like T-cell repertoire in adult TdT-deficient mice, as elegantly demonstrated by Gavin and Bevan (1995), where the TdT-deficient repertoire was also less peptide

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Figure 8 Examples of the germline features of mouse T-cell receptor (TCR) and B-cell receptor (BCR) canonical nucleotide sequences. (A) Lymphocytes with the invariant T cell receptor, using Vg3 and Dd2 with the J and D regions, are generated in fetal mouse thymus (in the absence of TdT activity) and then migrate into the skin, where they remain in the adult as dendritic epidermal T cells (DETCs). (B) A dominant B-1 cell clone (recognized by the idiotype marker T15) expresses the VHS107/Vk22 HþL chains and the D, JH, and Jk regions shown, is generated in neonatal life and dominates the response to bacterial phosphorylcholine in the adult. The V, D, and J assignments are shown on top of each rearranged TCR or BCR nucleotide sequence. P nucleotides palindromic to the first nucleotides at the end of germline V, D, and J elements are shown in italics. Short homologous regions (underlined) between V, D, and J segments guide the recombination process. There is no TdT-mediated N addition in these joins.

specific. In addition, the N-less repertoire of B cells is more polyreactive, because TdT-deficient mice exhibit an increase in the phosphorylcholine (PC) response and TEPC-15 idiotypeþ antibody production (Benedict et al., 2001), both of which cannot normally be derived from adult bone marrow precursors, where constitutive TdT activity prohibits the production of the canonical germline CDR3 nucleotide sequences. Thus, in the absence of adult TdT activity the fetal/neonatal windows of T and B cell development are extended for the life of the mouse. Although studies are limited, the TdT deficiency mutation appears to protect against rather than promote autoimmunity, because TdT-deficient mice develop a lower amount of anti-DNA

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antibodies and rheumatoid factors when immunized with lipopolysaccharide (LPS) (Weller et al., 1997). In addition, when crossed with nonobese diabetic mice or (NZB  NZW)F1 mice, hybrid mice show a reduced incidence of insulitis and autoimmune nephritis, respectively (Conde et al., 1998; Gilfillan et al., 1995a). Overall, the immunoglobulin and TCR repertoires of TdTdeficient mice resemble fetal repertoires: homology-directed recombination is enhanced, canonical joins are overused, CDR3 loops are short and less diverse, and response to PC is increased. More significantly, mice having fetal repertoires throughout life appear to fare normally and robustly against a variety of immunological insults, including viruses and bacteria. The TdT deficiency null mutation also affects human immunoglobulin rearrangement by influencing heavy chain variable (VH) and joining (JH) gene segment usage, and high-frequency recombination occurs at sites of short homologies (Tuaillon and Capra, 2000). It is apparent that TdT is not an essential gene; therefore, to understand the subtle role that TdT plays in modulating the immune repertoire, the next obvious question concerns what happens to fetal immunoglobulin and TCR repertoires if TdT is expressed during ontogeny. Like TdT-deficient mice, TdTS and TdTL1 transgenic mice do not suffer any obvious phenotypic abnormalities and thrive in normal housing conditions (Benedict and Kearney, 1999; Benedict et al., 2000; Bentolila et al., 1997; Conde et al., 1998). Moreover, TdTS and TdTL1 mice appear to sustain normal B and T cell development. As expected, N regions are present in fetal V(D)J joins and in adult L chains of TdTS transgenic mice; thus, fetal repertoires resemble those of adults. The consequences of this ontogenetically forced N addition are the failure, in adult TdTS transgenic mice, to produce the normally dominant B-cell clones expressing the hallmark canonical T15 immunoglobulin receptor and a subsequent failure to make a T15 anti-PC antibody response on challenges with PC-containing Streptococcus pneumoniae. Administration of sera obtained from adult TdTS transgenic mice immunized with S. pneumoniae failed to protect B-cell–deficient (xid) recipients from challenges with a virulent pneumoccal infection, whereas transfer of sera from normal adult donors immunized with S. pneumoniae provided complete protection to xid recipients against a virulent pneumococcal infection. Expression of TdT in the neonatal spleen reduces reading frame 1 (RF1) usage relative to littermate controls exhibiting 80% RF1 usage, and positive selection of VH81x-encoded H chains during fetal life is almost abolished (Marshall et al., 1998). In line with these observations, forced expression of N nucleotides in L chains that are predominantly N-less decreases VH81x clonal production, thereby negatively affecting positive selection in the spleen, specifically marginal zone B cells (Martin and Kearney, 2000). In contrast, TdTL1 transgenic mice display a normal anti-PC response; however, anti-protein

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antibody responses in these mice have not been determined. However, a report shows that forced TdT expression in fetal thymus decreases the number of gd T cells and that Vg3Vd1 T cells randomly disseminate in newborn, but not in adult, skin (Aono et al., 2000). Thus, the loss of innocence or the lack of maturity, although not detrimental to immunoglobulin and TCR repertoires, negatively modulates the immune system. 10. Biochemical Properties and Substrate Specificity of TdT Although bovine terminal deoxynucleotidyltransferase (bTdT) and hTdT appear to be more related evolutionarily than are mTdT and hTdT, the short isoform of bovine terminal deoxynucleotidyltransferase (bTdTS) and the short isoform of mouse terminal deoxynucleotidyltransferase (mTdTS) appear to use Zn2þ preferentially over Mg2þ as divalent cation (Bollum, 1974), whereas hTdTS, purified from leukemic cells, prefers Mg2þ (Deibel and Coleman, 1980). The activity of TdTS from all three species is completely abrogated by EDTA treatment (Bollum, 1974). Because bTdTL1/2 and hTdTL1/2 have not yet been thoroughly characterized, from this point forth, the long isoform of mouse terminal deoxynucleotidyltransferase 1 (mTdTL1) is exclusively described. Unlike mTdTS, mTdTL1 does not require Zn2þ for its Exo activity (Mg2þ appearing to be sufficient), and mTdTL1 is similarly sensitive to EDTA treatment. A pyrophosphorolytic dismutase activity has been ascribed to a calf thymus TdT preparation when Co2þ is used (Anderson et al., 1999). However, we know that all three bTdT isoforms can potentially be expressed in calf thymus (T.-H. Thai, unpublished observations); thus, it is plausible that the proposed pyrophosphorolytic dismutase activity is actually caused by Exo activity of bTdTL isoforms present in the preparation. bTdTS and mTdTS incorporate purines (A and G) more efficiently than pyrimidines (C and T) (Basu et al., 1983; Bollum, 1960; Gauss and Lieber, 1996). On the other hand, oligodeoxynucleotide primers composed of A or T residues [d(pA)n and d(pT)n, respectively] are better priming substrates for bTdTS and mTdTS than d(pC)n or d(pG)n (Bollum, 1960). mTdTL1 has similar substrate requirements. This preferential substrate usage is recapitulated using bona fide mouse D elements (DFL16.1 and DQ52), where mTdTL1 efficiently deletes nucleotides from the 30 end of the forward sequence, whereas the reverse sequence of the same D element suffers less nucleotide loss (H. Schroeder, personal communication). The minimum number of residues that a substrate can support priming is three (Bollum, 1974). These data suggest that the overrepresentation of G and C residues in N regions at V(D)J junctions may result from the concomitant activity of TdTS and TdTL1 (Thai et al., 2002).

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11. Expression of Human TdT in Human Leukemias Since the discovery of TdT in 1960, a large body of studies has been published documenting TdT overexpression in B- and T-cell acute lymphocytic leukemias (ALLs) and in acute myelocytic leukemias (AMLs) (Bertazzoni et al., 1982; Greaves et al., 1980; Hoffbrand et al., 1977; Kung et al., 1978; Oiwa et al., 1989). Historically, TdT has been used as a marker in the diagnosis of groups of human leukemias. Its expression in tumors, which often bear markers of multiple lineages, indicative of B, T, and myeloid origin, suggests that at least some of them are derived from an early multipotential progenitor. Acute lymphocytic leukemia encompasses a group of cancers that represent immature B or T lymphoblasts. Classification of ALL is based on the degree of differentiation of the cells isolated. Approximately 90% of patients with ALL express TdT in their blast cells (Hoffbrand et al., 1977; Janossy et al., 1980). The level of TdT expression varies widely, up to 1000-fold, in each cell and between each individual as detected by the standard tests now in use. It is not known whether all hTdT isoforms can be detected in these assays (Hutton et al., 1982), nor is the functional significance of the high TdT expression in these tumors known. Our preliminary studies show that multiple patterns of isoforms are expressed in each tumor (R. Schulz, T.-H. Thai, and J. F. Kearney, unpublished observations). Approximately 18% of (AMLs) express TdT. The highly variable reported expression of TdTþ AML cells (0 to >50%) may be due to detection methods and also may be due to the specific FAB (French–American–British) system used for classification (Drexler et al., 1993). TdT positivity appears to be higher in ‘‘pure’’ monocytic lines (M0–M1) compared with the monocytic subclasses (M5–M6) (Skoog et al., 1984). Thus, it appears that TdT expression decreases as the AML cells become more phenotypically monocytic (Paietta et al., 1993). Finally, approximately 30% of patients with blastic transformation in chronic myelocytic leukemia (CML) exhibit increased TdT activity (Hoffbrand et al., 1977; Hurwitz et al., 1995; Kung et al., 1978). Blast crisis in these patients portends death, usually within 6 months. Prognosis and long-term survival studies show that remission rates for patients with TdT AML were higher (61%) compared with TdTþ AML (36%). Median survival rates were also higher in those patients who had TdT-negative AML cells (Del Poeta et al., 1997). Because the signs and symptoms for ALL and AML are similar, accurate quantification of TdTþ cells is important for differentiating between ALL and AML, and more precise diagnoses will dictate the appropriate therapy. Although it is evident that TdT expression has been useful in the detection and classification of human lymphomas and leukemias, the use of TdT as

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a prognostic marker has met with mixed results (Shurin and Scillian, 1983). ALL remission rates in children exceed 90% with current chemotherapy regimens regardless of TdT expression. This is in contrast to AML, which represents less than 20% of all childhood acute leukemias, but represents more than 30% of childhood deaths from leukemia (Hurwitz et al., 1995). If differences in expression of the isoforms of huTdT can be ascertained more precisely, prognostic outcomes can be reevaluated to determine the role of TdT in therapeutic outcomes and survival. Furthermore, the potential role of TdT in transformations can be studied. Transformations herald a poor prognosis as observed in ALL, where the Philadelphia chromosome t(9;22) is seen in only 3% of childhood ALL but represents 25% of adult cases, possibly explaining why adults fare worse with this disease (Cline, 1994). Further studies on TdT isoforms in these leukemias are now warranted. 12. Possible Aberrant Activity of Human TdT in Leukemias Although there is extensive literature on the expression patterns of hTdT in leukemia, there are few studies that examine the functional activities that TdT may exert on the development, maintenance, or exacerbation of lymphoid neoplasia. There is abundant evidence for inappropriate rearrangements of TCRs in B-cell ALL (Dombret et al., 1992), and of both TCR and BCR genes in AML (Foa et al., 1987), although at the time of analysis these patterns did not always coincide with TdT expression. There are a few tantalizing clues that abnormal TdT polymerase activity may be responsible for extensive junctional N addition in ALL (Foa et al., 1987; Genevee et al., 1994; Kung et al., 1978; Langlands et al., 1993). In a more limited example of TALL, long CDR3 regions of TCRb rearrangements were also found (Yamanaka et al., 1997). Similar findings in AML have been published, in which BCR and TCR rearrangements with long N insertions can be detected together with deletions of V and J coding regions and multiple Dd segments (Przybylski et al., 1994; Schmidt et al., 1995). It is also of interest that the TCRd locus is often the target of unusual rearrangements in AML and ALL (Kimura et al., 1996). It is not known whether early and/or abnormal expression of TdT isoforms in hematopoietic progenitors affects normal lymphopoiesis and receptor-driven selection processes in the thymus and the bone marrow, thus giving rise to disordered development. It is assumed that TdT expression is merely a reflection of the stage of development at which a given tumor type arises. However, a reasonable alternative hypothesis is that unchecked or abnormal activity of TdT may have more widespread effects, and it is not known what effects abnormal expression of TdT isoforms might have on the maintenance and exacerbation of lymphoid malignancies. Indeed, it has been shown that

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TdT-accessible breaks appear to be scattered over the VH segment obtained from Ramos cells transfected with a TdT-encoding plasmid (Sale and Neuberger, 1998). N insertions, catalyzed by TdT, into the DNA breaks result in frameshift mutations in the VH region, which in turn generates IgM-loss variants in culture. These results suggest that accessible DNA breaks within the genome may serve as substrates for TdT isoforms to modify, either by N addition or N deletion, thus generating mutations. 13. Conclusions The limited diversity of the early fetal repertoire of B and T cells is guided by genetic mechanisms involved in the production of the lymphocyte receptors that are active during fetal development. The reduced expression and activity of the mTdT isoforms clearly distinguish lymphocyte development in the fetus from that in the adult. Further understanding the function of that in relation to RAG and other proteins in receptor gene formation is clearly of significance to our understanding of the generation of receptor diversity. In the future it will be necessary to determine the expression patterns of TdT isoforms during T- and B-cell development with respect to ontogeny, the cell cycle, and in relation to other key enzymes involved in recombination. This will involve analysis of in vivo effects of TdTS and TdTL1/2 isoform interactions with each other and with other DNA-modifying factors. There are already clues that substrate specificity preferences of V, D, and J targets exist for the polymerase and exonuclease activities of TdTS and TdTL1/2 isoforms. Future studies may reveal that these substrate preferences are the cause of GC nucleotide enrichment at CDR3 joins. TdT isoforms may also play a role in the secondary rearrangements involved in k locus inactivation and in receptor revision of autoreactive T and B cells. Further understanding of the normal structure–function relationship of huTdT isoforms will assist in determining their role in B and T cell development, and their involvement in human lymphoid malignancies. The new information obtained and the development of new isoform-specific reagents will provide unique tools to reevaluate these human tumors for TdT expression and assist in the diagnosis and classification of leukemias and may aid in the generation of better antileukemic drugs. Acknowledgments We thank Dr. Zeev Pancer for help in generating the phylogenetic tree, Tamer Mahmoud for reading of the manuscript, and Ms. Ann Brookshire for secretarial assistance. This work was supported in part by NIH grants AI14782, AI14594, AI51533, T32AI07051, and CA13148.

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Innate Autoimmunity Michael C. Carroll* and V. Michael Holers{ *CBR Institute for Biomedical Research, and Department of Pediatrics Harvard Medical School, Boston, Massachusetts 02115 { Departments of Medicine and Immunology, University of Colorado Health Sciences Center, Denver, Colorado 80217

1. 2. 3. 4. 5. 6. 7. 8.

Abstract .................................................................................................. Introduction.............................................................................................. Ischemia–Reperfusion Injury ......................................................................... Reperfusion Injury Mediation by Natural Antibody .............................................. Specificity of Natural IgM-Mediating Reperfusion Injury ....................................... Initiation of Reperfusion Injury by a Single IgM.................................................. Limition of Ischemia-Related Antigens ............................. ................................ Renal Ischemia–Reperfusion.......................................................................... Fetal Loss Syndromes.................................................................................. References ...............................................................................................

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Abstract The adaptive immune system has evolved highly specific pattern recognition proteins and receptors that, when triggered, provide a first line of host defense against pathogens. Studies reveal that these innate recognition proteins are also self-reactive and can initiate inflammation against self-tissues in a similar manner as with pathogens. This specific event is referred to as ‘‘innate autoimmunity.’’ In this review, we describe two classes of autoimmune responses, that is, reperfusion injury and fetal loss syndrome, in which the recognition and injury are mediated by innate immunity. Both disorders are common and are clinically important. Reperfusion injury (RI) represents an acute inflammatory response after a reversible ischemic event and subsequent restoration of blood flow. Findings that injury is IgM and complement dependent and that a single natural antibody prepared from a panel of B-1 cell hybridomas can restore injury in antibody-deficient mice suggest that RI is an autoimmune-type disorder. Fetal loss syndrome is also an antibody- and complement-dependent disorder. Although both immune and natural antibodies are likely involved in recognition of phospholipid self-antigens, inhibition of the complement pathway in rodent models can block fetal loss. As new innate recognition proteins and receptors are identified, it is likely that innate responses to self represent frequent events and possibly underlie many of the known chronic autoimmune disorders normally attributable to dysregulation of adaptive immunity.

137 advances in immunology, vol. 86 0065-2776/05 $35.00

ß 2005 Elsevier Inc. All rights reserved.

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1. Introduction The innate immune system is thought of as the host’s first line of defense against microbial infections (Baumgarth et al., 2000; Ochsenbein et al., 1999). Like adaptive immunity, it includes recognition proteins such as collectins (Epstein et al., 1996), serum complement (Reid and Porter, 1981), and natural antibody (Boes et al., 1998). The latter is produced primarily by a specialized subset of B cells termed B-1 and is included as a component of innate immunity (discussed in more detail below) (Hardy et al., 1994). Recognition of pathogens leads to a cascade of events resulting in initiation of inflammation and infiltration of inflammatory cells. The identification of toll-like receptors (TLR) has greatly expanded our view of the host innate recognition of pathogens (Janeway and Medzhitov, 1999), and the list of pattern recognition receptors (PRRs) and of pathogen-associated molecular patterns (PAMPs), both associated with TLRs (Barton et al., 2003) and other pathways (Aderem and Underhill, 1999), continues to grow. Activation of TLRs results in release of cytokines that also induce infiltration and activation of proinflammatory cells (Barton and Medzhitov, 2003). Thus, it is apparent that the innate immune system has evolved highly specific recognition molecules that provide a rapid response to infectious agents. Given the highly conserved nature of many of the known PAMPs, it is inevitable that cross-reactivity with host antigens such as heat shock protein 70 (HSP-70; Vabulas et al., 2002) occurs, leading to an inflammatory response against self. This type of response is termed innate autoimmunity, as the initial event is based on innate recognition of self. In this review, we discuss two common models of autoimmune response to self-antigens, that is, reperfusion injury and fetal loss syndrome. Both are examples of innate autoimmunity and represent clinically relevant disease in humans. Because the review focuses on early events in initiation of autoimmune injury, downstream events such as infiltration of leukocytes, mast cell activation, or injury due to the terminal components of complement are not discussed in depth. 2. Ischemia–Reperfusion Injury Ischemia–reperfusion injury (RI) represents an acute inflammatory response following a reversible ischemic event and subsequent restoration of blood flow (Cotran et al., 1994). It is potentially life threatening and is primarily responsible for the tissue injury following reperfusion that occurs in myocardial infarction, cerebral ischemic events, intestinal ischemia, renal ischemia, and other events such as vascular surgery, trauma, and transplantation. The response of cells to hypoxia is pleiotropic and includes alterations of cytoplasmic

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architecture, activation of enzymatic pathways, and initiation of gene transcription (Tatsumi et al., 2003). Mitochondria are particularly sensitive to changes in oxygen concentration (Liu et al., 2003). However, little is known regarding events outside of the cell that lead to the observed acute inflammatory response. One proposal is that that surface alterations following hypoxia are recognized by neutrophils, and this leads to release of reactive oxygen species (ROS) that promote tissue injury (Li and Jackson, 2002). Although neutrophils are a hallmark of acute inflammation and are observed in RI, their role in initiation of injury is not clear. In some examples of RI, injury occurs in the presence of a limited numbers of neutrophils (Briaud et al., 2001; Simpson et al., 1993). The first suggestion that the complement system might mediate RI came from elegant studies by Fearon and colleagues demonstrating protection in a rat model of coronary ischemia (Weisman et al., 1990). They found that pretreatment of animals with a soluble inhibitor of activated C3, that is, soluble CR1 (sCR1), dramatically reduced inflammatory injury to the myocardium. Thus, shutting off the complement cascade at the central step of C3 activation was protective. Subsequent studies in other animal models of RI, including models of cardiopulmonary bypass (CPB) (Chai et al., 2000) and stroke in the pig (Huang et al., 1999) and the mouse skeletal muscle model (Hill et al., 1992), confirmed and extended their observations that blocking of the complement system with sCR1 significantly reduced injury. Although these studies suggested that the complement system was important in RI in various tissues, they did not provide an explanation for how complement activation was initiated. That is because each of the three complement activation pathways (classical, alternative, and lectin) converge on C3 and require C3 cleavage to then generate C5a and the membrane attack complex (MAC). 3. Reperfusion Injury Mediation by Natural Antibody The early events in RI were first examined using mice bearing specific deficiencies in complement or innate immunity. Using a model of skeletal muscle RI, Weiser et al. (1996) demonstrated that mice totally deficient in C3 were protected to a similar level as mice pretreated with sCR1. Importantly, mice deficient in C4 were also protected in this model. These results not only confirmed the importance of the complement system in RI but suggested that initiation of the cascade was via the classical or lectin pathways. To test a role for antibody, RAG-1–/– mice, which are deficient in mature lymphocytes and thus do not express immunoglobulin, were treated in the RI model. Notably, the RAG-1–/– mice were protected from injury to a similar extent as complement-deficient animals. Importantly, reconstitution with normal mouse

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Figure 1 Proposed model of reperfusion injury mediated by natural IgM after recognition of neoepitopes exposed or expressed during ischemia. IgM binding activates the classical pathway of complement, leading to release of proinflammatory peptides C3a and C5a and deposition of the membrane attack complex C5–C9, leading to direct cell lysis.

serum restored injury, suggesting that the defect lies in the absence of serum immunoglobulin. These observations were interpreted to suggest that preexisting antibody recognized neoepitopes on hypoxic endothelium and initiated the classical pathway of complement (Fig. 1). The identification of IgM deposition on vessels within the reperfused hind limb muscle supported this hypothesis (Weiser et al., 1996). Williams et al. (1999) then extended these observations in an intestinal model of RI demonstrating that reconstitution of RAG-1
/
mice with purified serum IgM restored injury (Fig. 2). These results not only confirmed the importance of serum IgM but suggested a common mechanism for RI in at least two tissues, that is, skeletal muscle and intestine. 4. Specificity of Natural IgM-Mediating Reperfusion Injury The first indication of the specificity of natural IgM in RI derived from experiments with mice deficient in complement receptors CD21 and CD35 (Cr2
/
). Intriguingly, in independent experiments two groups reported that the Cr2
/
mice were protected from injury in an intestinal model of RI (Fleming et al., 2002; Reid et al., 2002). In both reports, reconstitution of the deficient animals with pooled IgM—prepared from wild-type mice—restored pathogenic injury to the small bowel as evaluated by histology, myeloperoxidase (MPO) generation, and vascular permeability analysis. By contrast, reconstitution of the deficient animals with IgM prepared from the

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Figure 2 Mice deficient in IgM are protected from intestinal reperfusion injury (RI). Reconstitution of mice deficient in recombinase-activating gene (RAG-1null) with pooled IgM from wild-type mice restores injury. RI is based on the permeability index (see Williams et al., 1999).

Cr2-deficient mice did not restore injury (Reid et al., 2002). In one report, a role for IgG was suggested on the basis of an increase in local generation of MPO when the deficient mice were reconstituted with wild-type IgG (Fleming et al., 2002). Because Cr2
/
mice express normal levels of serum IgM, yet were not injured, it was proposed that ischemia-specific IgM was missing from the repertoire. It was concluded that the complement system has an additional role in RI and is important in the development or maintenance of the subset of B cells that secrete the IgM involved in RI. Cr2
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mice also have an impaired response to T-dependent and -independent antigens, as the receptors are required for an effective humoral response (for review see Carroll, 1998; Fearon and Cartes, 1995; Hannan et al., 2002). To identify the population of B cells responsible for secretion of pathogenic IgM, Reid et al. (2002) engrafted Cr2
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mice with an enriched fraction of B-1 cells prepared from Cr2þ/þ wild-type mice. B-1 cells are a major source of natural antibody and differ from conventional B cells by several criteria such as cell surface markers, ability to self-replenish, and a limited repertoire that is biased toward microbial and highly conserved self-antigens (discussed further below). Characterization of the chimeric mice confirmed the presence of normal levels of Cr2þ/þ B-1 cells within the peritoneum within 4–6 weeks of engraftment (Fig. 3). More importantly, the engrafted mice developed full

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Figure 3 Engraftment of Cr2-deficient mice with wild-type peritoneal B-1 cells restores injury. (a) Analysis of peritoneal B-1 cells by FACS after staining with antibody specific for complement receptors indicates reduction in cell surface level of CD21 and CD35 in Cr2null mice, as expected. By contrast, analysis of peritoneal B cells isolated from Cr2null mice engrafted with wild-type B-1 cells 6 weeks previously confirms the presence of CD21þ CD35þ B cells. (b) Cr2null mice are protected from intestinal RI, but engraftment with wild-type B-1 cells restores injury (see Reid et al., 2002, for details).

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injury in the intestinal RI model. Thus, these results not only confirmed the importance of IgM in RI but supported a role for B-1 cells as a potential source of pathogenic antibody. 5. Initiation of Reperfusion Injury by a Single IgM The observations taken from the Cr2
/
experiments strongly suggested that natural IgM produced by B-1 cells was at least one source—if not the major source—of pathogenic antibody in RI. To test further the hypothesis that the antibody was specific and might directly recognize antigens on ischemic endothelium/tissues, a panel of IgM-expressing hybridomas was prepared from an enriched fraction of wild-type peritoneal B-1 cells. Supernatant was pooled from a group of 22 hybridomas that produced relatively high levels of IgM and injected into RAG-1
/
mice before treatment in the intestinal RI model (Fig. 4; Zhang et al., 2004). Notably, reconstitution with the total pool restored injury similar to that of wild-type IgM. Thus, the panel of 22 hybridomas included a clone or clones that secreted RI-specific IgM. To further identify the clone or clones, pools were prepared from groups of five or fewer hybridomas and tested in the RAG-1
/
mice. Using this in vivo approach, one clone, CM-22, was identified that could restore injury in the immunoglobulin-deficient mice in the intestinal RI model. Importantly, the other 21 monoclonal antibodies (mAbs) did not restore injury. Nucleotide sequence analysis of the immunoglobulin heavy and light chain cDNAs expressed by the pathogenic clone CM-22 identified the rearranged V(D)J as a VH Vm 3.2, DFL 16, and JH1, respectively. The VH–DH and DH– JH junctions bore limited N region addition, and the rearranged VH represented a germline-like sequence with no evidence of somatic mutation. The immunoglobul in light chain (Lc) was identified as a member of the Vk 21–12 (Jk2) subfamily and is 99% identical to an Lc previously identified as a natural antibody. Thus, the structure of the immunoglobulin heavy chain (Hc) and Lc support the origin of the IgM as a B-1 cell. Evidence that CM-22 IgM was specific for ischemic intestine came from histologic examination of tissues isolated from RAG-1
/
mice treated with IgM isolated from either CM-22 or CM-31 (one of the other mAbs from the panel). RAG-1
/
mice reconstituted with control IgM failed to develop injury and appeared similar to the saline controls. By contrast, CM-22-reconstituted mice developed significant injury to microvilli within the ischemic and reperfused jejunum (Fig. 5). Pathologic injury correlated with deposition of the CM-22 antibody, which colocalized with complement C4 and C3 within the injured microvillus (Fig. 6).

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Figure 4 Identification of pathogenic IgM that mediates intestinal RI. (A) In vivo screening approach used to identify B-1 cell hybridomas that secrete pathogenic IgM. (B) Reconstitution of RAG-1–/– mice with IgM prepared from clone CM-22 restores injury in the intestinal RI model (see Zhang et al., 2004, for details).

The identification of a single mAb that initiates RI in the intestinal model provides direct support for the hypothesis that recognition of ischemic tissue is specific and raises the question of the number of antigens involved and whether the same or related antigens are also involved in RI among other tissues. Similar experiments were performed in a hind limb model of skeletal muscle ischemia in which RAG-1
/
mice were reconstituted with IgM isolated from either CM-22 or a control mAb. Notably, mice reconstituted with CM-22 IgM developed RI in contrast to those receiving control IgM (W. Austen, M. Zhang, R. Chan, H. Hechtman, M. C. Carroll, and F. D. Moore,

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Figure 5 B-1 cell hybridoma clone CM-22 mediates RI in RAG-1–/– mice in the intestinal model. (A) Hematoxylin and eosin–stained cryosections prepared from intestinal tissue of RAG-1–/– mice reconstituted with (i) saline, (ii) wild-type IgM, (iii) clone CM-31, or (iv) clone CM-22, 1 h before treatment in the intestinal RI model. Arrowheads indicate subepithelial spacing and sloughing of microvilli tips in RI. (B) Injury scores of reconstituted RAG-1–/– mice after treatment in the intestinal RI model. Scores are based on pathology (see Zhang et al., 2004, for details).

unpublished results). Thus, in at least two distinct tissues, CM-22 IgM appears to recognize ischemia-related antigens and initiate injury. It will be important to extend these studies to other tissues such as myocardium and the CNS stroke model. 6. Limition of Ischemia-Related Antigens An inherent feature of B-1 cells—the major source of natural antibodies—is that they are limited in diversity relative to conventional B cells. One explanation for the limited repertoire is that B-1 cells develop primarily during the late fetal and neonatal stages. This period of B cell development differs from that of adults in that terminal deoxynucleotidetransferase (TdT), which is responsible for nucleotide addition to the CDR3 region of the rearranged immunoglobulin heavy chain, is not expressed, and VH usage is biased toward proximal genes (Hardy et al., 1994; Kantor et al., 1997; Malynn et al., 1990; Seidl et al., 1999). During this period, B-1 cells make up a major fraction of the B cells produced. Given the limited repertoire and the bias toward highly conserved

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Figure 6 Deposition of IgM and complement within microvilli correlates with induction of injury by pathogenic IgM. RAG-1–/– mice were reconstituted with either CM-31 or CM-22 hybridoma IgM and treated in the RI model as described in Fig. 5. Red, anti-mouse IgM; green, anti-mouse C4 (e, f, h, and i) or C3 (n, o, q, and r); yellow, colocalization of red and green (see Zhang et al., 2004, for details).

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antigens, it is likely that the antigen/antigens recognized by CM-22 IgM are also highly conserved and limited in number. Preliminary evidence in support of a limited number of RI antigens comes from two sets of experiments. First, CM-22 appears to precipitate a unique protein/proteins of high molecular weight compared with a control IgM. In these experiments IgM containing immune complexes (ICs) were prepared from lysates of intestinal tissues of RAG-1
/
mice reconstituted with the respective IgM mAbs before treatment in the intestinal RI model (Zhang et al., 2004). The second line of evidence comes from studies using a peptide inhibitor of CM-22. The peptide was shown to bind specifically to CM-22 in vitro and in vivo (M. Zhang, F. D. Moore, and M. C. Carroll, unpublished results). Importantly, reconstitution of RAG-1
/
mice with a mixture of CM22 and the peptide does not restore injury. Moreover, treatment of wild-type mice with the peptide before reperfusion in the intestinal model is also protective. Thus, these two lines of evidence suggest that the number of antigens involved in RI is limited. It will be important in future experiments to identify the specific antigen/antigens that are the target for natural IgM and compare them in various tissues. In this regard, it will also be relevant to determine whether lack of recognition of the antigen(s) targeted by CM-22 underlies the lack of RI in Cr2
/
mice. This is important because there are several intestinal antigens with other physicochemical properties that are differentially recognized by serum from Cr2þ/þ compared with Cr2
/
mice (L. Kulik and V. M. Holers, unpublished results). Thus, whereas results with CM-22 clearly illustrate the key role for this natural antibody recognition system in RI in the intestine and skeletal muscle, determination of how CD21 and CD35 themselves influence the evolution of the natural antibody repertoire requires additional evaluation. 7. Renal Ischemia–Reperfusion In addition to playing a major role in the development of natural antibodies that recognize antigen(s) and catalyze RI in intestine and skeletal muscle, complement as an innate immune mechanism through the alternative pathway can also function completely independently of natural antibody to identify certain other injured self-tissues. This unique capacity is best exemplified by ischemia– reperfusion injury of the kidney, the pathogenesis of which is reviewed below, in addition to the role of the alternative pathway of complement. Alternative pathway activation is promoted on surfaces that have neutral or positive charge characteristics and do not express or bind serum-derived complement inhibitors. This type of activation is directly catalyzed by a process termed ‘‘tickover’’ of C3, which occurs when C3 spontaneously undergoes a

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conformation change at a rate of 1%/h. This new conformation of C3, termed C3(H2O), can interact with factor B, allowing factor B to undergo a change that allows it to then be recognized and cleaved by protease factor D (MullerEberhard, 1988). The alternative pathway can, however, also be initiated or greatly amplified when antibodies block endogenous regulatory mechanisms (Ratnoff et al., 1983), when expression of complement regulatory proteins is either decreased (Linton and Morgan, 1999; Liszewski et al., 1996; Mizuno et al., 2001; Xu et al., 2000) or is absent such as in the disease paroxysmal nocturnal hemoglobinuria (PNH) (Nishimura et al., 1998), or when dysfunctional regulatory proteins are expressed as a result of genetic polymorphisms (Richards et al., 2002, 2003). RI of the kidney leads to acute renal failure, a condition that may be associated with a 40–50% mortality rate when it occurs in patients in the hospital setting (Levy et al., 1996; Liano et al., 1998; Thadhani et al., 1996). Hemodialysis and other supportive therapies are currently the only treatments for acute renal failure, although more specific mechanism-based therapies are under active investigation (Sheridan and Bonventre, 2000). Initial studies using animal models showed that mice deficient in C3 develop milder renal failure after ischemia–reperfusion, similar to the protective effects of C3 deficiency on intestinal injury. However, in sharp contrast to other organs such as the intestine, in the kidney neither C4 nor natural antibody is required to induce injury (Park et al., 2002; Zhou et al., 2000). Indeed, more recent studies have shown that mice deficient in the alternative pathway complement protein factor B ( f B
/
) are protected from ischemic acute renal failure (Thurman et al., 2003), thus directly implicating this pathway in recognition of ischemic tissue. The process of RI in the kidney is illustrative of several points relevant to complement. First, it is of some interest that there is normally a small amount of C3 that is deposited along Bowman’s capsule of the glomerulus and along the basolateral surface of the tubules. This C3 deposition has been suggested to represent activation of the alternative pathway by ammonia that is produced in the tubules, which can amidate C3 to create a molecule like C3(H2O), which can bind factor B and then allow factor B to serve as a substrate for factor D in the tubules (Nath et al., 1985). Consistent with a role for the alternative pathway in this basal activation process, mice lacking factor B do not exhibit basal C3 deposition around glomeruli or tubules (Thurman et al., 2003). After ischemia–reperfusion, C3 is deposited heavily along the basolateral surface of the tubules in the outer stripe of the outer medulla at the corticomedullary junction, which is also the primary region of histologic injury in the kidney in this condition. Complement activation must occur via the alternative pathway, as when f B
/
mice are subjected to ischemia–reperfusion, virtually no C3 is seen at this site.

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Complement activation by the alternative pathway is also causal in the kidney injury and dysfunction that follows ischemia–reperfusion. This is because f B
/
mice demonstrate milder functional impairment and morphological changes after reperfusion, with serum urea nitrogen (SUN) and histologic injury scores significantly lower compared with control mice. Of interest, the number of neutrophils per high-power field in the tubular region is also significantly lower in f B
/
mice compared with controls, suggesting that the chemotactic effects of C5a and other functionally similar peptides are under alternative pathway control in this setting. Thus, in the kidney the alternative pathway is capable of specifically recognizing ischemic tissue and activating complement, leading to markedly enhanced tissue injury and neutrophil infiltration in the absence of neoantigen recognition by natural antibodies. How this process unfolds specifically around tubules at this corticomedullary junction site in the kidney is under active investigation. One possibility is increased synthesis of alternative pathway components locally that overwhelm inhibitory mechanisms (Fig. 7). Enhanced synthesis of alternative pathway components could come from

Figure 7 Potential mechanisms by which the alternative pathway could be activated around renal tubules during IR injury. (1) Either enhanced levels of complement activators, (2) decreased levels of complement regulatory proteins, or (3) local production of alternative pathway components could promote C3 deposition and tubular injury in an antibody-independent manner.

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either upregulation in endogenous cells or from newly arriving inflammatory cells at that site. Because the alternative pathway is an enzymatically driven process that is actively inhibited, increasing substrate levels might account for increased activation. Another possibility is enhanced synthesis of ammonia that leads to increased amidation of C3, which is capable of interacting with factor B as outlined above. However, our own experiments using NaHCO3 loading ( J. M. Thurman and V. M. Holers, unpublished observations), as well as those of others using a similar approach to greatly decrease tubular ammonia production and subsequent amidation of C3 (Sporer et al., 1981), have both failed to protect mice from ischemia–reperfusion injury. The final possibility, one that we favor, is that alterations of endogenous regulatory proteins occur that lead to diminished inhibition and subsequent alternative pathway activation. In this regard, complement receptor 1–related gene/protein y (Crry), a membrane complement regulatory protein that has been extensively characterized (Foley et al., 1993; Kim et al., 1995; Li et al., 1993; Molina et al., 1992), is normally expressed along the basolateral aspect of renal tubular cells (Li et al., 1993). Crry is the only membrane complement inhibitor expressed by these cells (Li et al., 1993; Miwa et al., 2001; Qin et al., 2001). Thus, decreased expression of Crry, or alternatively the loss of polarity of Crry expression within the ischemic epithelial cells as the protein moves away from the basolateral site of initial complement deposition as a consequence of metabolic changes during ischemia, could allow alternative pathway activation by diminishing local complement inhibitory effects. This issue is under investigation, using a series of genetic and cell biologic approaches. One important question with regard to the role of innate immune recognition concerns why the kidney is different from the intestine and other vascularized organs. The answer is currently unknown, but there are two likely possibilities. The first and most straightforward is that the antigen(s) that are recognized by natural antibodies are not expressed in the kidney. In the absence of recognition by natural antibody, other innate immune recognition mechanisms become more apparent. The second is that the vascular supply in the kidneys to the tubules is complex. Blood first passes through the glomerulus and then to the tubules. Vascular tone through autonomic and soluble hormonal mechanisms plays an essential role in controlling perfusion of each region of the kidney, and thus these effects may alter the ability of innate immune recognition mechanisms to access the tubules. Whether additional target organs utilize a similar mechanism to activate complement, or whether the ischemic and reperfused kidney is unique in its ability to be directly recognized by this innate immune mechanism, is currently unknown. However, it is intriguing that RI in the intestine is also greatly diminished by experimental blockade of the alternative pathway (Stahl et al.,

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2003), which might not be expected to happen if the alternative pathway simply enhanced complement activation by 2-fold as suggested by serum studies in factor B–deficient mice (Matsumoto et al., 1997). Thus, it is possible that, in a setting in which the classical pathway is engaged by natural antibody, in addition to enhancement of complement activation by the amplification loop through C3b, direct recognition of injured tissue by the alternative pathway and engagement of this pathway using one of the mechanisms discussed above with regard to the kidney might also occur. After activation of the alternative pathway in the kidney, complementderived mediators can cause renal tubular injury. These factors include C5a through the C5a receptor (Arumugam et al., 2003; de Vries et al., 2003a,b) as well as the membrane attack complex (Zhou et al., 2000). Finally, in addition to complement, many other proinflammatory pathways are engaged after ischemia–reperfusion injury (reviewed in Sheridan and Bonventre, 2000). Thus, similarly to intestinal injury, defining what pathways are complement dependent and independent should provide important insights into the specific and causal roles that this system plays relative to other innate immune pathways. 8. Fetal Loss Syndromes There are several additional conditions in which the alternative pathway as an innate immune mechanism is likely to be a key component of the recognition and injury of tissues. One of the most intriguing classes of clinical syndromes in this regard is the fetal loss that occurs in pregnant women as the end result of many different types of immune recognition mechanisms that target the semiallogeneic fetus. The best defined syndrome to date is fetal loss that is caused by antiphospholipid antibodies. These autoantibodies can develop in patients with systemic lupus erythematosus as well as independently as the only manifestation of autoimmunity (reviewed in McIntyre et al., 2003). Although the specific patterns of lipid and lipid:protein recognition vary, what ties these autoantibodies together pathophysiologically is their ability to induce thrombosis, or clotting, in vivo. In nonpregnant individuals, the procoagulant effect of these autoantibodies results in arterial and venous clots with subsequent clinical effects such as stroke, deep venous thrombosis, and pulmonary embolism. In women who are pregnant, anti-phospholipid antibodies are additionally associated with early fetal death as well as growth restriction of surviving fetuses. A predilection for fetal complications appears to be due to the preferential recognition of trophoblast cells in the placenta, which are uniquely rich in phosphatidylserine on the external membrane leaflet, by anti-phospholipid autoantibodies.

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Although IgG anti-phospholipid antibodies are present in patients, antiphospholipid recognition is common in natural IgM antibodies (McIntyre et al., 2003). In addition, similar to the role of natural antibodies in intestinal ischemia–reperfusion injury, single monoclonal anti-phospholipid antibodies with germline sequences can cause tissue injury fetal loss when transferred into pregnant mice (Ikematsu et al., 1998). Therefore, anti-phospholipid antibody-mediated injury can also be considered a manifestation of innate immune recognition. For several decades the prevailing belief was that alterations of clotting factors in a prothrombotic direction and/or direct activation of endothelial or inflammatory cells, both mediated solely by the antigen-combining site of antiphospholipid antibodies, underlay the pathogenesis of this condition and mediated the profound in vivo effects (Espinosa et al., 2003; Rand, 2000). However, the realization that complement activation fragments also demonstrate procoagulant effects in vitro on endothelial cells, platelets, mast cells, macrophages, and polymorphonuclear cells (reviewed in Holers, 2001) led to the hypothesis that complement activation itself could mediate fetal loss (Holers et al., 2002). Consistent with this novel hypothesis, it was found that Crry-Ig, a C3 convertase inhibitor active in mice, or subsequently performing experiments in C3
/
mice, both resulted in complete reversal of the fetal injury phenotype (Holers et al., 2002). Thus, while anti-phospholipid antibodies can demonstrate procoagulant effects in vitro, whether these effects are indeed important to the pathogenesis of disease or whether the antigencombining site simply targets complement activation to sites of injury remains an unresolved question. Relevant to the alternative pathway and innate immunity in this condition, follow-up experiments using additional complement inhibitors and gene-targeted deficient mice revealed that C5a, the classical pathway, the alternative pathway and neutrophils, but not Fc receptors, are key intermediaries in anti-phospholipid antibody-mediated loss (Girardi et al., 2003). In addition, deposition of C3 in the placenta was correlated with the presence of neutrophils in the placenta after anti-phospholipid antibody treatment (Girardi et al., 2003). This result strongly suggests that alternative pathway components carried by these inflammatory cells into the placenta are necessary for tissue injury and that the protection afforded by alternative pathway deficiency is due to the lack of C3, factor B, and properdin brought in through this exogenous source (Girardi et al., 2003). Additional mechanism-based experiments are under way to further test this hypothesis, but in support of the conclusion that alternative pathway innate immune mechanisms are important in pathogenesis are results demonstrating that a novel inhibitory

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monoclonal antibody that blocks mouse factor B activity also protects mice from anti-phospholipid antibody-mediated fetal loss (Thurman et al., 2005). In addition to this model, studies have shown that the alternative pathway is activated in the placenta and is pathogenic in causing fetal loss in other circumstances. One example is the finding that mice with a complete absence of Crry expression caused by gene targeting are not viable. Specifically, heterozygous Crry-deficient mice are born in normal numbers and are healthy, but homozygous Crry-deficient mice die in utero (Xu et al., 2000). In this setting, initial Crry-deficient embryo numbers are normal, but after the deposition of C3 through alternative pathway recognition of the placenta and fetus, death ensues and no viable progeny are delivered (Mao et al., 2003). Finally, whereas results using anti-phospholipid antibodies have been illustrative of the problem related to those particular antibodies, and Crry-deficient mice illustrate the effects of complete loss of complement regulation, other studies have suggested that recurrent cellular immune-mediated fetal loss may also be primarily mediated by inappropriate alternative pathway complement activation (Mellor et al., 2001). Indeed, this area is receiving substantial attention, as it is possible that complement activation is the major pathway of fetal loss through multiple immune pathways that are initiated through either innate or adaptive immune recognition mechanisms (Cauchetaux et al., 2003). Thus, as the mechanisms of innate autoimmunity become better understood, it is likely that this pathway of tissue recognition and injury will be increasingly found to be central to the pathogenesis of human disease.

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Molina, H., Wong, W., Kinoshita, T., Brenner, C., Foley, S., and Holers, V. M. (1992). Distinct receptor and regulatory properties of recombinant mouse complement receptor 1 (CR1) and Crry, the two genetic homologs of human CR1. J. Exp. Med. 175, 121–129. Muller-Eberhard, H. J. (1988). Molecular organization and function of the complement system. Annu. Rev. Biochem. 57, 321–347. Nath, K. A., Hostetter, M. K., and Hostetter, T. H. (1985). Pathophysiology of chronic tubulointerstitial disease in rats: Interactions of dietary acid load, ammonia, and complement component C3. J. Clin. Invest. 76, 667–675. Nishimura, J., Smith, C. A., Phillips, K. L., Ware, R. E., and Rosse, W. F. (1998). Paroxysmal nocturnal hemoglobinuria: Molecular pathogenesis and molecular therapeutic approaches. Hematopathol. Mol. Hematol. 36, 119–146. Ochsenbein, A., Fehr, T., Lutz, C., Suter, M., Brombacher, F., Hengartner, H., and Zinkernagel, R. (1999). Control of early viral and bacterial distribution and disease by natural antibodies. Science 286, 2156–2159. Park, P., Haas, M., Cunningham, P. N., Bao, L., Alexander, J. J., and Quigg, R. J. (2002). Injury in renal ischemia–reperfusion is independent from immunoglobulins and T lymphocytes. Am. J. Physiol. Renal Physiol. 282, F352–F357. Qin, X., Miwa, T., Aktas, H., Gao, M., Lee, C., Qian, Y. M., Morton, C. C., Shahsafaei, A., Song, W. C., and Halperin, J. A. (2001). Genomic structure, functional comparison, and tissue distribution of mouse Cd59a and Cd59b. Mamm. Genome 12, 582–589. Rand, J. H. (2000). Antiphospholipid antibody-mediated disruption of the annexin-V antithrombotic shield: A thrombogenic mechanism for the antiphospholipid syndrome. J. Autoimmun. 15, 107–111. Ratnoff, W. D., Fearon, D. T., and Austen, K. F. (1983). The role of antibody in the activation of the alternative complement pathway. Springer Semm. Immunopathol. 6, 361–371. Reid, K. B. M., and Porter, R. R. (1981). The proteolytic activation systems of complement. Annu. Rev. Biochem. 50, 433–464. Reid, R. R., Woodcock, S., Shimabukuro-Vornhagen, A., Austen, W. G., Jr., Kobzik, L., Zhang, M., Hechtman, H. B., Moore, F. D., Jr., and Carroll, M. C. (2002). Functional activity of natural antibody is altered in Cr2-deficient mice. J. Immunol. 169, 5433–5440. Richards, A., Goodship, J. A., and Goodship, T. H. (2002). The genetics and pathogenesis of haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura. Curr. Opin. Nephrol. Hypertens. 11, 431–435. Richards, A., Kemp, E. J., Liszewski, M. K., Goodship, J. A., Lampe, A. K., Decorte, R., Muslumanoglu, M. H., Kavukcu, S., Filler, G., Pirson, Y., Wen, L. S., Atkinson, J. P., and Goodship, T. H. (2003). Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc. Natl. Acad. Sci. USA 100, 12966–12971. Seidl, K., Wilshire, J., MacKenzie, J., Kantor, A., Herzenberg, L., and Herzenberg, L. (1999). Predominant VH genes expressed in innate antibodies are associated with distinctive antigenbinding sites. Proc. Natl. Acad. Sci. USA 96, 2262–2267. Sheridan, A. M., and Bonventre, J. V. (2000). Pathophysiology of ischemic acute renal failure. Contrib. Nephrol. 132, 7–21. Simpson, R., Alon, R., Kobzik, L., Valeri, C., Shepro, D., and Hechtman, H. (1993). Neutrophil and nonneutrophil-mediated injury in intestinal ischemia–reperfusion. Ann. Surg. 218, 444–453. Sporer, H., Lang, F., Oberleithner, H., Greger, R., and Deetjen, P. (1981). Inefficacy of bicarbonate infusions on the course of postischaemic acute renal failure in the rat. Eur. J. Clin. Invest. 11, 311–315.

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Formation of Bradykinin: A Major Contributor to the Innate Inflammatory Response Kusumam Joseph and Allen P. Kaplan Division of Pulmonary/Critical Care Medicine and Allergy/Clinical Immunology, Medical University of South Carolina, Charleston, South Carolina 29425

1. 2. 3. 4. 5. 6. 7. 8.

Abstract .................................................................................................. Introduction ............................................................................................. Contact Activation ...................................................................................... Assembly on Cell Surfaces ............................................................................ Activation of the Kinin Cascade: The Role of Endothelial Cells ............................... Inhibition of Contact Activation ..................................... ................................ Inactivation of Bradykinin............................................................................. Relations of the Contact Factors to Other Systems .............................................. Considerations in Human Diseases ................................. ................................ References ...............................................................................................

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Abstract The plasma kinin-forming cascade can be activated by contact with negatively charged macromolecules leading to binding and autoactivation of factor XII, activation of prekallikrein to kallikrein by factor XIIa, and cleavage of high molecular weight kininogen (HK) by kallikrein to release the vasoactive peptide bradykinin. Once kallikrein formation begins, there is rapid cleavage of unactivated factor XII to factor XIIa, and this positive feedback is favored kinetically over factor XII autoactivation. Examples of surface initiators that can function in this fashion are endotoxin, sulfated mucopolysaccharides, and aggregated Ab protein. Physiological activation appears to occur along the surface of endothelial cells both by the aforementioned contact-initiated reactions as well as bypass pathways that are independent of factor XII. Factor XII binds primarily to cell surface u-PAR (urokinase plasminogen activator receptor); HK binds to gC1qR via its light chain (domain 5) and to cytokeratin 1 by its heavy chain (domain 3) and, to a lesser degree, by its light chain. Prekallikrein circulates bound to HK (as does coagulation factor XI), and prekallikrein is thereby brought to the surface as HK binds. All cell-binding reactions are dependent on zinc ion. Endothelial cells (HUVECs) have bimolecular complexes of u-PAR–cytokeratin 1 and gC1qR–cytokeratin 1 at the cell surface plus free gC1qR, which is present in substantial molar excess. Factor XII appears to interact primarily with the u-PAR–cytokeratin 1 complex, whereas HK binds primarily to the gC1qR–cytokeratin 1 complex and to free gC1qR.

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Release of endothelial cell heat shock protein 90 (Hsp90) or the enzyme prolylcarboxypeptidase leads to activation of the bradykinin-forming cascade by activating the prekallikrein–HK complex. In contrast to factor XIIa, neither will activate prekallikrein in the absence of HK, both reactions require zinc ion, and the stoichiometry suggests interaction of one molecule of Hsp90 (for example) with one molecule of prekallikrein–HK complex. The presence of factor XII, however, leads to a marked augmentation in reaction rate via the kallikrein feedback as well as to a change to classic enzyme–substrate kinetics. The circumstances in which activation is initiated by factor XII autoactivation or by these factor XII bypasses are yet to be defined. The pathologic conditions in which bradykinin generation appears important include hereditary and acquired C1 inhibitor deficiency, cough and angioedema due to ACE inhibitors, endotoxin shock, with contributions to conditions as diverse as Alzheimer’s disease, stroke, control of blood pressure, and allergic diseases.

1. Introduction The plasma pathway by which bradykinin is generated is closely linked to the pathways of coagulation, fibrinolysis, and inflammation and, by analogy with, the alternative complement pathway, the mannose-binding lectin (MBL) complement pathway, and Toll-like receptors; it represents part of the ‘‘innate’’ inflammatory response rather than a strictly innate ‘‘immune’’ response. The pathway consists of three requisite plasma proteins, namely, coagulation factor XII (Hageman factor), prekallikrein, and high molecular weight kininogen (HK), which interact as a plasma proteolytic cascade (Kaplan and Silverberg, 1987). However the most recent data indicate that there are important interactions of those proteins with cell surface ‘‘receptors’’ such that activation of the cascade and generation of bradykinin can occur along the cell surface. Key cell surface proteins thus far identified include qC1qR (the receptor for the globular heads of the C1Q subcomponent of complement) (Joseph et al., 1996), cytokeratin 1 (Hasan et al., 1998; Joseph et al., 1999b), and the urokinase plasminogen activation receptor (u-PAR) (Colman et al., 1997). These proteins fulfill binding functions on the surface of microvascular endothelial cells of the skin and lung, as well as human umbilical vein endothelial cells (HUVECs), astrocytes, and possibly smooth muscle cells. HK binding to neutrophils has been shown to be dependent on MAC-1 (CD11b/CD18) (Wachtfogel et al., 1994), whereas HK binding to platelets requires interaction with glycoprotein 1b (Bradford et al., 1997; Joseph et al., 1999a). Activation along the cell surface has been most extensively studied with HUVECs, and observations have dramatically altered our understanding of the biochemical mechanisms by

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which these proteins interact. New initiation mechanisms have been described that are dependent on cell-derived proteins, such as heat shock protein 90 (Hsp 90) (Joseph et al., 2002b) and prolylcarboxypeptidase (Shariat-Madar et al., 2002). In this article we first review the role of each of the plasma proteins in the generation of bradykinin, and then describe binding and activation at the plasma–cellular interface with consideration of physiological and pathologic consequences of these reactions. 2. Contact Activation The concept of contact activation was originally developed because it was found that addition of blood to a glass tube leads to coagulation. Thus ‘‘contact’’ with the silicate surface appeared to initiate a proteolytic cascade culminating in the conversion of fibrinogen of fibrin. At the same time, bradykinin is generated. These reactions that occur during activation in this fashion are shown in Fig. 1.

Figure 1 Pathway for bradykinin formation indicating the autoactivation of factor XII, the positive feedback by which kallikrein activates factor XII, cleavage of high molecular weight kininogen (HK) to release bradykinin, formation of factor XII fragment, and enzymatic activation of C1. The steps inhibitable by C1 INH are indicated.

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It has been shown that factor XII (Hageman factor) circulates as a b globulin, of molecular weight 80,000, which autoactivates on binding to surfaces bearing negative changes (Silverberg et al., 1980a). Because the zymogen has no detectable enzymatic activity (Silverberg and Kaplan, 1982), it has been proposed that trace quantities of the active enzyme (factor XIIa) actually circulate but that digestion of factor XII by factor XIIa occurs only on binding of factor XII to the surface. Thus the surface renders factor XII a substrate (Griffin, 1978) for traces of preformed factor XIIa. Prekallikrein (PK) and coagulation factor XI each circulate as a complex with high molecular weight kininogen (HK) (Mandle et al., 1976; Thompson et al., 1977) with a stoichiometry of 1:1 and 1:2, respectively (factor XI is a dimer). The binding sites for prekallikrein and factor XI on HK overlap (Tait and Fujikawa, 1986, 1987) to such a degree that HK can bind only one molecule of each, but never both. HK, however, is present in considerable molar excess. Thus we have separate complexes of prekallikrein–HK and factor XI–HK and the percentage bound to HK in each case is 85 and 95%, based on equilibrium considerations (Scott and Colman, 1980). High molecular weight kininogen is a key factor that regulates contact activation. It is also the link protein that allows assembly of the kinin-forming cascade along the surface of cells, and we therefore consider its structural features in some detail. Human plasma has two kininogens (Jacobsen, 1966) that are designated high molecular weight kininogen (HK) and low molecular weight kininogen (LK). They are assembled by alternative splicing of the terminal exons (Fig. 2) such that a large portion of their amino acid sequence is identical (Kitamura et al., 1985). The domain structure of the protein HK is shown in Fig. 3; it consists of six domains. At the N terminus are three domains (encoded by exons 1–9) that are homologous to cystatins and stefans (Kellermann et al., 1986a), including sulfhydryl proteases such as cathepsin B, H, and L, and domains 2 and 3 actually retain cysteine protease inhibitory activity (Gounaris et al., 1984; Higashiyama et al., 1986; Ishiguro et al., 1987; Muller-Esterl et al., 1985a). Domain 4 contains the bradykinin sequence plus the next 12 amino acids. Up to this point LK and HK have identical amino acid sequences. Then exon 10, which includes bradykinin plus domains 5 and 6, is added for HK (Fig. 2), whereas exon 11 is added for LK with the attachment at the C terminus of domain 4. When HK is cleaved by plasma kallikrein to release bradykinin (fast cleavage occurs at a C-terminal Arg–Ser bond, followed by cleavage at an N-terminal Lys–Arg bond) (Mori and Nagasawa, 1981; Mori et al., 1981), and the kinin-free HK is reduced and alkylated, one can isolate a heavy chain (domains 1–3) and a light chain (the C-terminal 12 amino acids of domain 4 plus domains 5 and 6) (Thompson et al., 1979). Thus the light chain of HK and LK are quite different (Kellermann et al., 1986b), and

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Figure 2 The gene for HK. Boxes 1–9 represent the exon encoding the heavy chain of both HK and LK. Exon 10 encodes the bradykinin sequence and the light chain of HK, whereas exon 11 encodes the light chain of LK. The mature mRNAs are assembled by alternative splicing events in which the light chain sequences are attached to the 30 end of the 13-amino acid common sequence C terminal to bradykinin. HMWK ¼ HK; LMWK ¼ LK.

this accounts for the difference in molecular weight and many of the functional properties of HK that are not shared by LK. It should be noted that plasma kallikrein preferentially cleaves HK (Reddigari and Kaplan, 1988, 1989), whereas tissue kallikrein (encoded by a separate gene from that of plasma kallikrein), cleaves both HK and LK, but with more favorable kinetics if LK is the substrate (Lottspeich et al., 1984; Muller-Esterl et al., 1985b). The function of HK in contact activation, as depicted in Fig. 1, are multiple. First, it accelerates the conversion of PK and factor XI to kallikrein and factor XIa, respectively (Griffin and Cochrane, 1976; Meier et al., 1977; Wiggins et al., 1977). This acceleration appears to be due to the ability of PK and factor XI to bind to HK; as a result each of them is in a more favorable conformation for activation than when they are tested unbound. In addition, HK provides the attachment to initiating surfaces and brings both PK and factor XI to the surface as a complex. If PK and factor XI bind to the surface in the absence of HK, activation by factor XIIa is markedly inhibited, even when compared with activation in the fluid phase. Thus the conformational effects of binding of PK and factor XI to HK are even more evident when activation along the

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Figure 3 The structure of HK. The heavy chain region consists of three homologous domains (1–3), of which the latter two are sulfhydryl protease inhibition sites. Domain 4 contains the bradykinin moiety. The light chain region contains the surface binding site (domain 5) and overlapping binding sites for prekallikrein and factor XI (domain 6).

surface is compared with fluid-phase activation (since factor XII is activated along surfaces, this comparison is made by adding preformed factor XIIa to PK or PK–HK either in solution or bound to a surface). Figure 1 also depicts a positive feedback in which kallikrein activates surface-bound factor XII to form factor XIIa (Cochrane et al., 1973; Meier et al., 1977; Silverberg et al., 1980b). In fact, factor XII that is bound undergoes a conformational change that renders it a substrate for factor XIIa (Dunn et al., 1982; Griffin, 1978). Thus autoactivation of factor XII can initiate the cascade once sufficient factor XIIa forms to overcome plasma inhibitors (Silverberg et al., 1980b; Tankersley and Finlayson, 1984), and only a few percent conversion to factor XIIa is required. Then the kallikrein formed activates the remaining surface-bound factor XII at a much more rapid rate. This positive feedback is also accelerated by the presence of HK (Griffin and Cochrane, 1976; Meier et al., 1977; Silverberg et al., 1980b). The factor XIIa formed remains attached to the initiating surface; further digestion of factor XIIa by kallikrein (Fig. 1) yields a 32.5-kDa factor XII fragment (factor XIIf) (Dunn and Kaplan, 1982; Kaplan and Austen, 1970, 1971) that retains the active site of factor XIIa but lacks the binding site to the surface. It is a doublet on sodium dodecyl sulfate (SDS) gels with bands at 30 and 28.5 kDa. Factor

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XIIf consists of the light chain of factor XIIa, containing the active site, disulfide linked to small C-terminal fragments of the heavy chain of 2000 or 500 corresponding to the two SDS bands. Factor XIIf lacks the binding site for the surface and is released into the fluid phase, where it can continue to activate prekallikrein until it is ultimately inactivated by plasma protease inhibitors. Factor XI is activated by factor XIIa in the presence of HK, but factor XIIf possesses only 2–4% of the coagulant activity of factor XIIa and HK does not augment its reaction rate. Thus factor XIIf can be important for bradykinin formation, but not for intrinsic coagulation. The mechanism by which HK catalyzes factor XII activation is multifactorial and indirect, since HK does not increase the enzymatic activity of kallikrein, nor does it interact with factor XII to render it a better substrate (Silverberg et al., 1980b). Its main effect is to allow dissociation of kallikrein from its complex with HK so that it can enzymatically activate factor XII along the cell surface (Cochrane and Revak, 1980). Kallikrein bound directly to the surface is much less effective and cannot disseminate the reaction (Silverberg et al., 1980b). Since HK is required for the formation of kallikrein, that is, activation of PK, the amount of kallikrein is increased when HK is present. Thus the effective ratio of kallikrein/factor XII in this enzymatic reaction is significantly augmented when HK is present. The percent augmentation of contact activation in the presence of a surface plus HK is estimated to be 3000- to 12,000-fold (Rosing et al., 1985; Tankersley and Finlayson, 1984). If one considers the rate of factor XI activation, HKdeficient plasma is almost as abnormal as factor XII-deficient plasma. HK increases the rate of formation of kallikrein, it facilitates factor XII conversion to factor XIIa by kallikrein, and it facilitates factor XI activation by factor XIIa. For comparison, it is of interest to consider the rate of factor XI activation in PK-deficient plasma, where the kallikrein feedback activation of factor XII is not possible, and the only role of HK is in conversion of factor XI to factor XIa. In this case contact activation of coagulation is very slow, but if the time of incubation of citrated plasma with the surface is increased prior to recalcification, the clotting time approaches normal (Saito et al., 1974; Weiss et al., 1974; Wuepper, 1973). This is due to gradual conversion of factor XI to factor XIa as a result of factor XII autoactivation on the surface. The next step, conversion of factor IX to factor IXa by factor XIa, is dependent on calcium, and thus a prolonged incubation allows the amount of factor XIa to increase toward normal. It should be evident from Fig. 1 that plasma that is deficient in either factor XII, PK, or HK cannot generate bradykinin via contact activation. Any bradykinin formed is then dependent on tissue kallikrein activation of LK. A detailed discussion of the structure of each protein, transcription and translational events involved in the synthesis of each protein, and mechanistic

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details regarding activation of factor XII, PK, and factor XI has been presented in this series previously (Kaplan et al., 1997), and the reader is referred to that publication for further details. 3. Assembly on Cell Surfaces 3.1. Binding of High Molecular Weight Kininogen to Human Umbilical Vein Endothelial Cells The initial studies of the interaction of proteins of the kinin-forming cascade with cells were performed with platelets (Greengard and Griffin, 1984; Gustafson et al., 1986) and then human umbilical vein endothelial cells (HUVECs) (Schmaier et al., 1988; van Iwaarden et al., 1988). In each instance, HK was shown to bind to each cell type in a zinc-dependent fashion. The binding was saturable and reversible; however, binding was found to be dependent on domains 3 and 5 (Hasan et al., 1995; Herwald et al., 1995; Reddigari et al., 1993a), so that both heavy chain and light chain were capable of similar ion-dependent interactions (Reddigari et al., 1993a). Although direct prekallikrein binding to HUVECs has been described (Mahdi et al., 2003), it is of doubtful physiological relevance, since prekallikrein is brought to the cell surface as a bimolecular complex as a result of its interaction with domain 6 of HK. Factor XII interacts with HUVECs in a similar fashion to HK; the interaction requires zinc ion and factor XII, and HK can compete for binding to the cell surface (Reddigari et al., 1993b). The latter observation suggests that they bind to similar cell surface proteins with comparable affinity. We therefore sought to purify and characterize this binding protein; the results (Joseph et al., 1996) are summarized below and correspond to a p33 endothelial cell protein isolated also by Herwald et al. (1996) and identified to be gC1qR, the receptor for the globular heads of C1q discovered by Ghebrehiwet et al. (1994). A solubilized endothelial cell membrane preparation was passed over an HK affinity column in the presence or absence of zinc ion and eluted with glycine-HCl (0.1 M, pH 2.5), and the fractions were neutralized. An aliquot of each eluate fraction (with or without zinc) was spotted onto nitrocellulose membrane and blotted with biotinylated HK, developed with alkaline phosphatase–streptavidin, followed by reaction with nitroblue tetrazolium/5-bromo-4-chloroindolyl phosphate. A prominent increase in HK binding was observed after elution in the presence of zinc. These fractions were then pooled, concentrated, and analyzed by SDS–PAGE. The main feature was the appearance of a new prominent band at 33 kDa that was visible with Coomassie stain. In addition, ligand blot experiments demonstrated that biotinylated HK bound only to the 33-kDa band. Based on this information, the

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33-kDa protein was subjected for N-terminal amino acid sequence analysis and the first 13 amino acids were found to be identical to the known NH2 terminus of gC1qR (Ghebrehiwet et al., 1994). We next performed a Western blot using anti-gC1qR monoclonal antibody 60.11 to further assess the identity of these two proteins. Monoclonal antibody 60.11, which interacts with an epitope at the N terminus of gC1qR, identified the 33-kDa HUVEC-derived membrane-binding protein. We next determined whether factor XII could also bind to gC1qR. HUVEC membrane–purified gC1qR or recombinant gC1qR (rgC1qR) at 1.0 to 2.0 mg was applied to nitrocellulose membranes and blotted with biotinylated HK or factor XII in the presence or absence of 50 mM zinc. Sufficient o-phenanthroline was added to bind the zinc in the purified (but not recombinant) gC1qR to allow zinc-independent binding to be assayed. We found that both HK and factor XII bind to either purified or rgC1qR in the presence of zinc. Additional controls included biotinylated IgG, which did not bind to gC1qR, and substituting prekallikrein for gC1qR to which biotinylated HK bound, as expected (Mandle et al., 1976). Addition of excess unlabeled HK reversed the ability of factor XII to bind to gC1qR by over 90% as quantitated by scanning, which suggests interaction with a common domain within the protein. Factor XII only partially (46%) reverses HK binding. This difference may be due to the relative affinity of the two ligands for the gC1qR molecule. Nevertheless, these data completely paralleled those observed on binding of factor XII and HK to endothelial cells. We next attempted to demonstrate that the interaction with gC1qR is indeed responsible for binding to the cell surface, which was addressed by inhibition experiments. HUVECs were incubated for 30 min with HK (8.7  108 M), or anti-gC1qR mAbs 74.5.2 and 60.11. Then, in the presence of 50 mM zinc, [125I]HK was added and incubated for 60 min at 37 8C, conditions known to saturate the binding sites (Hasan et al., 1995). For each condition, the percentage inhibition of [125I]HK binding was determined. Whereas a 100molar excess of nonradiolabeled HK inhibited subsequent [125I]HK binding, a comparable concentration of C1q did not, indicating that the binding sites of gC1qR for C1q and for HK do not overlap. Furthermore, although mAb 60.11 did not efficiently inhibit [125I]HK binding to HUVECs, mAb 74.5.2 did. It had been shown previously that antibody 60.11 inhibits C1q binding to gC1qR, whereas mAb 74.5.2 does not. Other mAbs recognizing epitopes in different regions of the molecule were also tested for their inhibitory activity. Of these, only 25.15, which is similar to 74.5.2, was able to inhibit [125I]HK binding to HUVECs. Herwald et al. (1996) also demonstrated that gC1qR, a major endothelial cell-binding protein for HK, used a domain 5-derived peptide from the light chain rather than whole HK as the ligand. In aggregate, these

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data indicate that gC1qR serves as a zinc-dependent binding protein for factor XII as well as for HK, and that binding to HK occurs via the light chain moiety. The specific location within HK for binding to endothelial cells is within domain 5 (Hasan et al., 1995; Reddigari et al., 1993a), and this also appears to be the site of interaction with gC1qR. It is also clear that the HK heavy chain also binds to endothelial cells. This interaction has been shown to require domain 3. The methods employed to identify the cell surface protein that interacts with heavy chain are analogous to those described above for isolation of gC1qR. Later, a second HK-binding protein was identified in HUVECs by affinity chromatography employing HK as ligand (Hasan et al., 1998; ShariatMadar et al., 1999) and was identified as cytokeratin 1. We reasoned that this protein might contribute to heavy chain interaction with cells and therefore prepared an affinity column by covalently coupling peptide LDC27 sequence to the matrix; this is a 27-amino acid peptide derived from domain 3, which has been identified as an HK-binding site (Herwald et al., 1995). When cell membranes derived from HUVECs were applied to the column in the presence and absence of 50 mM zinc and each was eluted with 0.1 M glycine-HCl, pH 2.5, a band was noted at molecular mass of 68 kDa in the zinc-containing eluate. A ligand blot with HK confirmed binding to this band. When we attempted to sequence it, the N terminus was blocked. We therefore next digested the protein with cyanogen bromide and subjected the mixture to mass spectrometry. A major peptide at molecular weight 2721.7 was identified, and its sequence was determined and shown to correspond to an internal peptide derived from cytokeratin 1 (Fig. 4) (Joseph et al., 1999b,c). Thus HK binding to HUVECs appeared to depend on interaction with two proteins, cytokeratin 1 and gC1qR, with binding to each by domains 3 and 5 of HK, respectively (i.e., binding of heavy chain to cytokeratin 1 and light chain to gC1qR). We demonstrated that gC1qR cannot bind heavy chain at all, whereas cytokeratin 1, when tested as a purified protein, can bind both heavy and light chains; however, binding to heavy chain clearly predominates. Factor XII is capable of binding to both proteins. To confirm that these proteins are important for binding to endothelial cells, we performed an inhibition experiment in which antibody to gC1qR and antibody to cytokeratin 1 were employed. As can be seen in Fig. 5, antibody to gC1qR inhibited zinc-dependent binding by 65%, antibody to cytokeratin 1 inhibited binding by 30%, whereas a combination of antisera inhibited binding by 85%. Since 15% binding corresponds to zinc-independent binding, our data suggest that we account for most, if not all, HK binding to endothelial cells by these two proteins. A third protein reported to be important for HK binding to HUVECs was identified to be u-PAR (the urokinase plasminogen activator receptor)

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Figure 4 Localization of the peptide fragment within the cytokeratin 1 sequence. Complete amino acid sequence of cytokeratin 1 indicates a 24-amino acid internal peptide (boldface, double underlined) corresponding identically to the sequence of the peptide isolated from the cyanogen bromide digest. Fragments were isolated by HPLC and the 2721.7 molecular weight peak was sequenced.

by inhibition of HK binding with antisera to domain 2/3 of u-PAR (Colman et al., 1997). However, we have not been able to isolate u-PAR from cell membranes (confirmed to contain considerable u-PAR) by HK affinity chromatography. One difference in the experiments is that the studies by Colman et al. employed cleaved HK lacking the bradykinin moiety; it is possible that cleaved HK binds more avidly to u-PAR than does native HK while native HK binds more avidly to gC1qR and cytokeratin 1 than it does to u-PAR. 3.2. Binding of Factor XII to HUVEC Early studies demonstrated that factor XII binds to both gC1qR and cytokeratin 1 and that it competes for the same binding sites as does HK (Fig. 6). The first study to attempt to identify the binding site on HUVECs (rather than testing purified proteins shown to bind HK) was a study by Mahdi et al. in which antisera to u-PAR, cytokeratin 1, and gC1qR were employed to inhibit cell binding of factor XII (Mahdi et al., 2002). A surprising result was that antibody to u-PAR inhibited best, although the other antisera were

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Figure 5 [125I]HK binding to HUVECs and its inhibition by monoclonal antibodies. (A) HUVECs were incubated with [125I]HK (20 nM) in the presence (Z) or absence (m) of 50 mM zinc. After the indicated times, the cells were washed and counted for bound radioactivity. (B) For inhibition studies, cells were preincubated with monoclonal antibodies or nonimmune mouse IgG for 30 min. After 30 min, [125I]HK was added and further incubated for 1 hr at room temperature. The lines represent HK-binding values after treatment with a monoclonal antibody to gC1qR (s), monoclonal antibody to u-PAR (Z), monoclonal antibody to cytokeratin 1 (“), and a combination of monoclonal antibodies to gC1qR and cytokeratin 1 (r). Antibody to gp1b (control) showed similar results to the control mouse IgG. Each point is a mean of three different experiments, performed in triplicate.

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Figure 6 High molecular weight kininogen competes with factor XII for the same binding sites on HUVECs. (A) HUVECs were incubated with [125I]FXII (1 mg/ml) in triplicate in the presence of incremental concentrations of unlabeled factor XII, HK, or normal human IgG for 120 min, and bound ligand was determined. The percentage bound in the presence of a competitor is plotted against the concentration of the competitor. (B) HUVECs were incubated with [125I]HK (1 mg/ml) in triplicate in the presence of increasing concentration of unlabeled factor XII, and bound ligand was determined.

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contributory. We therefore sought to corroborate the observation by isolation of factor XII–binding proteins directly from HUVEC-derived cell membrane preparations by affinity chromatography employing factor XII as ligand. The major zinc-dependent binding protein was clearly u-PAR; gC1qR was also isolated as well as small amounts of cytokeratin 1 (our unpublished data). We could also demonstrate more avid binding of factor XII to u-PAR than to either gC1qR or cytokeratin 1 by competitive displacement of factor XII bound to one protein, employing increasing quantities of either of the other two. Thus we began to develop a model for assembly of the kinin-forming cascade on endothelial cells in which zinc-dependent binding of factor XII is associated predominantly with u-PAR, and HK binds to gC1qR as well as cytokeratin 1 while prekallikrein is bound to the HK. How that binding occurs, particularly for HK, depends on the way these proteins are distributed within the cell membrane of HUVECs. 3.3. Interaction of gC1qR, Cytokeratin 1, and u-PAR with HUVEC Membranes One dilemma posed by antibody inhibition studies in which all three antisera were employed was that the total percent inhibition obtained when the three percentages were added exceeded 100%. One possible explanation was that these proteins might interact in some fashion within the cell membrane and a trimolecular complex containing all three was proposed (Colman and Schmaier, 1997). Thus antisera to one protein might sterically interfere with binding to the others and falsely influence the percent inhibition observed. A second, different alternative was proposed by another group, who reported that HK binding to HUVECs was not due to interaction with proteins at all, but that proteoglycans such as syndican and glypican were in fact responsible (Renne et al., 2000) and questioned whether gC1qR is truly demonstrable along the cell membrane surface (Dedio and Muller-Esterl, 1996; Dedio et al., 1998). It should be noted that u-PAR is known to be linked to cell membrane constituents by a phosphatidylinositol bond, but that gC1qR and cytokeratin 1 are not, and the latter two proteins lack typical transmembrane domains. Thus if they are present within cell membranes, the mode of attachment is unknown. Finally, the number of binding sites reported for gC1qR on the cell membrane varied from just under 1 million to 10 million (Motta et al., 1998; Reddigari et al., 1993a, van Iwaarden et al., 1988); questions were raised regarding such a high figure, although binding to a cell membrane proteoglycan could achieve such levels. We (and others) addressed each of these issues. Employing high-titer, monospecific antisera to gC1qR, the protein was clearly demonstrated to be

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at the HUVEC surface (Joseph et al., 1999c). Mahdi et al. then demonstrated the presence of all these proteins within cell membranes by immunoelectron microscopy (Mahdi et al., 2001, 2002). Cytokeratin 1 and u-PAR appeared to be colocalized while gC1qR was present throughout the cell membrane. We addressed the question of binding to proteoglycan by employing heparanases, which remove all heparan sulfate–containing structures from cell membranes and demonstrated that HUVECs treated so as to become unreactive to antisera to heparan sulfate (the major sulfate source associated with cell surface proteoglycans) bound HK normally, and the binding was then inhibited with antisera to gC1qR and cytokeratin 1 (Fernando, 2003a). We next solubilized purified cell membranes from HUVECs and demonstrated that gC1qR, cytokeratin 1, and u-PAR are all present, by immunoblot analysis. There was no significant contamination by other cell constituents, particularly mitochondria, which are known to contain large amounts of gC1qR (Dedio et al., 1998). We then addressed the interactions of these proteins with each other. First, we could show that gC1qR binds to cytokeratin 1 but not u-PAR while u-PAR also binds to cytokeratin 1, but not to gC1qR. Thus a trimolecular complex is not possible, but two bimolecular complexes seemed feasible. We then precipitated gC1qR and u-PAR from cell membrane preparations and analyzed the composition of the precipitated materials. Cytokeratin 1 was precipitated with both anti-gC1qR and anti-u-PAR; however, the gC1qR–cytokeratin 1-containing fraction had no u-PAR while the cytokeratin 1–u-PAR fraction contained no gC1qR. Our current view of the assembly of the proteins of the kinin-forming cascade on HUVECs envisions factor XII bound to a complex of u-PAR–cytokeratin 1 while HK binds to a complex of gC1qR–cytokeratin 1. We do not know whether HK binds to the complex by both domain 3 and domain 5 simultaneously or whether binding to one site affects binding to the other. Complicating this assessment is the fact that the number of gC1qR sites within the cell membrane is at least three times that of u-PAR or cytokeratin 1, and thus gC1qR unassociated with either cytokeratin 1 or u-PAR is likely present and can bind HK or factor XII. Given the relative affinities of factor XII, HK heavy chain, and HK light chain for gC1qR, we would anticipate preferential binding of the light chain (domain 5) of HK to gC1qR. Consistent with this is the prominent inhibition of HK binding to the cell when employing peptide HKH20 derived from domain 5 of the light chain (Nakazawa et al., 2002). 3.4. Binding to Other Cells The interaction of factor XII and HK with other cell types resembles that seen in HUVECs, although there are differences in the number of binding sites, the affinity of binding, and the nature of the binding proteins. We reported studies

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of microvascular endothelial cells derived from skin and lung and compared binding with that seen in HUVECs, since these cells are more likely to be ‘‘physiological’’ (Fernando, 2003b). We also compared binding to HUVECs with that seen with astrocytes, because upregulation of bradykinin B2 receptors has been demonstrated within the central nervous system of patients with Alzheimer’s disease (Jong et al., 2002), and aggregated Ab protein of Alzheimer’s disease is a potent activator of the kinin cascade (Shibayama et al., 1999). The results are shown in Table 1. It should be noted that a large number of HK-binding sites was demonstrated for HUVECs by two separate methods, including a fluid phase-based assay in which 850,000 sites per cell were documented. It has been suggested that values in the 10 million range (Motta et al., 1998) may have been due to ligand binding to the plates used rather than to the cells coating the plates, and thus the cell surface represented only a fraction of the total binding seen (Baird and Walsh, 2002, 2003). However, the inhibition of such binding, employing antisera to the cell surface ligands, suggests some other interpretation (Mahdi et al., 2003), and our values in the fluid phase are in close agreement with binding studies performed with microtiter plates (Table 1). Binding of HK and factor XII to neutrophils and platelets has also been studied. HK interacts with neutrophils in a zinc-dependent fashion, analogous to that seen with other cell types; however, the protein with which it interacts is MAC-1 (C3bi receptor; CD11b/CD18) (Wachtfogel et al., 1994). Zinc-dependent HK binding to platelets is dependent on interaction with glycoprotein 1b (Bradford et al., 1997; Joseph et al., 1999a). Since both prekallikrein and factor Table 1 Dissociation Constant and Number of HK-Binding Sites per Cell in Human Endothelial Cells and Astrocytesa Dissociation constant (Kd, nM)

Number of binding sites per cell (n)

HMVEC-D HMVEC-L HUVECs

1.86  0.56 4.50  1.48 10.35  1.02

Astrocytes

23.73  3.61

52,833  11,121 316,306  101,031 696,427  123,497b 771,666  175,000c 61,574  4887

Cell type

Abbreviations: HMVEC-D, dermal microvascular endothelial cells; HMVEC-L, lung microvascular endothelial cells; HUVECs, human umbilical vein endothelial cells. a Data represent means  SD, n ¼ 3. The apparent affinity and the number of binding sites (n) are calculated by Scatchard analysis of the saturation curve. b Cells attached to 96-well plate. c Cells in suspension.

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XI circulate as complexes with HK, HK–factor XI should bind to HUVECs in an analogous fashion, as does HK–prekallikrein (Shariat-Madar et al., 2001). However, Baird and Walsh (2003) reported that although preformed HK– prekallikrein binds to endothelial cells as a complex, HK–factor XI does not. This is in contrast to studies in which HK is bound, and factor XI is added separately. In response, Mahdi et al. agree that HK–prekallikrein predominates, but that HK–factor XI can bind with lower affinity (Mahdi et al., 2003). Binding to platelets differs because factor XI can interact with platelets in the absence of HK, and there appear to be separate receptors for factor XI and factor XIa (Sinha et al., 1984). Platelets also possess an intrinsic protein with factor XI activity that cross-reacts with plasma factor XI immunologically, but differs in molecular weight and isoelectric point (Hsu et al., 1998; Lipscomb and Walsh, 1979). This form of factor XI is present even in patients who are deficient in plasma factor XI (Tuszynski et al., 1982) and has been shown to be an alternatively spliced form of factor XI in which one exon is missing (Hsu et al., 1998). During blood coagulation by the extrinsic (tissue factor) pathway, these forms of factor XI are more likely activated by the thrombin feedback (Baglia and Walsh, 2000; Baglia et al., 2002; Gailani and Broze, 1991; Naito and Fujikawa, 1991) than by factor XIIa to augment clot formation. This has been shown to occur as a late event within a fibrin matrix (Bouma and Meijers, 2000; Rand et al., 1996). Thus activation on platelets involves factor XII–dependent (Brunnee et al., 1993; Walsh and Griffin, 1981) and independent (thrombin) pathways (von dem Borne et al., 1994), and the factor XI may be attached to the platelets via HK or by separate receptors. LK, which has a separate light chain from HK, interacts with platelets (as is true of endothelial cells) and, of necessity, does so solely via domain 3 (Herwald et al., 1995; Jiang et al., 1992a,b). 4. Activation of the Kinin Cascade: The Role of Endothelial Cells 4.1. Activation by Binding to the Cell Surface We have demonstrated that factor XII can slowly autoactivate when bound to endothelial cells and that addition of kallikrein can digest bound HK to liberate bradykinin at a rate proportional to the kallikrein concentration and with a final bradykinin level dependent on the amount of bound HK (Nishikawa et al., 1992). Thus, activation of the cascade along the endothelial cell surface is likely; bradykinin is liberated and then interacts with the B2 receptor to increase vascular permeability. Bradykinin can also stimulate cultured endothelial cells to secrete tissue plasminogen activator (Smith et al., 1985), prostaglandin I2 (prostacyclin), thromboxane A2 (Crutchley et al., 1983; Hong, 1980),

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Figure 7 Effect of gC1qR and zinc ion on factor XII–dependent conversion of prekallikrein to kallikrein. Each reaction mixture contained factor XII (1 mg/ml), HK (1 mg/ml), prekallikrein (1 mg/ ml), 0.6 mM S2302, and recombinant gC1qR (0–100 mg/ml) in HEPES-buffered saline with 50 mM zinc chloride (A) or without zinc chloride (B) at 25 8C. The rate of conversion of prekallikrein to kallikrein was monitored at 405 nm.

and nitric oxide (Zhao et al., 2001) and can thereby modulate platelet function and stimulate local fibrinolysis. We next questioned whether factor XII binding to gC1qR is capable of initiating this cascade. We therefore incubated purified factor XII with a wide concentration of gC1qR (0–100 mg/ml) for a 30-min time period and prepared replicate samples that were incubated in the absence of zinc ion. As shown in Fig. 7, the rate of prekallikrein conversion to kallikrein increased as the concentration of gC1qR increased (Joseph et al., 2001a,b) and there was no activation if zinc was eliminated from the reaction mixture. Purified cell membrane (native) gC1qR yields a response that is indistinguishable from a recombinant protein, indicating that gC1qR glycosylation does not affect its ‘‘surface’’ properties. If gC1qR is incubated directly with prekallikrein or with prekallikrein plus HK, there is no conversion of prekallikrein to kallikrein, again emphasizing the requirement for factor XII. We believe this to be a physiologic phenomenon that is controlled by C1 INH and a2-macroglobulin. This may be one source of the minute quantities of factor XIIa that escape inhibition and that are requisite for contact activation in plasma or during pathologic processes. Other data employing endothelial cells corroborate the aforementioned effect of gC1qR when endothelial cells are incubated with normal plasma, and the rate of kallikrein formation is compared with that seen with plasma deficient in factor XII, prekallikrein, or HK. There was no detectable activation in any plasma except normal plasma (Fig. 8A), and the activation was inhibited by antisera to gC1qR and cytokeratin 1 (Fig. 8B). When the reaction proceeds beyond 2 hr, the factor XII–deficient

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Figure 8 Prekallikrein activation on endothelial cells. (A) Endothelial cells were incubated with normal, prekallikrein-deficient, factor XII–deficient, or HK-deficient plasmas for 1 hr at 37 8C. After incubation, the cells were washed with HEPES-buffered saline containing 50 mM zinc chloride, and prekallikrein activation was monitored by the cleavage of a kallikrein-specific substrate, S2302 (0.6 mM), at 405 nm. In (B), endothelial cells were preincubated with antibodies to cytokeratin 1, gC1qR, or a combination of both for 30 min before addition of normal plasma.

plasma activates, but the HK-deficient plasma and prekallikrein-deficient plasma do not, and thus a cell-dependent activation of the prekallikrein in the presence of HK but the absence of factor FXII appeared possible. 4.2. Factor XII–Independent Activation of the Prekallikrein–HK Complex Studies have demonstrated that binding of the PK–HK complex to endothelial cells leads to activation in the absence of factor XII (Rojkjaer and Schmaier, 1999a,b; Rojkjaer et al., 1998) and that the kallikrein that forms can digest HK to liberate bradykinin and also initiate fibrinolysis (Lin et al., 1997). The latter reaction is dependent on kallikrein activating prourokinase (bound to cell membrane u-PAR) to urokinase, which in turn converts plasminogen to the fibrinolytic enzyme plasmin. Once such a reaction is set in motion, the addition of factor XII leads to a marked increase in reaction kinetics as a result of the conversion of factor XII to factor XIIa by kallikrein. These observations raise two important questions: (1) What is the nature of the prekallikrein activator? (2) When factor XII is present (the normal circumstance), is the cascade initiated by factor XII autoactivation or is the prekallikrein first activated by some cell-derived factor and kallikrein then activates the factor XII?

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We next sought to purify and characterize the cell-derived protein(s) responsible for prekallikrein activation in the absence of factor XII. We first noted that this activity was present in both the cell membrane fraction as well as the cytosol derived from endothelial cells and chose to isolate it from the cytosol. We took advantage of the fact that the prekallikrein-activating moiety appeared to be inhibitable by corn trypsin inhibitor (CTI) (as is factor XIIa); because a CTI affinity column could bind the activity, it was recoverable by eluting the column. A single-step purification, followed by sequence analysis of suspect bands seen on SDS gel electrophoresis, ultimately determined that heat shock protein 90 (Hsp 90) is responsible for the activity seen. Thus when cloned Hsp 90 was incubated with prekallikrein and HK, the prekallikrein was converted to kallikrein, and HK was cleaved to liberate bradykinin (Joseph et al., 2002a,b; Fig. 9). This is also demonstrable by binding prekallikrein and HK to endothelial cells and assessing the rate of conversion of prekallikrein to kallikrein. Both HK and zinc ion are requisite, and the rate is fast. However, this is in contrast to the slow and factor XII–dependent activation seen when whole plasma (Fig. 8) is employed. The reaction is readily demonstrable in the fluid phase as well as by assembly of components along the cell surface; however, it differs strikingly from that seen with factor XIIa. The most critical difference is that prekallikrein is not activated unless HK is present. Factor

Figure 9 Prekallikrein activation of Hsp 90. Purified Hsp 90 (2 mg) was incubated with prekallikrein (20 nM), HK (20 nM), zinc (50 mM), and S2302 (0.6 mM), and chromogenic activity was monitored. Controls were either in the absence of zinc, HK, or both.

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XIIa readily activates prekallikrein, although the presence of HK does augment the reaction rate. Second, the reaction is stoichiometric, that is, the amount of prekallikrein activated has a 1:1 molar ratio to the amount of Hsp 90 present. When we tried to determine the structural features of HK that are required, we found that individual heavy and light chains were inactive, and cleaved HK, with bradykinin removed (two-chain HK rather than single chain), lost about 70% of the activity (Fig. 10). Thus native HK is required. Addition of a peptide that prevents the interaction of prekallikrein with HK also completely inhibits the effect of adding Hsp 90. Hsp 90 is therefore a stoichiometric activator of the prekallikrein–HK complex and not a prekallikrein activator, as is factor XIIa. One of the interesting questions we might consider is whether Hsp 90 has enzymatic activity with the prekallikrein–HK complex as substrate. Hsp 90 does have ATPase activity (Richter et al., 2001), but it is not known to be a proteolytic enzyme. The prekallikrein-activating activity can be inhibited by diisopropylfluorophosphate (DFP), but it has been difficult to characterize the active site. Although DFP inhibits the reaction, we have been unable to incubate DFP with individual components, dialyze it out, and inhibit the reaction. In fact, if DFP is added to a mixture of Hsp 90, prekallikrein, and

Figure 10 Effect of HK on prekallikrein activation. Cytosol (20 mg) was incubated with 20 nM HK, low molecular weight kininogen (LK), cleaved HK (2C-HK), purified heavy chain of HK (HCHK), or light chain of HK (LC-HK) in the presence of 20 nM prekallikrein, 50 mM zinc, and 0.6 mM S2302. After 2 hr, the chromogenic activity was measured at 405 nm.

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HK, it inhibits conversion of prekallikrein to kallikrein; but if the DFP is then dialyzed out of the mixture, prekallikrein activation then proceeds normally. Thus DFP behaves as a reversible inhibitor instead of as an irreversible inhibitor and is not phosphorylating an active site serine as is its usual effect on serine proteases. We have considered alternative possibilities, for example, autoactivation of prekallikrein within the prekallikrein–HK complex on addition of Hsp 90, or even that HK becomes an enzyme that converts prekallikrein to kallikrein when Hsp 90 binds. Other studies have isolated yet another protein with similar functional capability. Shariat-Mader et al. isolated a membrane protein that converts prekallikrein to kallikrein within the prekallikrein–HK complex and identified it to be prolylcarboxypeptidase (Motta et al., 2001; Shariat-Madar et al., 2002). This is an exopeptidase that, if its enzymatic capability is relevant, is behaving as an endopeptidase. It is said to be active along the cell surface but not in the fluid phase, which differs from Hsp 90, but its mechanism of action is otherwise strikingly similar. Both require the presence of HK and zinc ion, the reaction in each case is stoichiometric, and each is inhibited by DFP. Although the prolylcarboxypeptidase is assumed to be the enzyme that activates prekallikrein within the prekallikrein–HK complex, we suspect that some other mechanism may be operative, perhaps common to both. The prolylcarboxypeptidase provides an interesting link of the kinin-forming cascade to the biology of angiotensin, since its function, when originally isolated, was to convert angiotensin II to angiotensin III, which inactivates it. Thus a molecule that can generate bradykinin, a vasodilator, inhibits another that is a vasoconstrictor. Hsp 90 is also of particular interest, since this is a protein that is constitutively present yet upregulated with tissue stress such as hypoxia or during an inflammatory response. 4.3. Considerations of Activation When Factor XII is Present Since the endothelial cell participates in the activation of the bradykininforming cascade, when all three components are present, factor XII might be activated by autoactivation on gC1qR, requiring trace amounts of factor XIIa that is present in plasma, or factor XII might be activated by kallikrein. In the latter scenario, the source of kallikrein would be the stoichiometric interaction of prekallikrein–HK with Hsp 90 and/or prolylcarboxypeptidase. Formation of factor XIIa then markedly accelerates activation of prekallikrein– HK, since this reaction has typical Michaelis–Menten kinetics. Of course, all these may be occurring simultaneously, but the evidence thus far suggests that the rate of activation of the prekallikrein–HK complex exceeds that of factor XII autoactivation. Thus it is possible that initiation of the cascade on the

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surface is actually kallikrein, whereas factor XII has the role of an accelerator. Some favor this possibility, employing endothelial cells and purified protein constituents (Rojkjaer et al., 1998; Schmaier, 1997, 1998; Schmaier et al., 1999). On a quantitative basis, the cascade remains factor XII dependent. However, the data in Fig. 8 suggest that factor XII may truly initiate when whole plasma is studied, that is, minimal dilution in the presence of all the plasma inhibitors. 5. Inhibition of Contact Activation Regulation of factor XII–dependent pathways occurs by both intrinsic and extrinsic controls. Cleavage of factor XIIa to XIIf (Fig. 1) is one example of an intrinsic control. The factor XIIf produced is not surface bound and is a poor activator of factor XIa. At the same time, the heavy chain moiety, which has no enzymatic activity, retains the surface-binding site and can compete with factor XII and HK for binding to the surface. Thus, the conversion of factor XIIa to factor XIIf will reduce the rate of the surface-dependent reactions of coagulation, whereas bradykinin generation via fluid-phase activation of prekallikrein continues. Similarly, digestion of kinin-free HK by factor XIa has been reported to limit its coagulant activity, (Scott et al., 1985) although, in this case, the kinetics appear to be too slow to be of physiologic importance (Reddigari and Kaplan, 1988). Extrinsic controls are provided by plasma inhibitors for each enzyme. Table 2 indicates the major inhibitors of each active enzyme and, where Table 2 Plasma Inhibitors of Enzymes of Contact Activation: Relative Percent Contributions to Inhibition in Normal Human Plasma Enzyme Inhibitor C1 inhibitor Antithrombin IIIc a2-Macroglobulin a1-Protease inhibitor a2-Antiplasmin

Factor XIIa

Factor XIIf

91.3 1.5 4.3 — 3.0

93 4 — — 3

Kallikrein 52 (84)b ND 35 (16)b ND ND

Factor XIaa 8 (47) 16 (5) — 68 (23.5) 8c (24.5)

Abbreviation: ND, not determined separately. a Data given are from kinetic studies and irreversible complexes formed in plasma are given in parentheses. b Data obtained from generation of kallikrein in situ. c Data are for results obtained in the absence of added heparin.

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known, their relative contributions to the total inhibition in plasma. Inhibition of the contact activation proteases is clearly different from that of the rest of the coagulation pathways in that antithrombin III (ATIII) appears to play only a minor role. Instead, contact activation appears to be limited mainly by C1 inhibitor (C1 INH), which is not active against any of the other clotting factors except for inhibition of factor XI. C1 INH is cleaved by the protease it inhibits and attaches to the active site in a covalent complex. It may remain in a stable form of the acyl enzyme intermediate that characterizes the normal serine protease mechanism (Travis and Salvesen, 1983). Thus, after a protease has reacted with C1 INH, it cannot digest protein substrates or hydrolyze small synthetic substrates, and the reaction of the active site serine with DFP is abolished. C1 INH is the only major plasma inhibitor of factor XII and factor XIIf (de Agostini et al., 1984; Forbes et al., 1970; Pixley et al., 1985; Schreiber et al., 1973). Although ATIII can inhibit activated factor XII (Cameron et al., 1989; de Agostini et al., 1984; Stead et al., 1976), its contribution to factor XIIa inhibition in plasma is apparently only a few percent of that caused by C1 INH (de Agostini et al., 1984; Pixley et al., 1985). Disagreement exists over the effect of heparin on the inhibition of activated factor XII by ATIII. Some investigators have observed little enhancement of the rate of factor XIIa inhibition (Pixley et al., 1991), whereas others have observed a significant increase (Cameron et al., 1989; Stead et al., 1976). Heparin can act as an activating surface for contact activation, and factor XII and factor XIIa can bind to it (Hojima et al., 1984; Silverberg and Diehl, 1987). This binding is a factor in the inhibition by ATIII, since inhibition of factor XIIf, which lacks the surface-binding site, is not augmented in the presence of heparin as much as that of factor XIIa (Cameron et al., 1989). Curiously, a2-macroglobulin, although thought of as a ‘‘universal’’ protease inhibitor (Barrett and Starkey, 1973), does not significantly inhibit either form of activated factor XII. The two major inhibitors of plasma kallikrein are C1 INH and a2-macroglobulin (Gigli et al., 1970; Harpel, 1974; Harpel et al., 1985; McConnell, 1972). Together they account for over 90% of the kallikrein inhibitory activity of plasma, with the remainder contributed by ATIII (Schapira et al., 1982c; van der Graaf et al., 1983a). When kallikrein is added to plasma, approximately one half is bound to C1 INH and one half to a2-macroglobulin (Harpel et al., 1985). a2-Macroglobulin does not bind to the active site of kallikrein but appears to trap the protease within its structure so as to sterically interfere with its ability to cleave large protein substrates (Barrett and Starkey, 1973). The degree of inhibition is greater than 95%, but the residual activity is detectable when assayed for lengthy incubation periods. In contrast, digestion of small synthetic substrates is much less affected, and approximately one third

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of the starting activity is retained. When a surface such as kaolin is added to plasma so that kallikrein is generated in situ, close to 70 or 80% of it is bound to C1 INH (Harpel et al., 1985). The reason for the difference between the patterns of inhibition of added kallikrein and of endogenously produced kallikrein is unknown. Interestingly, at low temperatures, most of the inhibition of added kallikrein is accounted for by a2-macroglobulin (Harpel et al., 1985); C1 INH appears to be ineffective in the cold (Cameron et al., 1989), and this may underlie the phenomenon of ‘‘cold activation’’ of plasma. The inhibition of kallikrein by ATIII is also enhanced by heparin (Vennerod et al., 1976) and may therefore become significant in heparinized plasma. The inhibition profile of factor XI is complicated by the involvement of several factors. In kinetic studies of purified components, a1-antiproteinase inhibitor (a1-antitrypsin) appears to be the most significant inhibitor of factor XIa (Heck and Kaplan, 1974; Scott et al., 1982b), whereas a1-antitrypsin is not a major inhibitor of other coagulation factors. When the generation of irreversible enzyme inhibitor complexes was assessed in plasma, however, C1 INH was found to be the key inhibitor (Wuillemin et al., 1995), with approximately equal contributions by a2-antiplasmin and a a1-antiproteinase inhibitor. ATIII is also an inhibitor of factor XI, with potential for augmentation by heparin, the magnitude of which is unclear (Beeler et al., 1986; Scott et al., 1982a). Physiologic glycosaminoglycans also augment inhibition by C1 INH (Wuillemin et al., 1995, 1996). The combined effects of ATIII and C1 INH may therefore be most significant at the surfaces, where these substances are plentiful. Finally, platelets secrete protease nexin-2 (a soluble form of amyloid b-protein precursor), which is an efficient but reversible inhibitor in the regulation of factor XIa activity when first generated, although the protective effect of HK against inactivation may also be important (Scandura et al., 1997; Zhang et al., 1997). The predominant role of C1 INH in the regulation of contact activation in human plasma is underscored by the fact that it alone is an efficient inhibitor of activated factor XII, kallikrein, and factor XIa. In plasma from patients with hereditary angioedema (HAE), in which C1 INH is absent, the amount of dextran sulfate required to produce activation is reduced 10-fold compared with normal plasma (Cameron et al., 1989); similar results are obtained in cold plasma. Because some surface was still required for activation under these conditions, we may surmise that the other inhibitors that are active against the contact factors do serve to limit their reactions, but that in normal plasma it is inhibition by C1 INH that forms the barrier to the initiation of contact activation. The plasma concentration of C1 INH is approximately 2 mM, and it is remarkable that its inhibition is ever overcome. That surfaces are able to induce activation must reflect the protection of the proteases at the surface

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from inhibition. It has also been proposed that kallikrein bound to HK is protected from inactivation by C1 INH (Schapira et al., 1981, 1982b) and a2-macroglobulin (Schapira et al., 1982b; van der Graaf et al., 1983a,b) and that factor XIa is similarly protected from a1-antiproteinase inhibitor (Scott et al., 1982b); this mechanism, however, has been ruled out in the case of kallikrein and C1 INH (Silverberg et al., 1986; van der Graaf et al., 1983a). 6. Inactivation of Bradykinin Bradykinin is an exceedingly potent vasoactive peptide that can cause venular dilatation, activation of arterial endothelial cells, increased vascular permeability, hypotension, constriction of uterine and gastrointestinal smooth muscle, constriction of the coronary and pulmonary vasculature, bronchoconstriction, and activation of phospholipase A2 to augment arachidonic acid metabolism. Its regulation is of prime importance, and a variety of enzymes in plasma contribute to kinin degradation. Carboxypeptidase N (Erdos and Sloane, 1962) removes the C-terminal Arg from bradykinin to leave an octapeptide, des-Arg9 bradykinin (Sheikh and Kaplan, 1986b), which is then digested by angiotensinconverting enzyme (ACE), acting as tripeptidase, to separate the tripeptide, Ser-Pro-Phe, from the pentapeptide Arg-Pro-Pro-Gly-Phe (Sheikh and Kaplan, 1986a). Enzymes that have not been characterized rapidly digest Ser-Pro-Phe to individual amino acids and more slowly convert the pentapeptide to Arg-Pro-Pro plus Gly and Phe. The final products of bradykinin degradation are the peptide Arg-Pro-Pro, plus 1 mol each of Gly, Ser, Pro, and Arg, and 2 mol of Phe (Sheikh and Kaplan, 1989a,b). The initial change of bradykinin to des-Arg9 bradykinin formed by this initial cleavage retains some but not all the various activities of bradykinin (Marceau and Bachvarov, 1998). It can, for example, interact with B1 receptors (Regoli and Barabe, 1980) induced by inflammation [e.g., interleukin-1 (IL-1) and tumor necrosis factor a (TNFa)] in the vasculature and cause hypotension, but the activities of bradykinin on the skin and the contraction of other smooth muscles are abolished. Bradykinin interacts with constitutively expressed B2 receptors to mediate all its functions. Selective B2 and B1 receptor antagonists have been synthesized (Beierwaltes et al., 1987; Stewart et al., 1999; Vavrek and Stewart, 1985). When blood is clotted and serum is studied, all of the reactions for bradykinin degradation occur as described, but the rate of initial Arg removal is accelerated 5-fold compared with plasma (Sheikh and Kaplan, 1989a). This is probably due to the action of a plasma carboxypeptidase that is distinct from carboxypeptidase N and is expressed (activated) as a result of blood coagulation. One such carboxypeptidase is the thrombin-activatable fibrinolysis inhibitor (TAFI) (Bajzar et al., 1995, 1996). It should also be noted that bradykinin

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degradation in vivo occurs largely along the pulmonary vasculature and that endothelial cells there have carboxypeptidase as well as angiotensin-converting enzyme activities. In the pulmonary circulation, the initial cleavage may occur by angiotensin-converting enzyme acting as a dipeptidase to remove first PheArg and then Ser-Pro (each of which is next cleaved to free amino acids), leaving the pentapeptide Arg-Pro-Pro-Gly-Phe. This is then metabolized further. The cough, wheeze, and angioedema sometimes associated with use of ACE inhibitors for treatment of hypertension or heart failure is likely due to inhibition of kinin metabolism leading to increased levels of bradykinin (Nussberger et al., 1998). Because bradykinin is a peripheral vasodilator, it has been considered to be a counterbalance to the vasopressor effects of angiotensin II. It is clear that the two peptides are also related in terms of metabolism, because ACE cleaves His-Leu from the C terminus of angiotensin I, a decapeptide, to leave the octapeptide angiotensin II. Thus, ACE creates a vasoconstrictor and inactivates a vasodilator. 7. Relations of the Contact Factors to Other Systems 7.1. Intrinsic Fibrinolytic Cascade A factor XII–dependent pathway leading to the conversion of plasminogen to plasmin was described in the 1960s and early 1970s (Iatridis and Ferguson, 1962; McDonagh and Ferguson, 1970; Ogston et al., 1969), and a defect in this pathway has been observed in plasma deficient in factor XII, prekallikrein, or HK (Colman et al., 1975; Donaldson et al., 1976; Saito et al., 1974; Weiss et al., 1974; Wuepper, 1973; Wuepper et al., 1975). The factor XII–dependent fibrinolytic activity is relatively weak and difficult to demonstrate in whole plasma, because large quantities of a potent plasminogen activator are not formed. Relatively little plasmin is generated, and this is rapidly inactivated by plasma inhibitors (a2-antiplasmin and a2-macroglobulin). Most studies have therefore used diluted, acidified plasma (Ogston et al., 1971) or chloroformtreated plasma (Ogston et al., 1969) in which inhibition of contact activation and plasmin is minimized, or have studied a euglobulin preparation that concentrates the plasma enzymes and cofactors but limits inhibition (Kluft, 1976), or have added organic compounds that destroy a2-antiplasmin and C1 INH (Kluft, 1977; Miles et al., 1981, 1983b). Such measures are not needed to study blood coagulation or the liberation of bradykinin. Plasminogen can be activated by kallikrein (Colman, 1969) and factor XIa (Mandle and Kaplan, 1979; Thompson et al., 1977). When purified preparations are compared, kallikrein and factor XIa are equipotent as direct plasminogen activators (Mandle and Kaplan, 1979). The plasma concentration

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of prekallikrein is approximately 10-fold higher than that of factor XI, however, and, in addition, factor XIIf can readily convert prekallikrein to kallikrein in the fluid phase (Kaplan and Austen, 1970; Tankersley et al., 1980), whereas it has minimal activity on factor XI (Kaplan and Austen, 1971). Furthermore, kallikrein can dissociate from surfaces and act in the fluid phase, whereas factor XIa cannot. For these reasons, kallikrein is more important in this pathway; nevertheless, it is possible to demonstrate a fibrinolytic abnormality in factor XI–deficient plasma (Saito, 1980). Activated factor XII (XIIa or XIIf) can also convert plasminogen to plasmin (Goldsmith et al., 1978), but its activity is only 5% that of kallikrein. These are all weak reactions in that the potencies of kallikrein and factor XIa as plasminogen activators are thousands of times lower than that of urokinase (Jorg and Binder, 1985; Mandle and Kaplan, 1979; Miles et al., 1983a). Thus, it can be argued that plasminogen is not a significant substrate for any of them. Although each of these proteins is capable of converting plasminogen to plasmin, the other blood-clotting enzymes—factors IXa, Xa, and VIIa, and thrombin—have no such activity, which argues against this activity being a contingent epiphenomenon. Later studies of contact-activated fibrinolysis demonstrated that kallikrein activates the trace quantity of prourokinase in plasma (Hauert et al., 1989; Ichinose et al., 1986; Miles et al., 1981) and that urokinase is the main plasminogen activator of plasma (Huisveld, 1985). Inhibition by antiurokinase antisera supports this notion (Miles et al., 1981) as do zymographic gel studies using plasma euglobulin preparations (Hauert et al., 1989). Other workers have suggested a role for plasma urokinase in factor XII–independent fibrinolysis (Kluft et al., 1984). Although urokinase is clearly a much more potent plasminogen activator than any of the enzymes associated with contact activation, the quantities of urokinase generated are small. If the effects of a2-antiplasmin and C1 INH are abrogated by addition of flufenamic acid derivatives, contact activation results in the formation of plasmin at approximately 35 ng/ml (Miles et al., 1983b), which represents activation of 0.05% of plasma plasminogen. These observations are shown diagrammatically in Fig. 11. Of particular interest are studies in which kinetically favorable prourokinase activation occurred at the surface of platelets or endothelial cells on addition of activated factor XII, HK, and prekallikrein (Lenich et al., 1995; Loza et al., 1994). Although the role in vivo of this cascade as a pathway for fibrinolysis is not yet clear, patients with abnormalities of contact activation proteins such as factor XII have died of thrombosis. It is now felt that a role for contact activation in fibrinolysis is more important physiologically than any role it has in blood coagulation (hemostasis), since factor-deficient patients do not bleed (Kaplan et al., 2002). Furthermore,

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Figure 11 Pathways by which plasminogen is converted to the fibrinolytic enzyme plasmin. The major activation pathway is dependent on kallikrein conversion of prourokinase to urokinase. However, kallikrein, factor XIa, and factor XIIa are all capable of directly converting plasminogen to plasmin.

factor XI is considered to be a contributor to the extrinsic (tissue factor) coagulation pathway via the thrombin feedback. An additional interaction between the kallikrein–kinin system and in vivo fibrinolysis is suggested by the observation that bradykinin is a potent stimulator of the release of tissue plasminogen activator (TPA) from endothelial cells (Smith et al., 1985). 7.2. Interaction with Other Plasma Proteases Factor XIIf has been reported to activate factor VII (Radcliffe et al., 1977; Seligsohn et al., 1979) and thereby initiate the extrinsic coagulation pathway. This reaction contributes significantly to the kaolin-activated partial thromboplastin time (PTT) as usually seen when plasma is exposed to the cold (Gjonnaess, 1972; Laake et al., 1974). This is accentuated in women who use oral contraceptives containing estrogen, apparently owing to an increased concentration of factor XII (Gordon et al., 1980, 1983; Jespersen and Kluft, 1985; Laake et al., 1974). It is theorized that this pathway might contribute to the increased incidence of thrombosis reported as a complication of oral contraceptive use. Factor XIIf (but not factor XIIa) can enzymatically activate the first component of complement when it is incubated with purified C1 or added to plasma

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(Ghebrehiwet et al., 1981). C1 activation is due to cleavage of the C1r subcomponent (Ghebrehiwet et al., 1983) by factor XIIf. Little complement activation is seen when kaolin is incubated with whole plasma, and significant complement activation may be seen only under conditions that result in substantial conversion of factor XIIa to factor XIIf. Once such circumstance is C1 INH deficiency (i.e., HAE), in which factor XII activation may contribute to complement consumption (Donaldson, 1968; Fields et al., 1983). Kallikrein can also cleave C1 subcomponents, but the net result is destruction rather than activation. On the other hand, kallikrein can activate factor B of the alternative complement pathway and thereby substitute for factor D (DiScipio, 1982). 7.3. Interaction with Leukocytes Kallikrein has been reported to interact with human leukocytes in a variety of ways. It is a chemotactic factor for neutrophils (Kaplan et al., 1972) and monocytes (Gallin and Kaplan, 1974), and it has been shown to cause neutrophil aggregation (Schapira et al., 1982a) and release of elastase (Klemperer et al., 1968). In a rabbit model, kallikrein stimulation of chemotaxis appeared to require cleavage of C5 and release of C5a chemotactic factor (Donaldson et al., 1977). Therefore C5 bound to the surface of neutrophils can possibly be cleaved in the aforementioned reactions. Anti-kallikrein serum was inhibitory, whereas anti-C5 serum had no effect; the authors therefore concluded that the effect of kallikrein on human neutrophils does not require complement. Furthermore, a degraded form of kallikrein (b-kallikrein), in which the heavy chain is partially digested, is enzymatically active on kininogen to form kinin but possesses a markedly attenuated reactivity with neutrophils (Colman et al., 1985). Factor XIIa has also been shown to stimulate neutrophils; because factor XIIf did not do this, a requirement for a binding site in the heavy chain was inferred (Wachtfogel et al., 1986). No studies have demonstrated a cell surface receptor for these enzymes or required cleavage of surface components. In each instance, the active site of the enzyme is required, and the proenzyme or DFP-treated enzyme is inactive (Kaplan et al., 1972; Schapira et al., 1982a). 8. Considerations in Human Diseases 8.1. C1 Inhibitor Deficiency Although C1 INH was defined as an inhibitor of the activated first component of complement, it is clearly a key control protein of the plasma kinin-forming cascade. The pathogenesis of the swelling in C1 INH deficiency is dependent

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on the plasma kinin-forming pathway rather than complement; however, it is germane to review the history of the complement data and to point out how it was first thought to be the key, and then to present the more recent data that suggest otherwise. Intracutaneous injection of C1 into normal individuals was reported to cause the formation of a small wheal reaction, whereas injection into patients with hereditary angioedema yields localized angioedema, that is, an augmented response because of low C1 inhibitor (Klemperer et al., 1968). A kinin-like peptide was isolated from such patients and its formation appeared to be inhibited in C2-deficient plasma. Thus C2 was considered to be the source of the pathogenic peptide (Donaldson, 1968). However, direct demonstration of such a kinin-like peptide on interaction of activated C1 and C4 and C2 or with C2 alone is lacking. Although it was originally reported that cleavage of C2b by plasmin generates a kinin (Donaldson et al., 1977), attempts to confirm this experiment have all failed (Colman et al., 1985; Fields et al., 1983). The only identifiable kinin seen in subsequent studies was bradykinin (Fields et al., 1983). On the other hand, the amino acid sequence of C2b is known, and Strang et al. (1988) synthesized peptides of various lengths and tested each for kinin-like activity. One such peptide was shown to cause edema when injected intracutaneously, reminiscent of the C2 kinin originally described. However, this peptide has not been shown to be a cleavage product of C2b, nor has it been shown to be present during attacks of swelling in patients with hereditary angioedema. Thus, at this point it seems unlikely that a kinin-like molecule is derived from C2b as a result of enzymatic cleavage. On the other hand, the presence of bradykinin has been documented as described below, and it is the likely cause of the swelling. In fact, when one of the proponents of the C2 kinin reexamined kinin formation in the plasma of patients with hereditary angioedema, only bradykinin was found (Shoemaker et al., 1994). It should be noted that 24-hr urine histamine excretion may also be increased during attacks of angioedema, suggesting that C3a, C4a, or C5a is being generated. Although the plasma levels of C3 and C5 are normal in this disorder, C3 turnover is clearly enhanced (Carpenter et al., 1969). The lesions, however, are not pruritic, and antihistaminics have no effect on the clinical course of the disease. Thus, complement activation is undoubtedly occurring, perhaps even during quiescent periods, to lead to a low level of C4, but the vasoactive consequences of augmented complement activation that occurs during attacks of HAE do not appear to be the cause of the swelling. C1 inhibitor inhibits all functions of factor XIIa (Gigli et al., 1970; Schreiber et al., 1973) and is one of the two major plasma kallikrein inhibitors, the other being a2-macroblobulin (Harpel et al., 1985), and all functions of kallikrein are

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thereby inhibited including the feedback activation of factor XII, the cleavage of HK, and the activation of plasma prourokinase (Ichinose et al., 1986) to lead to plasmin formation (Fig. 1). C1 inhibitor also inhibits the fibrinolytic enzyme plasmin, although it is a relatively minor inhibitor compared with a2-antiplasmin or a2-macroglobulin. Patients with hereditary angioedema appear to be hyperresponsive to cutaneous injections of kallikrein, as they are to C1 (Juhlin and Michaelsson, 1969), and elevated levels of bradykinin and cleaved kininogen have been observed during attacks of swelling (Nussberger et al., 1998; Talamo et al., 1969). There is also evidence that C1 activation observed in hereditary angioedema may also be factor XII dependent (Donaldson, 1968). Thus, a factor XII–dependent enzyme may be initiating the classic complement cascade. Plasmin is capable of activating C1s and may represent one such enzyme (Ratnoff and Naff, 1967). Ghebrehiwet et al. demonstrated that Hageman factor fragment (factor XIIf) can directly activate the classic complement cascade by activating C1r and to a lesser degree C1s (Ghebrehiwet et al., 1981, 1983). This may represent a critical link between the intrinsic coagulation–kinin cascade and complement activation (Fig. 1). The presence of kallikrein-like activity in induced blisters of patients with hereditary angioedema supports this notion (Curd et al., 1980), as does the progressive generation of bradykinin on incubation of hereditary angioedema plasma in plastic non-contact-activated test tubes (Fields et al., 1983), as well as the low prekallikrein HK levels seen during attacks (Schapira et al., 1983). More recent data support these indirect observations, favoring bradykinin as the critical pathogenic peptide for hereditary angioedema and—likely—acquired C1 INH deficiency as well. One unique family has been described in which there is a point mutation in C1 INH (Ala443 ! Val) leading to inability to inhibit the complement cascade but normal inhibition of factor XIIa and kallikrein (Zahedi et al., 1995, 1997). No family member of this type II mutation has had angioedema. Plasma bradykinin levels have been shown to be elevated during attacks of swelling of hereditary and acquired forms of C1 INH deficiency (Cugno et al., 1996; Nussberger et al., 1998), and local bradykinin generation has been documented at the site of the swelling (Nussberger et al., 1999). The role of fibrinolysis also needs to be considered a part of the pathogenesis of the disease, since antifibrinolytic agents such as e-aminocaproic acid and tranexamic acid appear to be efficacious (Frank et al., 1972; Lundh et al., 1968; Sheffer et al., 1972), and plasmin is generated during active disease (Cugno et al., 1993). Although kallikrein factor XIa, and even factor XIIa, have some ability to activate plasminogen directly, the plasma pathway via the prourokinase intermediate appears to be the major factor XII–dependent fibrinolytic mechanism (Fig. 11). Among the functions of plasmin are the activation of

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C1s, the ability to cleave and activate factor XII just as kallikrein can (Kaplan and Austen, 1971), and digestion of C1 inhibitor (Wallace et al., 1997). Each of these would serve to augment bradykinin formation and further deplete the levels of C1 inhibitor. Thus, the formation of plasmin may in this fashion contribute to the pathogenesis of the disease. 8.2. Contact Activation in Allergic Diseases By analogy with observations based on dextran sulfate, naturally occurring glycosaminoglycans or proteoglycans may be able to induce contact activation. We have tested heparin proteoglycan from the Furth murine mastocytoma for its ability to activate a mixture of factor XII and prekallikrein (Brunnee et al., 1993, 1997). There is progressive conversion of prekallikrein to kallikrein as the concentration of mast cell heparin is increased. The potency of heparin proteoglycan equals that of dextran sulfate, and its activity is inhibited by heparinase I or II, but not by heparitinase or chondroitinase ABC. Of the glycosaminoglycans we have tested, heparin, dermatan sulfate, keratin polysulfate, and chondroitin sulfate C are positive in the assay (in that order), whereas heparin sulfate and chondroitin sulfate A are negative. Collagen types I, III, IV, and V; laminin; fibronectin; and vitronection are also negative. Activation can then occur by release of heparin and/or other mucopolysaccharides secreted by mast cells and basophils on exposure to plasma proteins or via interaction of these proteins with exposed connective tissue proteoglycans during tissue injury. The proteins of the kinin-forming system have been shown to be present in interstitial fluid of rabbit skin; thus, the source may not solely be dependent on exudation and activation of plasma. Any aspect of inflammation that leads to dilution of plasma constituents or exclusion of inhibitors will augment contact activation, because inhibitory functions are dependent on concentration. Thus, the activatability of plasma can be shown to be related directly to dilution. Once levels of C1 INH are less than 25% of normal (i.e., equivalent to a 1:4 dilution), patients with HAE are prone to attacks of swelling. Activation of the plasma and tissue kinin-forming systems has been observed in allergic reactions in the nose, lungs, and skin, and include the immediate reaction as well as the late-phase reaction, although the contributions of the plasma and tissue kallikrein pathways to each aspect of allergic inflammation are likely quite different. Antigen challenge of the nose followed by nasal lavage revealed an increase in tosyl-l-arginine-O-methyl ester (TAME) esterase activity, which is largely attributable to kallikrein(s) (Proud et al., 1983). The activation was seen during the immediate response as well as during the late-phase reaction (Creticos et al., 1984). Both LK and HK

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were shown to be present in nasal lavage fluid (Baumgarten et al., 1985), and fractionation of nasal washings demonstrated evidence of both tissue kallikrein (Baumgarten et al., 1986b) and plasma kallikrein (Baumgarten et al., 1986a). Tissue kallikrein can be secreted by glandular tissue as well as by infiltrating cells, such as neutrophils, and will cleave LK to yield kallidin. Plasma kallikrein will digest HK to yield bradykinin directly. HPLC analysis of kinins in nasal washings revealed both kallidin (lysylbradykinin) and bradykinin. The latter can be formed from kallidin by aminopeptidase action; however, a portion of the bradykinin is also likely the direct result of plasma kallikrein activity. Studies of the allergen-induced late-phase reactions in the skin (Atkins et al., 1987, 1992) have demonstrated the presence of kallikrein–C1 INH and activated factor XII–C1 INH complexes in induced blisters observed during an 8-hr period. Elevated levels of these complexes were seen between 3 and 6 hr, coincident with the late-phase response and were specific for the antigen to which the patient was sensitive. 8.3. Regulation of Blood Pressure The possible relationship of the contact system to blood pressure regulation is an intriguing question. As already seen, ACE creates the hypertensive peptide, angiotensin II, and plays a major role in inactivating the hypotensive product of contact activation, bradykinin. An unfortunate circumstance has dramatized the kinin-forming capacity of factor XIIf; trauma patients given plasma protein fractions as plasma expanders that were contaminated with factor XIIf showed profound hypotension (Alving et al., 1978, 1980). The mechanism by which infused factor XIIf causes hypotension has been demonstrated to be due to bradykinin formation. A more tenuous connection exists between blood pressure regulation and the contact system in the factor XII–dependent activation of plasma prorenin. Prorenin is activated to renin by cold treatment of plasma or acidification to pH 3.3. With acid treatment, most of the renin activity is produced after reneutralization; this alkaline phase activation is mediated by kallikrein (Derkx et al., 1979; Sealey et al., 1979), as is the cold-induced activation (Brown and Osmond, 1984). Kallikrein is able to activate purified prorenin (Yokosawa et al., 1979), but when added to plasma, it does not cause prorenin activation in the absence of an acidification step. Although it has been supposed that the acid treatment serves to destroy kallikrein inhibitors, prorenin is not activated in plasma deficient in C1 INH or a2-macroglobulin (Purdon et al., 1985). Thus, some other unknown event must occur on acidification. The physiologic significance of this reaction is uncertain.

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8.4. Kinin in Vascular Disease and Blood Pressure Control The bradykinin-forming cascade may have a role in cardiovascular diseases including hypertension and diabetes. Numerous interactions exist between the angiotensin cascade, vascular endothelium, and the plasma kinin-forming cascade, as indicated in Fig. 12. Prorenin can be converted to renin by plasma kallikrein in an acidic milieu and is factor XII dependent (Alving et al., 1978, 1980). Once there is conversion of angiotensin I to angiotensin II, the interaction of angiotensin II with the AT1 receptor on endothelial cells activates NADPH oxidase and P42/44 MAP kinase (Lu et al., 1998), leading to upregulation of the B2 receptor. This may also lead to augmented liberation of Hsp 90 (Joseph et al., 2002b) and prolylcarboxypeptidase (Shariat-Madar et al., 2002), each of which interacts with the prekallikrein–HK complex to generate bradykinin. The interaction of bradykinin with the endothelial cell stimulates the formation of NO (Zhao et al., 2001) and prostacyclin (Crutchley et al., 1983; Hong, 1980). Angiotensin-converting enzyme, expressed on endothelial cells (Caldwell et al., 1976) and present in plasma as kininase II (Yang and Erdos, 1967), is responsible for conversion of angiotensin I to angiotensin II and also

Figure 12 Diagrammatic representation of the many interconnections between the renin– angiotensin pathway and the bradykinin-forming pathway.

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degrades bradykinin by first cleaving Phe-Arg from the C terminus, followed by Ser-Pro. Thus angiotensin I, a vasoconstrictor, balances the effects of bradykinin, NO, and prostacyclin as vasodilators. Of particular interest regarding the pathogenesis of diabetic vasculopathy is the ability of glucose to upregulate the B2 receptor (Tan et al., 2004) and to raise the levels of plasma prekallikrein (Jaffa et al., 2003). The latter effect appears specific, because no significant change in factor XII or kininogen levels is noted. 8.5. Other Disorders Endotoxic shock is associated with depletion of contact activation proteins (Hirsch et al., 1974; Mason et al., 1970; O’Donnell et al., 1976; Robinson et al., 1975), and serial HK levels have prognostic value because a drop to near zero usually indicates a fatal outcome, as do lower prekallikrein levels (O’Donnell et al., 1976). A monoclonal antibody to factor XII markedly diminished the mortality by 50% in a baboon model of endotoxic shock (Pixley et al., 1992, 1993), largely due to effects on hypotension and its sequelae. Parameters of disseminated intravascular coagulation (DIC) were unaffected and likely mediated via tissue thromboplastin, although DIC due to endothelial cell injury and/or endotoxemia is associated with diminished levels of factor XII, prekallikrein, and kallikrein-inhibiting activity. The synovial fluid of patients with rheumatoid arthritis has been shown to contain plasma kallikrein, which can activate stromelysin and convert procollagenase to collagenase (Nagase et al., 1982). Uric acid and pyrophosphate crystals can act as surfaces for contact activation (Ginsberg et al., 1980; Kellermeyer and Breckenridge, 1965) and may contribute to the inflammation seen in gout or pseudogout. However, at least one case of gout (Londino and Luparello, 1984) and one of rheumatoid arthritis (Donaldson et al., 1972) have been reported in factor XII–deficient subjects. Pancreatitis, particularly acute hemorrhagic pancreatitis, is associated with release of large quantities of tissue kallikrein; thus, kallidin and/or bradykinin may contribute to the pooling of fluid within the abdominal cavity and hypotension that can result. The causes of Alzhemer’s disease are not known, although it is associated with deposition of b-amyloid protein in the form of plaques as well as  fibril proteins within neurofibrillary tangles and paired helical filaments. A rare hereditary form of Alzheimer’s disease has been associated with a mutation of the amyloid precursor protein. We have demonstrated that when b-amyloid monomer aggregates as it does within plaques, it is a potent initiator of the plasma kinin-forming cascade. It does so by binding factor XII and HK (Shibayama et al., 1999) and in so doing activates the cascade in a

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zinc-dependent reaction. Furthermore, the aggregation of b-amyloid has been shown to be zinc dependent (Bush et al., 1994). In this fashion, b-amyloid resembles the binding we see to cell membranes, and to gC1qR specifically, because the latter protein also activates the cascade. However, activation by aggregated b-amyloid is more rapid than that seen with gC1qR and may have features that are reminiscent of both negatively charged surfaces (which are ion independent) and cell surface initiators. Whether any of the functional disturbances of neurons or glial cells seen in Alzheimer’s disease are attributable to the generation of bradykinin remains to be established. References Alving, B. M., Hojima, Y., Pisano, J. J., Mason, B. L., Buckingham, R. E., Jr., Mozen, M. M., and Finlayson, J. S. (1978). Hypotension associated with prekallikrein activator (Hageman-factor fragments) in plasma protein fraction. N. Engl. J. Med. 299, 66–70. Alving, B. M., Tankersley, D. L., Mason, B. L., Rossi, F., Aronson, D. L., and Finlayson, J. S. (1980). Contact-activated factors: Contaminants of immunoglobulins preparations with coagulant and vasoactive properties. J. Lab. Clin. Med. 96, 334–346. Atkins, P. C., Miragliotta, G., Talbot, S. F., Zweiman, B., and Kaplan, A. P. (1987). Activation of plasma Hageman factor and kallikrein in ongoing allergic reactions in the skin. J. Immunol. 139, 2744–2748. Atkins, P. C., Kaplan, A. P., von Allmen, C., Moskovitz, A., and Zweiman, B. (1992). Activation of the coagulation pathway during ongoing allergic cutaneous reactions in humans. J. Allergy Clin. Immunol. 89, 552–559. Baglia, F. A., and Walsh, P. N. (2000). Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation by factor XIIa or factor XIa. J. Biol. Chem. 275, 20514–20519. Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002). Factor XI binding to the platelet glycoprotein Ib–IX–V complex promotes factor XI activation by thrombin. J. Biol. Chem. 277, 1662–1668. Baird, T. R., and Walsh, P. N. (2002). Activated platelets but not endothelial cells participate in the initiation of the consolidation phase of blood coagulation. J. Biol. Chem. 277, 28498–28503. Baird, T. R., and Walsh, P. N. (2003). Factor XI, but not prekallikrein, blocks high molecular weight kininogen binding to human umbilical vein endothelial cells. J. Biol. Chem. 278, 20618–20623. Bajzar, L., Manuel, R., and Nesheim, M. E. (1995). Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J. Biol. Chem. 270, 14477–14484. Bajzar, L., Morser, J., and Nesheim, M. (1996). TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin–thrombomodulin complex. J. Biol. Chem. 271, 16603–16608. Barrett, A. J., and Starkey, P. M. (1973). The interaction of a2-macroglobulin with proteinases: Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem. J. 133, 709–724. Baumgarten, C. R., Togias, A. G., Naclerio, R. M., Lichtenstein, L. M., Norman, P. S., and Proud, D. (1985). Influx of kininogens into nasal secretions after antigen challenge of allergic individuals. J. Clin. Invest. 76, 191–197.

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Baumgarten, C. R., Nichols, R. C., Naclerio, R. M., Lichtenstein, L. M., Norman, P. S., and Proud, D. (1986a). Plasma kallikrein during experimentally induced allergic rhinitis: Role in kinin formation and contribution to TAME-esterase activity in nasal secretions. J. Immunol. 137, 977–982. Baumgarten, C. R., Nichols, R. C., Naclerio, R. M., and Proud, D. (1986b). Concentrations of glandular kallikrein in human nasal secretions increase during experimentally induced allergic rhinitis. J. Immunol. 137, 1323–1328. Beeler, D. L., Marcum, J. A., Schiffman, S., and Rosenberg, R. D. (1986). Interaction of factor XIa and antithrombin in the presence and absence of heparin. Blood 67, 1488–1492. Beierwaltes, W. H., Carretero, O. A., Scicli, A. G., Vavrek, R. J., and Stewart, J. M. (1987). Competitive analog antagonists of bradykinin in the canine hindlimb. Proc. Soc. Exp. Biol. Med. 186, 79–83. Bouma, B. N., and Meijers, J. C. (2000). Role of blood coagulation factor XI in downregulation of fibrinolysis. Curr. Opin. Hematol. 7, 266–272. Bradford, H. N., Dela Cadena, R. A., Kunapuli, S. P., Dong, J. F., Lopez, J. A., and Colman, R. W. (1997). Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib–IX–V complex. Blood 90, 1508–1515. Brown, C. F., and Osmond, D. H. (1984). Factor XII and prekallikrein–kallikrein–kinin in the cryoactivation of human plasma prorenin. Clin. Sci. 66, 533–539. Brunnee, T., La Porta, C., Reddigari, S. R., Salerno, V. M., Kaplan, A. P., and Silverberg, M. (1993). Activation of factor XI in plasma is dependent on factor XII [see comment]. Blood 81, 580–586. Brunnee, T., Reddigari, S. R., Shibayama, Y., Kaplan, A. P., and Silverberg, M. (1997). Mast cell derived heparin activates the contact system: A link to kinin generation in allergic reactions. Clin. Exp. Allergy 27, 653–663. Bush, A. I., Pettingell, W. H., Multhaup, G., d Paradis, M., Vonsattel, J. P., Gusella, J. F., Beyreuther, K., Masters, C. L., and Tanzi, R. E. (1994). Rapid induction of Alzheimer A b amyloid formation by zinc [see comment]. Science 265, 1464–1467. Caldwell, P. R., Seegal, B. C., Hsu, K. C., Das, M., and Soffer, R. L. (1976). Angiotensin-converting enzyme: Vascular endothelial localization. Science 191, 1050–1051. Cameron, C. L., Fisslthaler, B., Sherman, A., Reddigari, S., and Silverberg, M. (1989). Studies on contact activation: Effects of surface and inhibitors. Med. Prog. Technol. 15, 53–62. Carpenter, C. B., Ruddy, S., Shehadeh, I. H., Muller-Eberhard, H. J., Merrill, J. P., and Austen, K. F. (1969). Complement metabolism in man: Hypercatabolism of the fourth (C4) and third (C3) components in patients with renal allograft rejection and hereditary, angioedema (HAE). J. Clin. Invest. 48, 1495–1505. Cochrane, C. G., and Revak, S. D. (1980). Dissemination of contact activation in plasma by plasma kallikrein. J. Exp. Med. 152, 608–619. Cochrane, C. G., Revak, S. D., and Wuepper, K. D. (1973). Activation of Hageman factor in solid and fluid phases: A critical role of kallikrein. J. Exp. Med. 138, 1564–1583. Colman, R. W. (1969). Activation of plasminogen by human plasma kallikrein. Biochem. Biophys. Res. Commun. 35, 273–279. Colman, R. W., and Schmaier, A. H. (1997). Contact system: A vascular biology modulator with anticoagulant, profibrinolytic, antiadhesive, and proinflammatory attributes. Blood 90, 3819–3843. Colman, R. W., Bagdasarian, A., Talamo, R. C., Scott, C. F., Seavey, M., Guimaraes, J. A., Pierce, J. V., and Kaplan, A. P. (1975). Williams trait: Human kininogen deficiency with diminished levels of plasminogen proactivator and prekallikrein associated with abnormalities of the Hageman factor-dependent pathways. J. Clin. Invest. 56, 1650–1662.

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Colman, R. W., Wachtfogel, Y. T., Kucich, U., Weinbaum, G., Hahn, S., Pixley, R. A., Scott, C. F., de Agostini, A., Burger, D., and Schapira, M. (1985). Effect of cleavage of the heavy chain of human plasma kallikrein on its functional properties. Blood 65, 311–318. Colman, R. W., Pixley, R. A., Najamunnisa, S., Yan, W., Wang, J., Mazar, A., and McCrae, K. R. (1997). Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor. J. Clin. Invest. 100, 1481–1487. Creticos, P. S., Peters, S. P., Adkinson, N. F., Jr., Naclerio, R. M., Hayes, E. C., Norman, P. S., and Lichtenstein, L. M. (1984). Peptide leukotriene release after antigen challenge in patients sensitive to ragweed. N. Engl. J. Med. 310, 1626–1630. Crutchley, D. J., Ryan, J. W., Ryan, U. S., and Fisher, G. H. (1983). Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells: Studies with lower homologs and calcium antagonists. Biochim. Biophys. Acta 751, 99–107. Cugno, M., Hack, C. E., de Boer, J. P., Eerenberg, A. J., Agostoni, A., and Cicardi, M. (1993). Generation of plasmin during acute attacks of hereditary angioedema. J. Lab. Clin. Med. 121, 38–43. Cugno, M., Cicardi, M., Coppola, R., and Agostoni, A. (1996). Activation of factor XII and cleavage of high molecular weight kininogen during acute attacks in hereditary and acquired C1-inhibitor deficiencies. Immunopharmacology 33, 361–364. Curd, J. G., Prograis, L. J., Jr., and Cochrane, C. G. (1980). Detection of active kallikrein in induced blister fluids of hereditary angioedema patients. J. Exp. Med. 152, 742–747. de Agostini, A., Lijnen, H. R., Pixley, R. A., Colman, R. W., and Schapira, M. (1984). Inactivation of factor XII active fragment in normal plasma. Predominant role of C-1-inhibitor. J. Clin. Invest. 73, 1542–1549. Dedio, J., and Muller-Esterl, W. (1996). Kininogen binding protein p33/gC1qR is localized in the vesicular fraction of endothelial cells. FEBS Lett. 399, 255–258. Dedio, J., Jahnen-Dechent, W., Bachmann, M., and Muller-Esterl, W. (1998). The multiligandbinding protein gC1qR, putative C1q receptor, is a mitochondrial protein. J. Immunol. 160, 3534–3542. Derkx, F. H., Bouma, B. N., Schalekamp, M. P., and Schalekamp, M. A. (1979). An intrinsic factor XII–prekallikrein-dependent pathway activates the human plasma renin–angiotensin system. Nature 280, 315–316. DiScipio, R. G. (1982). The activation of the alternative pathway C3 convertase by human plasma kallikrein. Immunology 45, 587–595. Donaldson, V. H. (1968). Mechanisms of activation of C0 1 esterase in hereditary angioneurotic edema plasma in vitro. J. Exp. Med. 127, 411–429. Donaldson, V. H., Glueck, H. I., and Fleming, T. (1972). Brief recordings: Rheumatoid arthritis in a patient with Hageman trait. N. Engl. J. Med. 286, 528–530. Donaldson, V. H., Glueck, H. I., Miller, M. A., Movat, H. Z., and Habal, F. (1976). Kininogen deficiency in Fitzgerald trait: Role of high molecular weight kininogen in clotting and fibrinolysis. J. Lab. Clin. Med. 87, 327–337. Donaldson, V. H., Rosen, F. S., and Bing, D. H. (1977). Role of the second component of complement (C2) and plasmin in kinin release in hereditary angioneurotic edema (H.A.N.E.) plasma. Trans. Assoc. Am. Physicians 90, 174–183. Dunn, J. T., and Kaplan, A. P. (1982). Formation and structure of human Hageman factor fragments. J. Clin. Invest. 70, 627–631. Dunn, J. T., Silverberg, M., and Kaplan, A. P. (1982). The cleavage and formation of activated human Hageman factor by autodigestion and by kallikrein. J. Biol. Chem. 257, 1779–1784. Erdos, E. G., and Sloane, G. M. (1962). An enzyme in human plasma that inactivates bradykinin and kallidins. Biochem. Pharmacol. 11, 585.

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Pixley, R. A., De La Cadena, R., Page, J. D., Kaufman, N., Wyshock, E. G., Chang, A., Taylor, F. B., Jr., and Colman, R. W. (1993). The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia: In vivo use of a monoclonal antifactor XII antibody to block contact activation in baboons. J. Clin. Invest. 91, 61–68. Proud, D., Togias, A., Naclerio, R. M., Crush, S. A., Norman, P. S., and Lichtenstein, L. M. (1983). Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen. J. Clin. Invest. 72, 1678–1685. Purdon, A. D., Schapira, M., De Agostini, A., and Colman, R. W. (1985). Plasma kallikrein and prorenin in patients with hereditary angioedema. J. Lab. Clin. Med. 105, 694–699. Radcliffe, R., Bagdasarian, A., Colman, R., and Nemerson, Y. (1977). Activation of bovine factor VII by hageman factor fragments. Blood 50, 611–617. Rand, M. D., Lock, J. B., van’t Veer, C., Gaffney, D. P., and Mann, K. G. (1996). Blood clotting in minimally altered whole blood. Blood 88, 3432–3445. Ratnoff, O. D., and Naff, G. B. (1967). The conversion of C0 IS to C0 1 esterase by plasmin and trypsin. J. Exp. Med. 125, 337–358. Reddigari, S., and Kaplan, A. P. (1988). Cleavage of human high-molecular weight kininogen by purified kallikreins and upon contact activation of plasma. Blood 71, 1334–1340. Reddigari, S., and Kaplan, A. P. (1989). Studies of the cleavage of human high molecular weight kininogen by purified plasma and tissue kallikreins, and upon contact activation of plasma. Adv. Exp. Med. Biol. 247B, 317–324. Reddigari, S. R., Kuna, P., Miragliotta, G., Shibayama, Y., Nishikawa, K., and Kaplan, A. P. (1993a). Human high molecular weight kininogen binds to human umbilical vein endothelial cells via its heavy and light chains. Blood 81, 1306–1311. Reddigari, S. R., Shibayama, Y., Brunnee, T., and Kaplan, A. P. (1993b). Human Hageman factor (factor XII) and high molecular weight kininogen compete for the same binding site on human umbilical vein endothelial cells. J. Biol. Chem. 268, 11982–11987. Regoli, D., and Barabe, J. (1980). Pharmacology of bradykinin and related kinins. Pharmacol. Rev. 32, 1–46. Renne, T., Dedio, J., David, G., and Muller-Esterl, W. (2000). High molecular weight kininogen utilizes heparan sulfate proteoglycans for accumulation on endothelial cells. J. Biol. Chem. 275, 33688–33696. Richter, K., Muschler, P., Hainzl, O., and Buchner, J. (2001). Coordinated ATP hydrolysis by the Hsp90 dimer. J. Biol. Chem. 276, 33689–33696. Robinson, J. A., Klondnycky, M. L., Loeb, H. S., Racic, M. R., and Gunnar, R. M. (1975). Endotoxin, prekallikrein, complement and systemic vascular resistance: Sequential measurements in man. Am. J. Med. 59, 61–67. Rojkjaer, R., and Schmaier, A. H. (1999a). Activation of the plasma kallikrein/kinin system on endothelial cell membranes. Immunopharmacology 43, 109–114. Rojkjaer, R., and Schmaier, A. H. (1999b). Activation of the plasma kallikrein/kinin system on endothelial cells. Proc. Assoc. Am. Physicians 111, 220–227. Rojkjaer, R., Hasan, A. A., Motta, G., Schousboe, I., and Schmaier, A. H. (1998). Factor XII does not initiate prekallikrein activation on endothelial cells. Thromb. Haemost. 80, 74–81. Rosing, J., Tans, G., and Griffin, J. H. (1985). Surface-dependent activation of human factor XII (Hageman factor) by kallikrein and its light chain. Eur. J. Biochem. 151, 531–538. Saito, H. (1980). The participation of plasma thromboplastin antecedent (factor XI) in contactactivated fibrinolysis. Proc. Soc. Exp. Biol. Med. 164, 153–157. Saito, H., Ratnoff, O. D., and Donaldson, V. H. (1974). Defective activation of clotting, fibrinolytic, and permeability-enhancing systems in human Fletcher trait plasma. Circ. Res. 34, 641–651.

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Interleukin-2, Interleukin-15, and Their Roles in Human Natural Killer Cells Brian Becknell*,{ and Michael A. Caligiuri*,{,{,x,} *Medical Scientist Program, Integrated Biomedical Graduate Program, { Department of Internal Medicine, x Division of Hematology/Oncology, } Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210 {

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Abstract ......................... ........................................................................ Introduction.................... ........................................................................ IL-2 and IL-15 Signaling .... ........................................................................ Role of IL-2 versus IL-15 in Human NK Cell Development . ................................ Low-Dose IL-2 Therapy Expands the CD56bright NK Subset................................. IL-2/IL-15 and NK Cell Survival................................... ................................ IL-2/IL-15 and NK Cell Proliferation ............................. ................................ IL-2/IL-15 and NK Cell Apoptosis ................................ ................................ IL-2/IL-15 and NK Cell Cytotoxicity .............................. ................................ IL-2/IL-15 and NK Cell Cytokine Production ................... ................................ NK Cell–Immune Cell Interactions and Modulation by IL-2/IL-15 ......................... Conclusions .................... ........................................................................ References ..................... ........................................................................

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Abstract Natural killer (NK) cells are CD56þCD3 large granular lymphocytes that constitute a key component of the human innate immune response. In addition to their potent cytolytic activity, NK cells elaborate a host of immunoregulatory cytokines and chemokines that play a crucial role in pathogen clearance. Furthermore, interactions between NK and other immune cells are implicated in triggering the adaptive, or antigen-specific, immune response. Interleukin2 (IL-2) and IL-15 are two distinct cytokines with partially overlapping properties that are implicated in the development, homeostasis, and function of NK cells. This review examines the pervasive effects of IL-2 and IL-15 on NK cell biology, with an emphasis on recent discoveries and lingering challenges in the field. 1. Introduction The innate immune system represents the human body’s essential first line of defense against infectious disease and malignant transformation. In the immune competent host, innate immune effectors act rapidly to restrict the

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dissemination of disease, as well as to trigger the adaptive, or antigen-specific, immune system. Human natural killer (NK) cells are CD56þCD3 large granular lymphocytes that constitute one component of the innate immune system. In addition to their potent cytolytic activity, NK cells elaborate a host of immunoregulatory cytokines and chemokines that play a crucial role in pathogen clearance. In particular, NK cells produce abundant quantities of interferon-g (IFN-g), a critical cytokine for the clearance of infectious pathogens as well as for tumor surveillance (Lieberman and Hunter, 2002; Shankaran et al., 2001). In rodent models, NK cells have been proved essential for the clearance of certain tumors, as well as bacterial, fungal, viral, and parasitic infections (Carayannopoulos and Yokoyama, 2004; Kim et al., 2000). Furthermore, in rare cases of human congenital immune deficiencies, the absence of NK cells produces a clinical spectrum that parallels classic severe combined immunodeficiency (SCID) syndromes (Gilmour et al., 2001). The importance of NK cells is magnified in a host of clinical scenarios in which the adaptive immune system is compromised. These states include congenital immune disorders, iatrogenic immune suppression following organ transplantation, and the acquired immune deficiency syndrome (AIDS). NK cells represent an attractive target for therapeutic manipulation to fight the rampant opportunistic infections and virus-induced cancers that arise under these states of adaptive immunoparalysis. Indeed, this is the rationale underlying ultralow-dose interleukin-2 therapy to potentiate the antitumor effects of NK cells in AIDS-associated malignancies (Bernstein et al., 1995; Jacobson et al., 1996; Smith, 1988). This approach is further substantiated by the advent of NK cell transplantation, in which alloreactive NK cells have been shown to mediate a potent graft-versus-tumor effect in patients with acute myeloid leukemia (Ruggeri et al., 2002). Based on these advances, it is anticipated that a greater mechanistic understanding of NK cells and the innate immune system will provide new means to enhance the function of these cells for the benefit of the immunocompromised patient. The study of interleukin-2 (IL-2) and IL-15 has served as a paradigm for our current understanding of NK cell function and homeostasis. These two cytokines—more than any others—are implicated in the development, survival, proliferation, apoptosis, and effector functions of NK cells. In addition, IL2 and IL-15 mediate and/or potentiate interactions between NK cells and other immune cells in secondary lymphoid organs and the periphery. This review examines these pervasive effects of IL-2 and IL-15 on NK cell biology. We begin with a discussion of their signal transduction mechanisms. Next, we highlight the roles of IL-2 and IL-15 in NK cell ontogeny, homeostasis, and function. Finally, we conclude by discussing the roles of these two cytokines in mediating interactions between NK cells and other leukocytes in the context of the human immune response.

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2. IL-2 and IL-15 Signaling 2.1. Receptors for IL-2 and IL-15 Interleukin-2 was initially described as a T cell–derived cytokine, or lymphokine, and is chiefly appreciated for its crucial role in T-cell activation, proliferation, and cell death (Waldmann et al., 2001). Interleukin-15 was first identified on the basis of its ability to mimic IL-2–induced T-cell proliferation (Burton et al., 1994; Grabstein et al., 1994). This proliferative effect of IL15 could be neutralized with antibodies specific to the b and g subunits of the previously characterized IL-2 receptor (IL-2R), demonstrating that these cytokines share these receptor subunits. In contrast, neutralizing antibodies to the high-affinity IL-2Ra chain did not inhibit IL-15–induced T-cell proliferation, suggesting that IL-15 possessed its own private high-affinity receptor (Carson et al., 1994a). Indeed, subsequent cloning and characterization of the IL-15Ra chain revealed that it possesses extremely high affinity for IL-15 (Kd, 1011 M)—even in the absence of the IL-2Rbg subunits (Anderson et al., 1995; Giri et al., 1995). In contrast, the IL-2Ra subunit binds IL-2 with low affinity (Kd, 108 M) in the absence of the IL-2Rbg, and a ternary IL-2Rabg complex is requisite for high-affinity binding of IL-2 (Kd, 1011 M; Smith, 1988). In the absence of their private a subunits, both IL-2 and IL-15 are capable of binding an intermediate-affinity IL-2Rbg heterodimer and initiating cytoplasmic signal transduction cascades. 2.2. Signal Transduction Cascades Initiated by IL-2/IL-15 In the absence of IL-2/IL-15, two tyrosine kinases associate with the cytoplasmic tails of the IL-2Rbg heterodimer: Jak1 with b and Jak3 with g. Binding of IL-2/IL-15 to the bg complex results in tyrosine phosphorylation of STAT3 and STAT5 by Jak1 and Jak3, respectively (Miyazaki et al., 1994). These phosphorylated STATs then dimerize and translocate to the nucleus, where they serve as transcription factors (Leonard, 2001). Consistent with the critical role of STAT5 in IL-2/IL-15 signal transduction, knockout mice lacking STAT5a/b, STAT5b, and Jak3 all have NK cell defects (Imada et al., 1998; Nosaka et al., 1995; Park et al., 1995; Teglund et al., 1998). In addition to Jak/STAT signaling, IL-2/IL-15 binding results in the phosphorylation of tyrosine residues on the cytoplasmic portion of the IL-2Rb chain. These phosphotyrosine residues serve as docking sites for SH2, the domain of the adaptor protein Shc, which initiates a Ras/Raf/MAPK cascade that culminates in AP-1 activation (Zhu et al., 1994). IL-2/IL-15 ligation also results in activation of the phosphatidylinositol-3-kinase (PI-3 kinase)/Akt pathway (Gu et al., 2000), stimulation through src family cytoplasmic tyrosine kinases (Miyazaki

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et al., 1995), and activation of the nuclear factor kB (NF-kB; McDonald et al., 1998). Finally, IL-2/IL-15 binding results in induction of BCL-2, a mitochondrial protein that promotes cell survival (Miyazaki et al., 1995). The importance of these signal transduction pathways in NK cells is highlighted throughout this review. 2.3. Divergent Functions of IL-2 and IL-15 In Vivo, Despite Similarities In Vitro Despite the shared signal transduction properties of IL-2 and IL-15 in vitro, studies of mice and occasional human patients deficient for these cytokines— or their private a chains—indicate that their in vivo functions are quite distinct. Indeed, genetic ablation of IL-2 or of the IL-2Ra chain in mice results in normal NK cell numbers and function (Kundig et al., 1993; Schorle et al., 1991; Willerford et al., 1995). Rather, these mice develop a severe inflammatory disease as a consequence of unabated T-cell proliferation, highlighting the role of IL-2 and its private a chain in activation-induced cell death of T lymphocytes. Likewise, rare patients have been identified with genetic deficiencies of IL-2 or IL-2Ra; yet NK cell numbers and function are preserved in these individuals (DiSanto et al., 1990; Sharfe et al., 1997). Although humans with deficiencies in IL-15 or IL-15Ra have not been reported, genetic ablation of IL-15 in mice results in a drastic diminution in NK cell number, which can be rescued by exogenous administration of IL-15 (Kennedy et al., 2000). Likewise, early experiments revealed a profound NK cell deficiency in IL-15Ra knockout mice (Lodolce et al., 1998); however, as we discuss below, NK cell expression of IL-15Ra is not required for most aspects of NK development and mature NK function (Koka et al., 2003). Rather, NK cell–independent IL-15Ra serves to present this cytokine in trans to mature NK cells in the periphery, and this action is critical for NK cell survival (Koka et al., 2003). Thus, in contrast to their shared signal transduction machinery, IL-2 and IL-15 occupy different physiologic niches. 2.3.1. Resolving the IL-2 versus IL-15 Paradox: Can the IL-15Ra Chain Signal? How can we resolve this paradox between shared in vitro signal transduction and disparate in vivo function? Given the unique a subunit of each cytokine’s receptor, one hypothesis states that the IL-15Ra subunit is capable of eliciting its own signal transduction cascades. Support for this hypothesis is largely restricted to a series of in vitro studies of IL-15Ra signaling. For example, association of IL-15 with its IL-15Ra subunit has been reported to initiate the recruitment of tumor necrosis factor (TNF) receptor–associated

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factor 2 (TRAF2) to the short cytoplasmic tail of IL-15Ra in fibroblasts (Bulfone-Pau et al., 1999). This association between TRAF2 and IL-15Ra, as demonstrated by immunoprecipitation and Western blotting, protects these cells from TNF-a–induced apoptosis and leads to activation of NF-kB. Similarly, this interaction of IL-15Ra and TRAF2, and IL-15-inducible NFkB nuclear translocation, have been demonstrated in an erythrocyte cell line lacking the IL-2Rb subunit (Giron-Michel et al., 2003). In addition to TRAF2 and NF-kB activation, IL-15 association with IL-15Ra results in activation of Syk, a cytoplasmic tyrosine kinase, in B and T lymphocytes (Bulanova et al., 2001). This leads to the Syk-dependent phosphorylation of the IL-15Ra cytoplasmic domain at residue 227 as well as phosphorylation and activation of phospholipase Cg. This Syk-dependent signal transduction pathway, initiated by association of IL-15 with the IL-15Ra chain, results in calcium influx as well as a substantial survival benefit for the cells. This can occur in the complete absence of the IL-2Rb chain and in the presence of neutralizing antibodies to the gc chain (Bulanova et al., 2001). Whereas these studies have successfully documented IL-15Ra–specific signaling pathways, their relevance in NK cells and physiologic significance in vivo remain unresolved. 2.3.2. Resolving the IL-2 versus IL-15 Paradox: A Matter of Distribution? An alternative hypothesis proposes that the distribution of the IL-2Ra and IL-15Ra subunits, as well as the distribution of IL-2 and IL-15 themselves, are the most likely determinants of the distinct physiologic functions of these cytokines. Indeed, the expression patterns of each cytokine and its private a subunit are certainly distinct. Expression of IL-2 mRNA and protein is generally restricted to activated T lymphocytes (Smith, 1988), although some data indicate that this cytokine can be expressed by dendritic cells in response to lipopolysaccharide (LPS) (Granucci et al., 2001, 2003). In contrast to IL2 mRNA, the levels of IL-15 transcript are plentiful and ubiquitous across nearly all cell types; however, IL-15 protein expression is far less abundant and generally restricted to monocytes, dendritic cells, and stromal fibroblasts (Fehniger and Caligiuri, 2001). Competitive binding experiments on human NK cells, using radiolabeled IL-15 and IL-2, reveal no difference in the preferential binding of one cytokine over the other, with equal inhibition of binding in the presence of anti-IL2Rb monoclonal antibody (mAb) (Carson et al., 1994a). This is consistent with the existence of a shared receptor for these cytokines on NK cells. Scatchard analysis of total NK cells has identified high and low-affinity binding sites for both cytokines (Carson et al., 1994a). The few high-affinity binding sites for IL-2 likely represent cells within

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the CD56bright NK subset, which exclusively expresses the private high-affinity IL-2Ra (Caligiuri et al., 1990). The distribution of high- and low-affinity IL15-binding sites among human NK cells is unknown; however, the significance of IL-15Ra expression on these cells is highlighted by the ability of ultralowdose IL-15 to promote their survival in serum-free medium (Carson et al., 1994a). 2.4. New Concepts in IL-15 Signaling: IL-15 Is Presented in Trans Our understanding of IL-15Ra distribution has taken an unexpected twist, as genetic and biochemical lines of evidence have converged in elegant demonstrations of trans signaling between an IL-15/IL-15Ra high-affinity complex on a ‘‘presenting cell’’ and IL-15bg on a neighboring T or NK cell (Dubois et al., 2002; Koka et al., 2003). Prior to this discovery, our understanding of IL-15 signaling was based entirely on the model depicted in Fig. 1A. In this model, IL-15 engages one or more receptor components on a single cell type, initiating signal transduction cascades within this particular cell. This is signaling in cis. However, studies of IL-15Ra knockout animals have definitively shown that a complex of IL-15 and IL-15Ra on one cell can present cytokine and signal through the intermediate-affinity bgc heterodimer on a neighboring cell, i.e., signaling in trans (Fig. 1B) (Koka et al., 2003; Schluns et al., 2004). Moreover, biochemical studies indicate that an IL-15/IL-15Ra complex on monocytes is capable of multiple rounds of endocytosis followed by cytokine presentation at the cell surface (Dubois et al., 2002). The various physiologic roles of cis and trans signaling are only now being delineated. In NK cells, the strongest evidence points toward a role for trans signaling in cell survival, which is highlighted later in this review (Koka et al., 2003; Schluns et al., 2004). In CD8þ T cells, trans signaling is required for IL-15–induced proliferation (Schluns et al., 2004). The discovery of trans signaling raises the possibility that the regulation of IL-15 presentation may represent a key point of IL-15/IL-15Ra distribution in vivo. Given the transient nature of IL-15 protein expression, in vivo studies have failed to identify its physiologic triggers. In vitro, stimulation of human monocytes and monocyte-derived cell lines with interferon-g and LPS elicits IL-15 presentation (Carson et al., 1995; Dubois et al., 2002; Musso et al., 1999). Human and murine monocyte-derived dendritic cells (DCs) produce IL-15 and IL-15Ra in response to treatment with IFN-a (Mattei et al., 2001; Santini et al., 2000). The only negative regulator of IL-15 presentation thus far described is IL-15 itself, which induces a rapid receptor internalization (Dubois et al., 2002).

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Figure 1 Cis versus trans signaling by IL-15. (A) Soluble IL-15 is capable of signaling in cis through its intermediate-affinity receptor (bgc heterodimer; Kd, 108) or the high-affinity receptor (abgc heterotrimer). Arrows denote the proven/potential ability of the each receptor chain to initiate signaling through its cytoplasmic domain. (B) Alternatively, a complex of IL-15/ IL-15Ra on one cell can present cytokine and signal through the intermediate affinity bgc heterodimer on a neighboring cell, i.e., signaling in trans. What is the precise nature of the physiologic IL-15–presenting cell? The answer is unknown, but dendritic cells, monocytes, and stromal fibroblasts are the leading candidates.

3. Role of IL-2 versus IL-15 in Human NK Cell Development 3.1. Historical Work: Before IL-15 Early studies of human NK cell development focused on the ability of stromal cells to support the differentation of small numbers of NK cells from bone marrow (BM) progenitors in long-term culture lacking exogenous cytokines

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(Pollack et al., 1992). Later, investigators observed that exogenous IL-2 could promote NK differentiation of CD34þCD38 hematopoietic progenitors in stromal-free cultures, and with higher efficiency in the presence of stroma (Lotzova et al., 1993; Shibuya et al., 1995; Miller et al., 1992). Furthermore, administration of recombinant IL-2 to mice or human patients results in NK cell expansion (Caligiuri et al., 1993; Piguet et al., 1986; Rosenberg et al., 1987). Despite these encouraging findings, their physiologic significance to normal NK development remained a mystery, given the limited production of IL-2 by antigen-activated T cells in the periphery, as well as the persistence of NK cells in mice lacking T cells (as well as in knockout animals lacking the Il-2 gene or the private Il-2Ra subunit; Dorshkind et al., 1985; Kundig et al., 1993; Schorle et al., 1991; Willerford et al., 1995; ). A valuable hint to resolve this paradox was provided by the observation that mice and human patients lacking either component of the IL-2Rbg complex are severely difficient in NK cells—suggesting a molecule other than IL-2 may be responsible for signaling through the intermediate-affinity IL-2R during NK development (Cao et al., 1995; DiSanto et al., 1995; Gilmour et al., 2001; Noguchi et al., 1993; Suzuki et al., 1997). 3.2. IL-15 Promotes Human NK Cell Development from Hematopoietic Progenitors Shortly after its codiscovery by the Grabstein and Waldmann laboratories in 1994, numerous studies have implicated IL-15 in the process of human NK cell development. Studies by Mrozek et al. first demonstrated the ability of IL15 alone to promote differentiation of CD34þ hematopoietic progenitor cells (HPCs) to CD56þ NK cells with large granular lymphocyte (LGL) morphology, although this occurred without appreciable cell expansion over the 21-day culture (Mrozek et al., 1996). In seeking to expand the number of NK cells derived from CD34þ HPCs, two stromal-derived cytokines have been identified: c-Kit ligand (KL) and Flt3 ligand (FL). Culture in either KL or FL results in significant expansion in absolute cell number, but these cells failed to acquire CD56 expression or LGL morphology. However, the combination of IL-15 with KL and/or FL increases the absolute cell number while maintaining the cell percentage adopting an NK fate (Mrozek et al., 1996; Yu et al., 1998). Thus, whereas IL-15 alone induces CD56 expression and LGL morphology, its coupling with FL or KL increases the number of these NK cells. NK cells derived in combinations of IL-15 with FL or KL are equally cytotoxic as those derived in IL-15 alone. In addition, CD56bright NK cells derived in the presence of IL-15, or the combination of IL-15 and KL, are capable of cytokine production [IFN-g, TNF-a, and granulocyte-macrophage

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colony-stimulating factor (GM-CSF)] and chemokine production (macrophage inflammatory protein-1a, MIP-1a) in response to the monocyte-derived cytokines IL-12 and IL-15. In contrast, cells expanded exclusively in the presence of FL or KL fail to exhibit cytolytic activity or elaborate cytokines, in keeping with the inability of these cytokines to promote NK differentiation in the absence of IL-15 (Mrozek et al., 1996; Yu et al., 1998). 3.2.1. Mechanism of Synergy Between IL-15 and Flt3 Ligand/c-Kit Ligand By what mechanism do FL and KL exert their action during human NK cell development? One possible explanation is that these cytokines increase the frequency of NK precursors among CD34þ HPCs. This hypothesis was addressed by limiting dilution analysis (LDA) of NK precursor frequency among CD34þ HPCs cultured for 21 days in FL versus KL, compared with freshly isolated CD34þ HPCs. Indeed, 3 weeks of culture in either FL or KL significantly increased the NK precursor frequency when the cells were placed for 14 days in IL-15 alone. Moreover, FL significantly outpaced KL in this regard. These data suggest that FL and KL exert some qualitative effect on the NK precursor pool, rendering it more responsive to IL-15. In support of this hypothesis, a CD122þCD34bright intermediate cell population is detectable on 10 days of culture in FL or KL (although to a lesser extent). Isolation of this CD122þ population from FL-cultured CD34þ HPCs and LDA in IL-15 reveals an NK cell precursor frequency that is 65- to 235-fold higher than that observed among freshly isolated CD34þ HPCs (Yu et al., 1998). In support of this concept, parallel studies in mouse BM have demonstrated synergistic interactions between FL or KL and IL-15 in promoting development of NK cells (Williams et al., 1997). Culture of murine BM progenitors in FL, KL, IL-6, and IL-7 results in the detection of a CD122þ population with heightened NK precursor potential in the presence of IL-15. Furthermore, murine BM contains a CD122þ NK precursor population that differentiates ex vivo into mature NK cells in the presence of IL-15 or high-dose IL-2 (Rosmaraki et al., 2001). 3.2.2. Differences between In Vitro–Derived NK Cells and Circulating NK Cells In Vivo Notably, NK cells derived from in vitro cultures of progenitor cells with IL15 or high doses of IL-2 are CD56bright with little or no expression of CD2 or CD16, in contrast to the majority of NK cells in peripheral blood (Yu et al., 1998). Presumably, additional factors are required for acquisition of these additional surface markers of peripheral blood (PB) NK cells. One

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soluble factor with an apparently key role in this late maturation of NK is IL-21 (Parrish-Novak et al., 2000). This T-cell–derived cytokine has been shown to promote the acquisition of CD16 and killer immunoglobulin receptor (KIR) repertoire by NK cells during their development from CD34þ cord blood precursors cultured in FL, KL, IL-7, and IL-15 (Sivori et al., 2003). 3.3. Endogenous Pools of IL-15 for Human NK Cell Development What is the specific cell within bone marrow that is responsible for IL-15 production, and in response to what physiologic cues does IL-15 production or surface presentation occur? The answers to both questions are currently unknown, but the leading candidates for the IL-15–producing/presenting cell are stromal fibroblasts, monocytes, and dendritic cells. Analysis of longterm cultures of human BM stromal fibroblasts demonstrates detectable IL-15 transcript by reverse transcription-polymerase chain reaction (RT-PCR) and IL-15 protein by enzyme-linked immunosorbent assay (ELISA), whereas IL2 transcript and protein are consistently undetectable (Cluitmans et al., 1995; Mrozek et al., 1996). In addition, splenic fibroblasts have been shown to express surface IL-15, which is necessary and sufficient for cells to support the differentiation of autologous CD34þ progenitors to NK cells (Briard et al., 2002). CD14þ peripheral blood monocytes have been shown to produce soluble IL-15 that is detectable by ELISA in response to stimulation with IFN-g and lipopolysaccharide (LPS; Carson et al., 1995). In addition, flow cytometric analysis reveals that stimulation of CD14þ monocytes with IFN-g and LPS induces the surface expression of IL-15 in complex with IL-15Ra (Dubois et al., 2002; Musso et al., 1999). Although IFN-g and LPS are improbable stimuli of IL-15 expression in the context of development, monocytes maintain a substantial intracellular pool of this cytokine, and it is certainly conceivable that alternative, currently unknown physiologic cues prompt these cells to present IL-15 in trans to developing NK cells. Like monocytes, dendritic cells are capable of IL-15 production in response to pathologic cues such as LPS, double-stranded RNA, or type I interferons (Mattei et al., 2001; Santini et al., 2000), and these cells also upregulate expression of the IL-15Ra protein in response to these stimuli (Jinushi et al., 2003). Further study is required to implicate one or more of these cell types in IL15 presentation/secretion during NK cell development and to identify the mechanisms that regulate this process in vivo.

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3.4. How Does IL-15 Promote Human NK Cell Development? 3.4.1. Does IL-15 Initiate an NK Cell Developmental Program? How does IL-15 contribute to human NK cell development? Does it promote proliferation/survival of an already committed NK precursor, or does it initiate a developmental program that is required for NK ontogeny? To address this critical question, Yu and colleagues examined the cell cycles status, apoptotic index, and total cell number of CD34þ HPCs cultured first in FL or KL for 3 weeks, followed by the addition of IL-15 for another 14 days (Yu et al., 1998). Whereas the absolute cell number did not change significantly over the 2 weeks in IL-15, the percentage of CD56þ cells rose from <1 to >80%. However, analyses of cell cycle and apoptosis revealed that a constant fraction of cells was proliferating and undergoing programmed cell death during this time. Thus, these authors argued, the increase in NK cells can be explained only by the ability of IL-15 to promote the differentiation of an NK precursor population to mature NK cells. These data support the hypothesis that IL-15 initiates a developmental program that is required for NK cell development. 3.4.2. Does IL-15 Promote Survival/Outgrowth of a Committed NK Precursor? However, an alternative hypothesis is that some factor apart from IL-15 is responsible for commitment to the NK lineage and that IL-15 promotes the selective survival and outgrowth of a small fraction of NK precursor cells. In support of this hypothesis, small numbers of NK cells can be found in mice lacking IL-15 (Kawamura et al., 2003). In further support, NK cell development proceeds when NK cell–deficient IL-2/15Rb knockout mice are modified to express a ubiquitous Bcl2 transgene—suggesting that IL-15 is important for survival of the developing NK cell but not lineage commitment (Minagawa et al., 2002). Indeed, as we discuss below, the homeostatic role of IL-15 as a key survival factor for mature NK is absolutely essential for the maintenance of NK progeny (Cooper et al., 2002; Ranson et al., 2003). However, the same Bcl2 transgene fails to rescue NK cell deficiency in gc knockout mice (Kondo et al., 1997), implicating either IL-15 itself (signaling through agc but not b) or another gc cytokine (IL-2, IL-4, IL-7, IL-9, or IL-21) in lineage commitment. 3.4.3. Can IL-2 Influence NK Cell Development? Studies of IL-15Ra–/– mice suggest that IL-2 and IL-15 may perform nonredundant roles in guiding the acquisition of the repertoire of activating and inhibitory receptors by developing NK cells (Kawamura et al., 2003).

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IL-15Ra expression by developing NK cells is critical for acquisition of all Ly49 receptors in the mouse, and mice deficient for IL-15 or IL-15Ra possess an incomplete Ly49 repertoire. Whereas IL-15 is unable to trigger Ly49 receptor acquisition in the absence of IL-15Ra, IL-2 is able to rescue expression of certain Ly49 receptors in the IL-15Ra–/– cells. This may occur through expression of the unique high-affinity IL-2Ra chain by certain murine NK precursors, much akin to the selective expression of IL-2Ra by CD56bright NK in humans (Caligiuri et al., 1990). In this regard, it is interesting that low-dose IL-2 induces acquisition of killer immunoglobulinlike receptors (KIRs) by CD56bright NK from secondary lymphoid organs (Ferlazzo et al., 2004). Further studies are required to delineate the relative contributions of IL-2 and IL-15 to NK receptor acquisition by murine and human NK cells. 4. Low-Dose IL-2 Therapy Expands the CD56bright NK Subset 4.1. IL-2Ra and c-Kit Are Uniquely Expressed by the CD56bright Human NK Subset Studies in human peripheral blood have documented the existence of two distinct NK populations based on cell surface density of the CD56 antigen (neural cell adhesion molecule, N-CAM) (Lanier et al., 1989; Nagler et al., 1989). Indeed, the majority (85–90%) of human peripheral blood NK cells are CD56dim and express high levels of FcgRIII (CD16). The remaining 10–15% of NK cells are CD56brightCD16dim/. Studies of these subsets at the time of their discovery focused exclusively on their cytotoxicity. This work demonstrated that the CD56dim subset displays heightened cytolytic activity when compared with the CD56bright subset (Lanier et al., 1986). CD56bright NK cells uniquely express the IL-2Ra chain on their surface, conferring high affinity for IL-2 (Caligiuri et al., 1990). In contrast, CD56dim NK cells express the intermediate-affinity IL-2Rbg. This distinction permits CD56bright NK cells to selectively proliferate in response to low-dose (picomolar) quantities of IL-2. In addition, CD56bright cells uniquely express the c-Kit (CD117) receptor tyrosine kinase on their surface (Matos et al., 1993). This finding led to studies demonstrating a prosurvival effect of c-Kit ligand (KL, stem cell factor) on CD56bright NK cells via increased expression of the antiapoptotic BCL-2 protein (Carson et al., 1994b). These laboratory findings formed the rationale for therapeutic administration of low-dose IL-2 (alone and more recently in combination with KL) in cancer and AIDS patients with advanced disease to promote the expansion and survival of CD56bright NK cells (Caligiuri et al., 1993; and M. A. Caligiuri, unpublished observations).

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Why low-dose IL-2 in particular? Although IL-2 therapy at intermediate to high doses (nanomolar serum concentrations) has produced significant clinical responses in the setting of metastatic renal cell carcinoma and metastatic melanoma, life-threatening side effects including hypotension and a capillary leak syndrome have prevented its widespread use (reviewed by Rosenberg, 2000). In contrast, regimens of low- and ultralow-dose IL-2 (picomolar serum concentrations) have yielded expansions of CD56bright NK cells in phase I/II studies of patients with cancer and/or human immunodeficiency virus (HIV) infection without significant toxicity (reviewed by Fehniger et al., 2003). Proof of efficacy for this regimen in the clinical setting of immune deficiency and/or cancer must await the development and implementation of randomized phase III clinical trials, results of which are pending at this time. 4.2. Why Expand CD56bright NK Cells in Patients with Cancer or Immunodeficiency? The ability of IL-2–expanded CD56bright NK cells to lyse tumor and virusinfected target cells in an MHC-independent manner, together with their capacity to elaborate cytokines that promote activation of other immune cells, provide a rationale for their expansion in these clinical settings. For example, during HIV infection, depletion of CD4þ T cells results in drastic reductions in IL-2 and IFN-g, two cytokines that are normally required to stimulate CD8þ T cells and initiate an effective cellular immune response. This is one mechanism responsible for increased incidence of opportunistic infections and malignancy in patients with AIDS (Fauci, 1993). IL-2–expanded CD56bright NK cells produce significant quantities of IFN-g, permitting these cells to serve as surrogates for the CD4þ helper T-cell type 1 (Th1) response (Khatri et al., 1998). 4.3. Mechanisms of CD56bright NK Cell Expansion in Patients Receiving Low-Dose IL-2 Administration of low-dose IL-2 to patients results in picomolar concentrations of this cytokine that selectively expand CD56bright NK cells within 4–6 weeks of therapy, in accordance with the expression of the IL-2 high-affinity receptor by this population (Bernstein et al., 1995; Caligiuri et al., 1990, 1993). Studies by Fehniger and colleagues focused on the mechanism for the specific outgrowth of this population, specifically whether IL-2 acts on mature CD56bright NK cell homeostasis or whether it supports the differentiation of NK cells from progenitor populations in the bone marrow (Fehniger et al., 2000a). Whereas a percentage of CD56bright NK cells proliferates modestly in low-dose IL-2 in vitro, they exit the cell cycle within 6 days. Moreover, NK

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cells from patients treated with low-dose IL-2 fail to transit the cell cycle. In contrast, low concentrations of IL-2 do promote survival of CD56bright NK cells, in accordance with earlier observations. The presence of a functional high-affinity IL-2 receptor is required for both processes—proliferation and survival. Whereas proliferation in response to IL-2 is limited, the survival benefit of IL-2 is long lasting and will maintain CD56bright NK cells for weeks at a time. Strikingly, in vitro culture of bone marrow CD34þ progenitor cells in low-dose IL-2 significantly enhances their differentiation to mature NK cells. Thus, the most significant effect of IL-2 on NK cell homeostasis may be its ability to induce the differentiation of NK cells from progenitor populations. The kinetics of IL-2–mediated NK expansion in patients in vivo parallel those of IL-2–mediated differentiation from bone marrow progenitors in vitro. Significantly, CD56bright cells cultured in low-dose IL-2 show enhanced cytokine production (interferon-g) and cytotoxicity compared with cells cultured in medium alone (Fehniger et al., 2000a). Thus, low-dose IL-2 likely exerts three major effects on NK cells in patients receiving this cytokine: (1) increased differentiation from progenitor populations, (2) increased survival, and (3) increased effector function. 5. IL-2/IL-15 and NK Cell Survival 5.1. IL-15 Specifically Promotes NK Cell Survival In Vitro and In Vivo When cultured under serum-free conditions, NK cells rapidly undergo programmed cell death, or apoptosis (Carson et al., 1997a). This finding suggests that extracellular factors are responsible for sustaining NK cell survival in vivo. Despite the persistence of NK cells in IL-2–deficient mice, treatment with an antibody to IL-2Rb resulted in elimination of murine NK cells (Kundig et al., 1993; Schorle et al., 1991; Tanaka et al., 1993). The discovery of IL-15 offered a potential explanation for these paradoxical findings. Indeed, addition of low (picomolar) concentrations of IL-15 sustains human NK cell survival for over 1 week in serum-free medium (SFM) (Carson et al., 1997a). As noted previously, IL-2 also exhibits a prosurvival effect at low doses (Carson et al., 1997a; Fehniger et al., 2000a). In contrast, other cytokines that share the IL-2Rg chain (i.e., IL-4, IL-7, IL-9, and IL-13) fail to promote NK survival in SFM (Carson et al., 1997a). These results are consistent with the ability of the IL-2Rb subunit to promote cell survival (Miyazaki et al., 1995). Although these in vitro results suggest a model whereby soluble IL-15 interacts with its highaffinity IL-15R on NK cells, elegant experiments in IL-15Ra–deficient mice

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suggest that non-NK cells expressing IL-15Ra, in complex with IL-15, are responsible for presentation of this cytokine in trans to NK cells expressing the IL-2/15Rb and gc chains in vivo. This model is supported by the ability of IL-15Ra–/– NK cells to persist on adoptive transfer in wild-type mice, whereas wild-type NK cells fail to survive on transfer to IL-15Ra–/– hosts (Koka et al., 2003). 5.2. IL-15 Promotes Survival Through Increased Expression of Bcl-2 How does IL-15 promote cell survival? Culture in SFM and IL-15 maintains levels of the anti-apoptotic BCL-2 protein in human NK cells, and treatment of these cells with Bcl-2 antisense oligonucleotide results in downregulation of BCL-2 protein levels and decreased cell viability (Carson et al., 1997a). Consistent with these observations, adoptive transfer of NK cells from wildtype mice into IL-15–deficient mice results in their rapid disappearance within 5 days, whereas transplantation of Bcl-2 transgenic NK cells into IL-15–deficient recipients results in enhanced survival (Cooper et al., 2002; Ranson et al., 2003). Moreover, when wild-type NK cells are examined after transfer into IL-15–deficient mice, their BCL-2 protein expression is decreased (Ranson et al., 2003). Thus, the prosurvival effects of IL-15 are at least partially attributed to its ability to enhance BCL-2 levels. The signaling events responsible for increased BCL-2 in response to IL-15 have not been elucidated. 5.3. Can Enforced Expression of BCL-2 Serve as a Proxy for IL-2/IL-15 In Vivo? With the implication of BCL-2 as one downstream target of IL-15 in NK cells, several investigators have sought to determine whether restoration of BCL-2 expression is sufficient to rescue the NK cell deficiences observed in mice genetically deficient in the shared components of the IL-15R/IL-2R. Indeed, enforced expression of BCL-2 rescues the deficiency in NK cell numbers observed in mice lacking the IL-2Rb chain. Although IL-2Rb–/– Bcl-2tg NK cells are no different from wild-type NK cells in their receptor repertoire and IFN-g production, they completely lack cytotoxic activity (Minagawa et al., 2002). This underlines the pleiotropic roles of IL-2/IL-15 in NK cell ontogeny, homeostasis, and effector function. However, enforced expression of BCL-2 in the absence of the gc chain does not rescue the NK cell deficiency observed in g= mice (Kondo et al., 1997). c

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5.4. IL-2 and IL-15 Transmit BCL-2–Independent Survival Signals Another way in which IL-15 and IL-2 influence NK cell survival appears to be through activation of the cytoplasmic tyrosine kinase, Syk (Jiang et al., 2003). Syk-binding sites are found in the IL-2Rb chain and IL-15Ra chain (Bulanova et al., 2001; Minami et al., 1995; Qin et al., 1994). IL-2–mediated activation of Syk initiates a PI-3 kinase–dependent transduction cascade that culminates in activation of Akt/protein kinase B (PKB). Consistent with this pathway, pharmacologic and dominant-negative inhibitors of Syk or PI-3 kinase result in impaired IL-2–mediated survival and initiation of apoptosis (Jiang et al., 2003). However, mice deficient in Syk maintain normal NK cell numbers (Colucci et al., 1999). These experiments underline the potential differences between human and murine NK cells, as well as the existence of multiple, functionally redundant pathways to promote NK cell survival. 6. IL-2/IL-15 and NK Cell Proliferation 6.1. In Vitro Evidence Supports a Role for IL-2/IL-15 in NK Cell Proliferation The role of IL-2 in proliferation of NK cells was appreciated long before the discovery of IL-15 through studies of mice transgenic for IL-2 or its highaffinity Tac chain that showed selective proliferation of NK cells but not T cells (Biron et al., 1990; Ishida et al., 1989). In vitro studies of human NK cells revealed that, at low concentrations, IL-2 selectively induces proliferation of the CD25þCD56bright NK subset, because of the selective expression of the high-affinity IL-2Rabg ternary complex by this population (Caligiuri et al., 1990; Nagler et al., 1990). Increasing IL-2 concentration to full saturation of the high-affinity IL-2R and partial saturation of the intermediate-affinity IL-2Rbgc maximize this proliferation. These effects of IL-2 are diminished in the presence of neutralizing antibodies to IL-2Ra or IL-2Rb (Carson et al., 1994a). With the discovery of IL-15 and its shared signaling through components of the IL-2R, it became necessary to compare its influence on NK proliferation with that of IL-2. Not unexpectedly, IL-15 promotes the proliferation of CD56bright NK cells, and this activity is decreased with coincubation with neutralizing antibodies to IL-2Rb. Consistent with the private usage of IL-2Ra by its cognate cytokine, neutralizing antibodies to IL-2Ra have no consequence on IL-15–mediated proliferation of CD56bright NK cells (Carson et al., 1994a). In contrast to these observations in CD56bright NK cells, CD56dim cells exhibit little to no proliferation in response to a broad range of IL-2 or IL-15. (Heterogeneous expression of CD16 within the CD56bright

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NK subset permits further stratification of its proliferation in response to low-dose IL-2: CD16dimCD56bright NK cells exhibit greater proliferation in response to activation of the high-affinity IL-2R, relative to CD16neg CD56bright NK cells; Carson et al., 1997b.) 6.2. NK Proliferation In Vivo: Fully Independent of IL-2/IL-15? Despite the significant impact of IL-2 and IL-15 on CD56bright NK cell proliferation in vitro, cell cycle analysis of CD56bright NK cells obtained from patients receiving low-dose IL-2 in vivo demonstrates that these NK cells are not actively dividing (Fehniger et al., 2000a). These observations, together with the observation that low-dose IL-2 promotes the development of CD56bright NK cells from CD34þ progenitors, have led to the hypothesis that low-dose IL-2 exerts its actions chiefly through enhanced differentiation of NK cells from a progenitor pool and enhanced NK survival, rather than via in vivo proliferation of mature NK cells (Fehniger et al., 2000a). Thus, the precise roles of IL-2 and IL-15 in human NK cell proliferation in vivo remain unclear at this time. Indeed, it is conceivable that these cytokines do not play a role in NK cell proliferation and that they serve critical roles in promoting NK differentiation and survival. This hypothesis is supported by studies of NK cell homeostasis in mice. In lymphocyte-replete mice, bromodeoxyuridine (BrdU) labeling experiments indicate that mature NK cell turnover is virtually nonexistent and comparable to that of memory T cells (Jamieson et al., 2004). Whereas adoptive transfer to NK cell–replete hosts results in little or no proliferation, NK cells undergo a profound ‘‘homeostatic’’ proliferation on transplantation to alymphoid hosts (Jamieson et al., 2004; Ranson et al., 2003). Whereas adoptive transfer to IL-15–/– hosts profoundly mitigates NK cell recovery, the degree of their homeostatic proliferation is unaffected by the absence of IL-15 (Jamieson et al., 2004). These findings argue against a role for IL-15 in homeostatic proliferation but reinforce the essential, nonredundant role of this cytokine in NK cell survival. 7. IL-2/IL-15 and NK Cell Apoptosis Whereas IL-15 promotes the survival of resting NK cells in the periphery, the combination of IL-15 (or IL-2) with IL-12 results in NK cell activation, with considerable elaboration of cytokines, such as IFN-g and TNF-a, followed by programmed cell death, or apoptosis (Ross and Caligiuri, 1997). The mechanism of apoptosis in response to IL-12 and IL-15 appears to be via the production of TNF-a by NK cells, since neutralizing this cytokine or its association with the p80 TNF-a receptor partially abrogates this process (Ross

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and Caligiuri, 1997). The in vivo significance of these findings is unknown, but they likely represent a homeostatic mechanism to downregulate the immune response and thereby prevent the establishment of damaging proinflammatory positive feedback circuits between NK cells and other innate effectors. 8. IL-2/IL-15 and NK Cell Cytotoxicity 8.1. IL-2 and IL-15 Enhance NK Cell Cytolytic Activity NK cells are capable of a wide range of cytotoxic mechanisms to lyse target cells (for a review, see Djeu et al., 2002). These range from the release of intracellular granules containing cytotoxic proteins (e.g., perforin, granzymes), to the triggering of apoptotic cascades within target cells via the release of cytotoxic cytokines (e.g., TNF-a) or the direct engagement of death domain receptors (e.g., Fas) on target cells. Although an in-depth description of these cytotoxic mechanisms is beyond the scope of this review, it is important to note the key roles of IL-2 and IL-15 in enhancing NK cell cytolytic activity. Indeed, exposure of NK cells to IL-2 concentrations that saturate the intermediateaffinity IL-2 receptor results in lymphokine-activated killer (LAK) activity toward tumor cells that otherwise resist NK cell–mediated lysis (Caligiuri et al., 1990). This LAK activity is inhibited in the presence of neutralizing antibodies to the IL-2Rb subunit (Phillips et al., 1989). Preincubation of CD56dim NK cells with IL-15 triggers cytolysis of NK cell–resistant COLO 205 target cells in a dose-dependent manner, which is abrogated in the presence of anti-IL-2Rb mAb (Carson et al., 1994a). In this study, identical results were obtained with IL-2. 8.2. Molecular Basis of IL-2–Enhanced NK Cell Cytotoxicity Advances in our knowledge of the signal transduction pathways initiated by IL-2 have shed considerable light on the molecular basis of this LAK activity. One mechanism for IL-2–enhanced NK cell cytotoxicity is via upregulation of the pore-forming cytolytic effector molecule, perforin. This occurs in part via STAT5 association with two enhancer elements in the perforin promoter (Zhang et al., 1999). In addition to STAT5 activation, IL-2 promotes the cytoplasmic stabilization and nuclear translocation of NF-kB p50/p65 complexes, which also associate with and upregulate perforin promoter activity (Zhou et al., 2002). The importance of NF-kB for perforin expression is underscored in patients with hypohidrotic ectodermal dysplasia (HED), who possess mutations in the NEMO/IKK-g gene and display impaired NK cell cytolytic activity, leading to an increased susceptibility to opportunistic

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infections (Doffinger et al., 2001). The NEMO protein is one component of the complex responsible for I-kB phosphorylation and NF-kB activation, and NK cells from HED patients display reduced NF-kB activity. Treatment of one HED patient with IL-2 partially rescued NF-kB activation and NK cytolytic activity (Orange et al., 2002). 8.3. Role of IL-2 and IL-15 in the Developmental Acquisition of Cytolytic Function Studies of human NK cell development point to a vital role for IL-2/IL-15 in the acquisition of cytolytic activity. Barao and colleagues derived NK cells in vitro using IL-7, which lack cytotoxicity toward K562 targets. Perforin and granzyme B levels in these IL-7–derived NK cells were comparable to those found in cytotoxic counterparts derived in the presence of IL-2 or IL-15, suggesting that the role of IL-2/IL-15 is not merely to upregulate the expression of these two proteins. In contrast, IL-7–derived NK cells fail to express the surface molecule, LFA-1. LFA-1 is a cell adhesion molecule on NK cells that engages ICAM-1 on target cells to establish a critical interaction, called conjugate formation, that must exist prior to the release of cytotoxic granules by the NK cell. Exposure of IL-7–derived NK cells to IL-2 or IL-15 results in their acquisition of LFA-1 surface expression and cytotoxicity that is neutralized by antibodies to LFA-1 subunits CD11a and CD18 (Barao et al., 2003). Thus, in addition to enhancing cytotoxicity in mature NK cells, IL-2/ IL-15 may serve an essential role in the acquisition of NK cell cytolytic machinery during NK cell development. This hypothesis is supported by the findings of Minagawa and colleagues, who sought to rescue the NK cell deficiency observed in IL-2Rb–/– mice through the overexpression of BCL-2. These investigators found that, whereas enforced BCL-2 expression restored NK cell numbers in IL-2Rb–/– mice, these NK cells completely lacked cytolytic activity (Minagawa et al., 2002)—an observation that is consistent with an absolute and nonredundant role for IL-2/IL-15 signaling in the acquisition and/or maintenance of NK cell cytotoxicity. 9. IL-2/IL-15 and NK Cell Cytokine Production 9.1. IL-2 and IL-15, in Synergy with Monokines, Elicit Significant Cytokine Production by NK Cells Numerous studies have documented the roles of IL-2 and IL-15 in eliciting the production of various immunoregulatory cytokines by human NK cells, including IFN-g, TNF-a, and GM-CSF (for a review, see Cooper et al., 2001).

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Whereas IL-2 or IL-15 can elicit little IFN-g production alone, both synergize equally well with IL-12 to produce substantial quantities of this cytokine (Carson et al., 1994a). In addition to IFN-g production, IL-12 synergizes with IL-15 or IL-2 in the synthesis of other cytokines, such as TNF-a and GM-CSF. Subtle differences between the ability of IL-15 and IL-2 to elicit cytokine production by NK cells have been noted. For example, Carson and colleagues observed that GM-CSF production in response to IL-2 alone was routinely twice that observed with IL-15, even when both cytokines were administered at concentrations that rule out selective use of private highaffinity receptors (Carson et al., 1994a). 9.2. CD56bright Subset is Chiefly Responsible for Cytokine Production by Human NK Cells Subsequent studies have demonstrated that the CD56bright NK subset produces significantly greater quantities of IFN-g and GM-CSF than CD56dim NK cells in response to stimulation with IL-2/IL-15 and IL-12 in vitro (Cooper et al., 2001b; Fehniger et al., 1999, 2003). Moreover, because of its unique expression of the HA IL-2R, CD56bright NK cells produce IFN-g in the presence of low (picomolar) concentrations of IL-2. This raises the possibility that this NK subset may participate in cross-talk with T cells in secondary lymphoid organs, where these cells exist in close proximity (Fehniger et al., 2003). Together, these findings are consistent with a model in which CD56bright NK cells serve to elaborate cytokines that regulate the response of other immune cells—such as monocytes, dendritic cells, and other lymphocytes (Cooper et al., 2001a, 2004). IL-2/IL-15 treatment of CD56bright NK cells results in the production of different cytokines, depending on the local milieu (Cooper et al., 2001b). For example, the combination of IL-15 and IL-18 results in optimal production of GM-CSF but minuscule expression of IL-10; in contrast, treatment with IL-15 and IL-12 results in optimal production of IL10 but lower production of GM-CSF (Cooper et al., 2001b). 10. NK Cell–Immune Cell Interactions and Modulation by IL-2/IL-15 10.1. CD56bright NK Cells Are Enriched in Secondary Lymphoid Organs, Where T-Cell–Derived IL-2 Can Influence Their Function CD56bright NK cells express two homing molecules, L-selectin and CCR7, that direct the migration of these lymphocytes to peripheral lymph nodes (LNs) via high endothelial venules (Campbell et al., 2001; Fehniger et al., 2003; Frey et al., 1998). Accordingly, CD56bright NK cells are enriched in secondary

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lymphoid organs, particularly LNs and tonsils (Fehniger et al., 2003; Ferlazzo et al., 2004). Immunohistochemical analysis demonstrates that CD56bright NK cells localize to the parafollicular regions of LNs, where they are in close proximity to T cells and activated DCs. This observation suggests that CD56bright NK cells, which possess the HA IL-2R, may respond to T-cell–derived IL-2 in vivo (Fehniger et al., 2003). In support of this model, coculture of CD56bright NK cells with T cells and APCs indicates that endogenous T-cell–derived IL-2 is capable of acting through the HA IL-2R and costimulates IFN-g production by CD56bright NK cells in the presence of IL-12. These findings suggest that CD56bright NK cells, through their elaboration of IFN-g, may provide an important bridge between innate and adaptive immunity (Cooper et al., 2004). 10.2. Low-Dose IL-2 Promotes the Maturation of CD56bright NK Cells from Secondary Lymphoid Organs In addition to IFN-g production, low-dose IL-2 may serve to promote the functional maturation of LN CD56bright NK cells (Ferlazzo et al., 2004). This hypothesis is suggested by the finding that administration of low-dose IL-2 to purified LN CD56bright NK cells causes these cells to acquire the surface phenotype of a CD56dim NK cell: CD16þKIRþNCRþ. In addition, treatment of LN or tonsillar NK cells with low-dose IL-2 induces these cells to express perforin and to exhibit cytolytic activity toward NK cell–sensitive targets (Ferlazzo et al., 2004). In contrast, treatment of peripheral blood CD56bright NK cells with low-dose IL-2 does not induce comparable changes. This difference may reflect the fact that peripheral blood CD56bright NK cells have yet to enter secondary lymphoid organs. This model of NK cell maturation is supported by the finding that circulating CD56bright NK cells express lymph node–homing molecules (CCR7 and L-selectin), whereas CD56bright NK cells in secondary lymphoid organs do not express these proteins (Fehniger et al., 2003; Ferlazzo et al., 2004). Murine bone-marrow–derived dendritic cells (mBMDCs) are capable of IL-2 production on stimulation with LPS (Granucci et al., 2001). DCs from IL-2–/– mice exhibit a severe impairment in their ability to stimulate CD4þ and CD8þ T-cell proliferation in a mixed lymphocyte reaction assay, and these IL-2–deficient DCs are reportedly inefficient in activating NK cell responses (Granucci et al., 2003). Thus, migration of CD56bright NK cells from peripheral blood to secondary lymphoid organs may influence the development and function of these cells, under the influence of T-cell– and/ or DC-derived IL-2.

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10.3. Dendritic Cells and Monocytes Produce IL-15, Which Influences NK Cell Cytolytic Function and Cytokine Production In addition to IL-2, dendritic cells produce IL-15 in response to type I interferons (IFN-a and IFN-b), which can increase NK cell activity. For example, human and murine monocyte-derived DCs produce IL-15 and IL15Ra in response to IFN-a treatment (Mattei et al., 2001; Santini et al., 2000). This IL-15/IL-15Ra signals in autocrine fashion to elicit additional production of IFN-a and upregulation of MICA on the DCs, which associates with the activating NKG2D receptor on NK cells to promote their cytolytic function and cytokine production (Jinushi et al., 2003). This mechanism is important for activation of NK cells by DCs, as underlined by impaired NK activity in patients with hepatitis C, whose DCs are unable to produce IL-15 in response to type I interferon treatment (Jinushi et al., 2003). Flow cytometric analysis of monocytes after stimulation with LPS and IFN-g reveals surface expression of IL-15 in complex with IL-15Ra (Dubois et al., 2002; Musso et al., 1999). Coculture experiments of macrophages and autologous NK reveal that LPS is sufficient to promote robust IFN-g production— particularly by CD56bright NK cells—which can be inhibited by neutralizing antibodies to IL-12 and IL-15 (Carson et al., 1995; Cooper et al., 2001b). Thus, macrophages produce IL-15 (along with other monokines, such as IL-12, IL-18, and IL-1b) in response to LPS stimulation, which costimulate the production of IFN-g by NK cells. This IFN-g greatly increases the sensitivity of the macrophage to LPS, such that subsequent exposure results in cytokine-induced shock and mortality, because of the unhindered release of proinflammatory cytokines. This cycle of sequential priming with IFN-g and LPS exposure is termed the generalized Schwartzman reaction (Brozna, 1990). Neutralizing antisera to IL-15 or the IL-2Rb subunit significantly reduce IFN-g levels and subsequent lethality in a murine model of the Schwartzman reaction, suggesting that this cytokine plays an instrumental role in this process (Fehniger et al., 2000b). Thus, monocyte-derived IL-15 serves a critical role in potentiating IFN-g production by NK cells both in vitro and in vivo, and therapeutic strategies aimed at disrupting this proinflammatory loop may prove efficacious. 11. Conclusions In this review, we have summarized our current understanding of the roles of IL-2 and IL-15 in human NK cell development, homeostasis, and function, as well as the potential physiologic roles of these two cytokines in orchestrating the interactions between NK cells and other immune cells. We have summarized these findings in Figs. 2 and 3.

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Figure 2 IL-2/IL-15 facilitate NK cell differentiation and contribute to mature NK cell homeostasis. IL-2 and IL-15 have the ability to promote differentiation of mature NK cells from NK precursors and also contribute to the homeostasis of mature NK cells, acting variously to promote survival, proliferation, or apoptosis. The physiologic role of these cytokines in proliferation and apoptosis of NK cells is unknown. See text for details.

Figure 3 IL-2/IL-15 contribute to mature NK cell function. IL-2 and IL-15 act at various levels to impact human NK cell function. They increase the cytolytic activity of CD56bright and CD56dim NK cells. They greatly enhance cytokine secretion by the CD56bright human NK subset. Specifically in secondary lymphoid tissues (e.g., lymph nodes), IL-2 has been shown to promote the acquisition of KIR and CD16 expression by CD56bright NK cells. See text for details.

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Although our comprehension of these topics has grown tremendously since the discovery of IL-15, several unresolved questions remain to challenge this field for years to come. The discovery of IL-15 trans presentation by IL-15Ra, together with the observation that IL-15Ra can signal independently of IL2Rbg, offer the possibility of reciprocal communication between NK cells and IL-15–presenting cells, such as DCs and monocytes. A more detailed inspection of IL-15Ra signaling and IL-15 presentation (cis versus trans) will undoubtedly aid our understanding of the specific effects of this cytokine on NK cell ontogeny, function, and homeostasis. Moreover, although IFN-a, IFN-g, and LPS may trigger IL-15 presentation in the context of infectious disease, these are not the likeliest physiologic cues that elicit IL-15 during NK cell development or NK cell survival in the periphery, and defining novel positive and negative regulators of IL-15 presentation is a major challenge for the future of this field. Finally, the discovery of secondary lymphoid organs as havens for CD56bright NK cells, together with the unexpected demonstration that low doses of IL-2 promote their differentiation to CD56dim KIRþ CD16þ cells, suggest that IL-2 and the microenvironment of these tissues may play a qualitative role in the development and maturation of human NK cells. Acknowledgments This work was supported by P01 CA95426 and R01CA68458.

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Regulation of Antigen Presentation and Cross-Presentation in the Dendritic Cell Network: Facts, Hypothesis, and Immunological Implications Nicholas S. Wilson and Jose A. Villadangos Immunology Division and The Cooperative Research Center for Vaccine Technology, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia

1. 2. 3. 4. 5. 6. 7. 8.

Abstract .................................................................................................. Introduction: Dendritic Cells at the Crossroads of Immunity and Tolerance ................ Commonalities and Diversity in the DC Network ................................................ Antigen Presentation Pathways....................................... ................................ Mechanisms and Regulation of Antigen Capture in DCs ....................................... Antigen Presentation by MHC II Molecules ...................... ................................ Control of MHC II Antigen Presentation in DCs ................................................ DCs and Cross-Presentation.......................................................................... Conclusions and Future Directions ................................. ................................ References ...............................................................................................

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Abstract Dendritic cells (DCs) are central to the maintenance of immunological tolerance and the initiation and control of immunity. The antigen-presenting properties of DCs enable them to present a sample of self and foreign proteins, contained within an organism at any given time, to the T-cell repertoire. DCs achieve this communication with Tcells by displaying antigenic peptides bound to MHC I and MHC II molecules. Here we review the studies carried out over the past 15 years to characterize these antigen presentation mechanisms, emphasizing their significance in relation to DC function in vivo. The life cycles of different DC populations found in vivo are described. Furthermore, we provide a critical assessment of the studies that examine the mechanisms controlling DC MHC class II antigen presentation, which have often reached contradictory conclusions. Finally, we review findings pertaining to the biological mechanisms that enable DCs to present exogenous antigens on their MHC class I molecules, a process known as crosspresentation. Throughout, we highlight what we consider to be major knowledge gaps in the field and speculate on possible directions for future research. 1. Introduction: Dendritic Cells at the Crossroads of Immunity and Tolerance T cells are generated from bone marrow precursors that migrate into the thymus, where they mature into naive T cells. The maturing thymocytes are interrogated and selected based on the quality of the interaction of their

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T-cell receptors (TCRs) with major histocompatibility complex (MHC)– peptide complexes displayed on the surface of thymic antigen-presenting cells. The thymocytes are selected in a two-pronged process: ‘‘positive’’ selection, which chooses thymocytes that express TCRs capable of some interaction with the host’s MHC molecule/self-peptide complexes; and ‘‘negative’’ selection, which deletes thymocytes that recognize self-MHC–peptide combinations too strongly (autoreactive). Since negative selection is responsible for the elimination of most anti-self T cells, the outcome of this process is referred to as the generation of central tolerance (Starr et al., 2003). Naive T cells that emerge from the thymus have the potential to interact strongly with (‘‘recognize’’) foreign peptides bound to host MHC molecules and become effector T cells in a process referred to as priming. However, it is important to note that the repertoire of naive T cells that emerges from the thymus is not ignorant of the peripheral self-MHC–peptide complexes; every T-cell reacts with self-complexes at a basal strength, a feature that may be required to maintain the peripheral T-cell compartment (Sprent and Surh, 2003). Indeed, the process of central tolerance allows some autoreactive T cells to escape to the periphery (Bouneaud et al., 2000). These autoreactive T cells must be eliminated or held in check (tolerized) to avoid autoimmunity in a process that is referred to as peripheral tolerance. Obviously, a healthy immune system should be efficient at priming antiforeign T cells while maintaining the self-reactive T cells tolerized. The problem is that the structural basis of the interaction between anti-foreign TCRs and anti-self TCRs with their respective MHC–peptide combinations is the same. Thus, autoreactive T cells cannot themselves discern whether they are recognizing self or foreign peptides via their TCRs. It is the context in which this interaction takes place that determines whether a given T-cell will be primed or tolerized. Research conducted on dendritic cells (DCs) has revealed that this rare and heterogeneous population of leukocytes is in charge of providing both the immunogenic and tolerogenic contexts for naive T cells (Banchereau and Steinman, 1998; Steinman, 1991; Steinman et al., 2003). But if DCs have the dual (and paradoxical) role of inducing priming and tolerance, how do they decide between these two opposing outcomes? It has been suggested that this decision depends on the developmental stage of the DCs (‘‘maturity’’) (Finkelman et al., 1996; Hawiger et al., 2001; Steinman and Nussenzweig, 2002). Although we are still far from understanding what makes a DC tolerogenic or immunogenic, characterizing the antigenpresenting properties of DCs at the different stages of their development has thus emerged as a primary goal to understand their roles in immunosurveillance and tolerance induction. Those properties are the main subject of this review.

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 243 2. Commonalities and Diversity in the DC Network 2.1. What Is DC Maturation? Articles on DCs usually begin with a description of a life cycle modeled on the findings of studies performed first on Langerhans cells (LCs, a type of DC found in the epidermis) (Romani et al., 1989; Schuler and Steinman, 1985; Steinman, 1991) and later reproduced using DC models grown in vitro (Cella et al., 1997b; Pierre et al., 1997; Winzler et al., 1997). According to this ‘‘LC paradigm,’’ DCs are present in peripheral tissues in a so-called immature state dedicated to sampling their environment (Fig. 1). Immature DCs are highly endocytic, but express low levels of MHC class II (MHC II) molecules on their surface, although these molecules appear abundant in endocytic compartments. Another feature of immature DCs is that they are inefficient at activating naive T cells. In the presence of inflammatory compounds expressed by foreign pathogens [lipopolysaccharide (LPS), double-stranded RNA, DNA rich in CpG motifs, etc.] or released by damaged tissues (e.g., tumor necrosis factor a, TNF-a), DCs become ‘‘activated’’ and migrate via the afferent lymphatics to the local draining lymph node (LN) (Larsen et al., 1990; Stoitzner et al., 2003). Simultaneously, DCs acquire a ‘‘mature’’ phenotype characterized by a reduced capacity to capture antigens, high expression of MHC II at the cell surface, and high expression of T-cell costimulatory molecules such as CD40, CD80, and CD86, which enable mature DCs to stimulate naive T cells (Sharpe and Freeman, 2002). Mature DCs show prolonged presentation of antigens they captured just before or at the time of receiving their activation signal, but they no longer process and present newly encountered antigens. The ability of mature DCs to sustain the presentation of the antigens they captured in their immature state has been referred to as antigenic memory. 2.2. DC Subtypes According to the LC paradigm, DC maturation is linked to tissue localization, with immature cells in the peripheral tissues and mature DCs in the lymphoid organs. However, the LC represents just one of the multiple types of DC contained in the body, and it has become apparent that the LC paradigm of DC life cycle and function does not apply to all DC types (Manickasingham and Reis e Sousa, 2001; Wilson and Villadangos, 2004). For researchers interested in antigen presentation but not familiar with the DC field, the subdivision of DCs into subpopulations can sometimes appear confusing, arbitrary, and irrelevant. However, we consider that a review of the mechanisms employed by DCs to control antigen presentation would not be complete

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Figure 1 Dendritic cell (DC) populations of the spleen and lymph nodes. (A) The spleen (left) receives only blood-derived DCs (red and blue), whereas the lymph nodes (right) receive both blood-derived and tissue-derived (green) DCs. (B) FACS analysis of the spleen and lymph node DC populations (see Table 1). The plots show the expression of CD4 vs CD8 (spleen, left), and of

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 245 without a description of the ‘‘life styles’’ for different DC types and how they agree with, or deviate from, the LC paradigm. Moreover, because our knowledge of the antigen-presenting properties of DCs comes from studies using different types of DC grown in vitro or purified from tissues, it is important to understand how the conclusions of the different DC models correlate with the functions of DCs in vivo. Because of the heterogeneity of the DC system, and the changes undergone by DCs during their development, definition of the DC lineage is usually based on a set of common phenotypic and functional features rather than a single surface marker or capability. Common features of DCs include the expression of CD11c, the ability to endocytose a large variety of antigens and present them via MHC II molecules, and a high capacity to stimulate naive T cells. Each DC population can then be further defined based on its expression of a combination of surface markers. In this section we summarize the most common markers used to discriminate the major DC subtypes known to date. We stress that this is not by any means an exhaustive description of phenotypic and functional differences among DC subtypes, which have been extensively reviewed elsewhere (Ardavin, 2003; Hart, 1997; Shortman and Liu, 2002; Wilson and Villadangos, 2004). DC heterogeneity has been characterized best in the mouse system, so we will start with murine DC subsets, followed by human DCs. 2.2.1. Tissue-Derived DCs A major subdivision of DC types can be made according to the paths used to access the lymphoid organs: the lymph or the blood (reviewed in Itano and Jenkins, 2003; Wilson and Villadangos, 2004). The term ‘‘tissue-derived DCs’’ refers to those present in the interstitial spaces of all tissues and in epithelial surfaces of the gut, the airways, and the skin. These DCs migrate via the

CD205 vs CD8 (lymph node, right) in CD11chigh DCs. The blood-derived DCs comprise three populations: CD8+CD4
CD205+ (CD8+ DCs, red), CD4+CD8
CD205
(CD4+ DCs, blue), and CD4
CD8
CD205
DCs (CD4
CD8
, also blue). Note that the CD4+ and CD4
CD8
DC populations appear as a single CD8
CD205
group in the lymph node (blue). The tissue-derived DCs can be distinguished as a CD205+CD8low population (green). These tissue-derived DCs constitutively migrate from the periphery to the draining lymph nodes (A, top right), where they have a mature phenotype, as shown in the immunofluorescence confocal microscopy image shown in (C). In contrast, the blood-derived DCs contained in the spleen and lymph nodes in the steady state are immature, as illustrated by the accumulation of MHC II molecules in endosomal (Lamp+) compartments (C, top left). The blood-derived DCs can be induced to mature in the lymphoid organs in response to inflammatory compounds such as LPS or CpG and then acquire a mature phenotype (C, bottom left).

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afferent lymphatics into their local LN, where they eventually die (Fig. 1) (Brand et al., 1993; Bujdoso et al., 1989; Geissmann et al., 2002; Howard et al., 1996; Larsen et al., 1990; Lukas et al., 1996). Tissue-derived DCs can be distinguished from other DC types by their low expression of CD8 and the intermediate to high expression of the C-type lectin CD205 (also known as DEC-205) (Table 1) (Anjuere et al., 1999; Henri et al., 2001; Kamath et al., 2002; Ruedl et al., 2000; Salomon et al., 1998; Stoitzner et al., 2003; Vermaelen et al., 2001; Wilson et al., 2003). LCs belong to this DC group, but of course the only LNs where they can be found are the subcutaneous. Thus, the subcutaneous LNs receives two types of DC via lymph: the ‘‘interstitial DCs’’

Table 1. Mouse Dendritic Cell Populations Blood derived Conventional CD4
CD8


pDCs

CD4+

CD8+

Spleen Subcutaneous LNs Visceral LNs Thymus

U U U U

U U U ?

U U U U

CD11c CD45RA CD4 CD8 CD205 Langerin CD11b

þþ þþþ var. var.

þþþ

B. Markers þþþ þþþ

Maturitya Costimulatoryb Antigen processingc MHC II a

im þ þ þ

A. Location U U U ?

Tissue derived LCs

iDCs

In Blood DCs

pDCs

þþ þþþ

U

U U

þþþ

þþþ

þþþ

þ þ þþþ

þþþ

mat þþ þ/– þþþ

im þ þþþ þ

þþþ þþþ þþ þ þþþ

þþþ

þþþ þþþ þþþ

im þ þþþ þþ

C. Maturity im im þ þ þþþ þþþ þþ þþ

mat þþ þ/– þþþ

im þ þ þ

im, immature; mat, mature. T-cell–costimulatory molecules (CD80, CD86, CD40). c The capacity to endocytose and present new antigens. Abbreviations: pDC, plasmacytoid dendritic cell; LC, Langer hans cell; iDC, immature DC; LN, lymph node. Key: ?, CD4þDCs can be identified, but this could be due to ‘‘pick-up’’ from thymocytes (Vremec et al., 2000); þ, low; þþ, intermediate; þþþ, high; var., heterogeneous (pDCs can be CD4
CD8þ, CD4þCD8
, CD4
CD8þ, or CD4þCD8þ; O’Keeffe et al., 2002a). b

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 247 of the dermis, which are the skin equivalent of the tissue-derived DCs present in all other tissues, and LCs from the epidermis (Table 1). These two DC types can be distinguished from each other by the higher expression of CD205, the intracellular expression of langerin, and the presence of Birbeck granules in the LCs (Henri et al., 2001; Kamath et al., 2002; Stoitzner et al., 2003; Valladeau et al., 2000; Wilson et al., 2003). Virtually all the tissue-derived DCs contained in the LNs have a mature phenotype: they express high levels of MHC II and T-cell costimulatory molecules and can activate naive T cells, but they are inefficient at processing and presenting newly encountered antigens (Anjuere et al., 1999; Henri et al., 2001; Kamath et al., 2002; Ruedl et al., 2000; Salomon et al., 1998; Stoitzner et al., 2003; Vermaelen et al., 2001; Wilson et al., 2003, 2004). Several studies have demonstrated that tissuederived DCs contained in the subcutaneous LNs, mesenteric LNs, and mediastinal LNs come from immature DCs that migrated from the skin, the gut, and the airways, respectively (Henri et al., 2001; Huang et al., 2000; Itano et al., 2003; Macatonia et al., 1987; Vermaelen et al., 2001). So in general this DC group behaves according to the LC paradigm, but there are two exceptions to this general rule. First, it has been reported that tissue-derived DCs can traffic to the LNs while retaining an immature phenotype (Geissmann et al., 2002), although this may only happen in pathological conditions such as chronic skin inflammation. Second, some DCs activated in the peripheral tissue may remain at the site of activation, where they might play an important role in driving inflammatory reactions (Huh et al., 2003; Luft et al., 2002; Vermaelen and Pauwels, 2003). It is also important to note that the migration and maturation of the tissue-derived DCs may not always be triggered by activatory signals but be part of their normal life cycle even in the absence of infections (in the steady state). 2.2.2. Blood–Derived DCs Another major group of DCs can be found in the LNs and also in the spleen. The spleen can only be accessed via the blood, so probably this is the pathway used by this DC group to access both the spleen and the LNs (Fig. 1) (reviewed in Itano and Jenkins, 2003; Wilson and Villadangos, 2004). One of the DC types belonging to this group of ‘‘blood-derived DCs’’ are the so-called interferon-producing cells or plasmacytoid DCs (pDCs), which are characterized by expression of CD45RA and lower levels of CD11c than other DC types (Table 1; Asselin-Paturel et al., 2001; Bjorck, 2001; Martin et al., 2002; Nakano et al., 2001; O’Keeffe et al., 2002a). It is unclear whether pDCs play a major role in presenting antigens for the initiation of immune responses because their antigen presentation and naive T-cell–stimulatory capacities are much

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poorer than those of the other DC types (Krug et al., 2003). Rather, the main function of this DC population appears to be the secretion of interferons against viruses as part of the innate immune response (Colonna et al., 2002). Because this group of DCs is quite unusual and may not be involved in priming, in this review we use the term ‘‘blood-derived’’ only to refer to the ‘‘conventional’’ CD45RA
nonplasmacytoid DCs. These conventional DCs can be subdivided into three populations: one expresses high levels of CD8 and CD205 (‘‘CD8þ DCs’’ hereafter), the other expresses CD4 but not CD205 (‘‘CD4þ DCs’’), and the third expresses neither of these three markers (‘‘CD4
CD8
DCs’’) (Table 1) (Anjuere et al., 1999; Henri et al., 2001; Vremec et al., 2000; Wilson et al., 2003). These populations most likely emerge from three independent developmental streams within the DC lineage (Kamath et al., 2002; Naik et al., 2003; Shortman and Liu, 2002), although some reports suggest that the CD8þ DCs are derived from one of the CD8
DC populations (Ardavin, 2003). In the steady state, the CD8þ and CD8
DCs are located in different areas within the lymphoid organs: the CD8þ DCs are present in the T-cell areas, and the CD8
DCs are found in the marginal zones (De Smedt et al., 1996; Pulendran et al., 1997). The functional significance of this segregation is unclear. In contrast to the tissue-derived DCs, virtually all the blood-derived DCs contained in the LN and the spleen in the steady state have an immature phenotype (Fig. 1; Wilson et al., 2003). This statement may appear paradoxical, because the LC paradigm predicts that the DCs reaching the lymphoid organs should have a mature phenotype. Indeed, the relatively high expression of MHC II in blood-derived DCs, and their capacity to activate naive T cells in mixed lymphocyte reactions (MLRs) in vitro, led to their consideration as mature DCs (Ruedl et al., 2000; Salomon et al., 1998; Vremec et al., 2000). However, as we discuss later, the level of surface expression of MHC II is a misleading parameter when assessing DC maturity. More importantly, the experiments that measured the capacity of blood-derived DCs to activate naive T cells in MLRs had an important caveat: even though freshly isolated DCs are immature, they become spontaneously activated in culture and quickly acquire a mature phenotype, thus gaining the capacity to activate naive T cells (De Smedt et al., 1996; El-Sukkari et al., 2003; Inaba et al., 1992; Wilson et al., 2003, 2004). This process of ‘‘spontaneous maturation’’ also happens to LCs extracted from the skin and cultured in vitro; indeed, the original definition of DC maturation was based on the phenotypic changes undergone by skin LCs during culture (Schuler and Steinman, 1985). In fact, blood-derived DCs freshly isolated from the spleen or the LN fulfill the definition of immaturity: most of their MHC II molecules are contained in endosomal compartments rather than exposed on the cell surface (Fig. 1); they

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 249 have the capacity to capture, process, and present newly encountered antigens; they express low levels of T-cell costimulatory molecules; and if they are fixed to prevent their maturation in culture, they are unable to activate naive T cells (Henri et al., 2001; Inaba et al., 1992; Ruedl et al., 2000; Salomon et al., 1998; Vermaelen et al., 2001; Wilson et al., 2003, 2004). Moreover, these DCs can be converted into mature DCs, similar to the tissue-derived DCs present in the LNs, by injecting stimulatory compounds in vivo or by culture in vitro (Fig. 1) (De Smedt et al., 1996; Inaba et al., 1992; Reis e Sousa and Germain, 1999; Schulz et al., 2000; Wilson et al., 2003, 2004). This implies that in the steady state virtually all the DCs contained in the spleen, and half of all the DCs contained in the LN, are immature (Wilson et al., 2003). Does this mean that the blood-derived DC types migrate from the peripheral tissues without acquiring a mature phenotype? The answer is, probably not. The three blood-derived DC subsets turn over quickly; for instance, they are completely replaced in the spleen within 3 to 5 days (Kamath et al., 2000, 2002). Yet none of these three subsets has been identified in peripheral tissues or circulating in the blood (O’Keeffe et al., 2002b, 2003). The blood does contain plasmacytoid DCs and a different DC population that has some features in common with the CD4þ, CD8þ, and CD4
CD8
DCs, but this blood DC type is probably not the precursor of the three types contained in the lymphoid organs (Table 1) (O’Keeffe et al., 2002b, 2003). This suggests that the three blood-derived DC populations contained in the spleen and the LNs arise directly in the lymphoid organs from earlier hematopoietic precursors. There, the vast majority spend their entire life span in an immature state unless they are activated in situ. Clearly, the life cycle of the blood-derived DC types is different from that of the tissue-derived DCs (Fig. 1). Why? We suggest this enables them to play a similar function to the tissue-derived DCs, and perhaps in addition two distinct functions. The similar function is immunosurveillance. When one thinks of areas of the body exposed to pathogen infection, the epithelial surfaces of the skin, the airways, the gut, or the genitourinary tract quickly spring to mind, but not the blood. However, the blood provides a major pathway for the entry and spreading of insect-borne pathogens, which include the causal agents of major diseases: malaria, leishmaniosis, Lyme disease, yellow fever, several forms of encephalitis, etc. The blood-derived DCs are ideally located to detect and initiate immune responses against these pathogens (Henri et al., 2002; Konecny et al., 1999; Perry et al., 2004). In addition, blood-derived DCs may play two additional functions. First, they may provide MHC–peptide ligands for recognition by naive T cells in the steady state, which may be required to maintain T-cell homeostasis (Sprent and Surh, 2003). Second, they may be responsible for the induction of peripheral

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tolerance by presenting self-antigens in an immature state (Finkelman et al., 1996; Hawiger et al., 2001; Steinman and Nussenzweig, 2002). However, we wish to point out that this does not discard the possibility that the tissuederived DCs that constantly reach the LNs in the steady state may also play a role in tolerance induction (Albert et al., 2001). Perhaps both (immature) blood-derived DCs and (mature) tissue-derived DCs can induce peripheral tolerance in the steady state, and only when the maturation process has been triggered by an inflammatory signal associated with infection or tissue damage do the DCs acquire an immunogenic phenotype. If this were the case, the phenotypic feature distinguishing tolerogenic from immunogenic DCs would not be the maturational status, but rather the ‘‘expression’’ of an additional signal induced only by activatory stimuli (Albert et al., 2001; Shortman and Heath, 2001). 2.3. Correlations and Gaps Between the Human and Mouse DC Systems For obvious reasons, the best-studied DC types in humans are those circulating in the blood and not those found in the lymphoid organs. Human blood contains two major DC groups equivalent to those found in mouse blood (Hart, 1997; Shortman and Liu, 2002). The human lymphoid organs also contain tissue-derived DCs (in the LNs) (Geissmann et al., 2002; Takahashi et al., 1998) and the two major groups of blood-derived DC: pDCs and conventional DCs (Bendriss-Vermare et al., 2001; McIlroy et al., 2001; Schmitt et al., 2000; Summers et al., 2001; Vandenabeele et al., 2001). However, whether the human conventional DCs comprise the equivalents of mouse CD4þ, CD8þ, and CD4
CD8
DCs is unknown. As in the mouse, the vast majority of the blood-derived DCs contained in the lymphoid organs of noninfected individuals have an immature phenotype (Bendriss-Vermare et al., 2001; McIlroy et al., 2001; Summers et al., 2001), and the number of mature DCs increases dramatically in patients suffering bacterial infections or multiple trauma (McIlroy et al., 2001). Therefore it is likely that blood-derived DCs in mice and humans follow a similar life cycle. 2.4. In Vitro-Generated DCs The development of methods to produce DCs in vitro from precursors found in mouse bone marrow (BMDCs) (Inaba et al., 1992) or spleen (D1DCs) (Winzler et al., 1997), and from monocyte precursors found in human blood (MoDCs) (Sallusto and Lanzavecchia, 1994), represented a major breakthrough for studies of DC biology and the development of DC-based immunotherapies. These methods enabled maintenance of large numbers of DCs

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 251 in culture in an immature state and provided the capacity to follow their developmental changes during maturation triggered by activatory signals added to the cultures. Most of the studies on antigen presentation by DCs have been performed on these DC models. However, the in vivo equivalent of the in vitro–generated DCs are unknown, so it is important to verify whether the findings of studies employing these DC models are applicable to DCs generated in vivo. 3. Antigen Presentation Pathways 3.1. Some Antigen Presentation Terminology To avoid confusion and to enable an informative discussion on the mechanisms that make DCs highly efficient antigen-presenting cells (APCs), we considered it would be important to define a number of often incorrectly used terms to categorize antigens. The term self will be used to refer to antigens that exist in a normal, uninfected individual, such as the proteins encoded by the genome or the products of the host’s metabolism. Model proteins such as ovalbumin (OVA) or hen egg lysozyme (HEL) expressed in transgenic mice fall into this category. We refer to antigens expressed by an infectious agent or to proteins injected in mice as foreign. We use the term endogenous to refer to components synthesized by the APCs themselves, as opposed to exogenous components, which are taken chiefly by endocytosis. Thus, viral antigens synthesized by an infected APC are ‘‘endogenous and foreign,’’ whereas a tissue antigen endocytosed by a DC is ‘‘self and exogenous,’’ and an endocytosed bacterium or injected ovalbumin (OVA) is ‘‘foreign and exogenous.’’ The terms cytosolic and endosomal, which should only be used to refer to the location where proteins normally perform their function within the cell, are sometimes used as synonymous with endogenous and exogenous, respectively, but we think this is misleading. It is true that most cytosolic proteins are endogenous, but it is incorrect to assume that all the contents of the endosomes are exogenous. In fact, these contents are predominantly endogenous, comprising membrane proteins that are delivered to lysosomal compartments at the end of their life span, or normal components of the endocytic route such as proteases, ATPases, etc. 3.2. Two Antigen Presentation Systems To obtain antigenic peptides for MHC presentation, APCs utilize the two major systems employed by eukaryotic cells to dispose of proteins. The first system is found in the endocytic route and comprises a large collection of

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proteases with variable substrate specificity and pH requirements (McGrath, 1999). Most of the endosomal proteases are collectively known as cathepsins. Endocytosed proteins, whether self or foreign, endogenous or exogenous, are degraded by these proteases in the endosomal compartments (Honey and Rudensky, 2003; Villadangos and Ploegh, 2000). In APCs, the resulting peptides can be loaded into the peptide-binding groove of MHC class II (MHC II) molecules and then presented on the plasma membrane for recognition by CD4þ T cells. When DC researchers talk about ‘‘developmental control of antigen presentation,’’ normally they are refering to the MHC II presentation pathway. This is one of the major themes in the sections that follow. The second major proteolytic system used by eukaryotic cells is the proteasome, a multimeric complex found in the cytosol that is composed of several proteolytic and regulatory subunits (Rock et al., 2002). The peptides generated by the proteasome can be translocated by the TAP transporter into the endoplasmic reticulum (ER), where they are loaded into the binding groove of newly synthesized MHC class I (MHC I) molecules. The resulting MHC I–peptide complexes then follow the default secretory pathway through the Golgi complex and are displayed on the plasma membrane for inspection by CD8þ T cells. With a few exceptions, all cells are capable of presenting antigens via MHC I molecules. In most APCs, the polypeptides that access the cytosol are synthesized by the APC itself, so most of the antigens presented by MHC I molecules are endogenous. A notorious exception to this rule is the cross-presentation pathway, which enables DCs and macrophages to present exogenous antigens via MHC I molecules. The process of antigen degradation by the proteasome (Rock et al., 2002), and the formation of MHC I–peptide complexes in the ER (Cresswell et al., 1999; Purcell, 2000), have been extensively reviewed elsewhere. It can be stated that DCs do not exhibit specific mechanisms for the control of presentation of endogenous antigens via MHC I molecules, so we refer the reader to those reviews for detailed discussion of the ‘‘classic’’ MHC I presentation pathway. In contrast, cross-presentation has special significance in the DC system and is described in detail after we review the mechanims that control MHC II antigen presentation. 4. Mechanisms and Regulation of Antigen Capture in DCs Whether it is for MHC II presentation or for MHC I cross-presentation, sampling of the environment plays a central role in the capacity of DCs to maintain peripheral tolerance to tissue-specific antigens and to activate immune responses against foreign pathogens. In this section we first describe the antigen-capture capabilities of immature DCs, followed by a review of the mechanisms suggested to regulate endocytosis during DC maturation.

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 253 4.1. Antigen Uptake in DCs Antigens can be endocytosed by a variety of mechanisms (Fig. 2) (Watts, 1997). Large particulates (bacteria, cells, or artifical beads) are often recognized by membrane receptors that trigger the formation of large endocytic vesicles (phagosomes), a process known as phagocytosis. Formation of the phagosome requires recruiting the actin cytoskeleton, which is necessary to mold the plasma membrane around the phagocytosed particle. The process of macropinocytosis is mechanistically similar to phagocytosis and also requires actin, but in this case the vesicle (a macropinosome) does not form around a particle; instead it simply engulfs a large portion of extracellular medium. Another mechanism to engulf extracellular medium is micropinocytosis, which does not require actin polymerization for vesicle formation (a pinosome), but instead requires the recruitment of the cytosolic protein clathrin to generate clathrin-coated pits. Finally, receptor-mediated endocytosis consists of the internalization of molecules recognized by specific membrane receptors, which also trigger the formation of clathrin-coated pinosomes. Uptake of material by macro- and micropinocytosis is often referred to as ‘‘fluid-phase’’ endocytosis to indicate that it is nonspecific rather than being triggered by particular molecular cues intrinsic to the endocytosed material. DCs were initially believed to have a low endocytic capacity because, as discussed above, the protocols used to isolate DCs from tissues stimulated their spontaneous maturation in vitro, in turn causing the downregulation of endocytosis (reviewed by Steinman and Swanson, 1995). It is now clear that DCs are highly efficient at all forms of endocytosis in their immature state. 4.1.1. Phagocytosis DCs can phagocytose bacteria (Rescigno, 2002), yeasts, hyphae (d’Ostiani et al., 2000), protozoans (Konecny et al., 1999), portions of live cells (Harshyne et al., 2001), and whole dying cells. Phagocytosis of apoptotic and necrotic cells has received particular attention in studies of DC function (for a review, see Heath et al., 2004). Whereas apoptosis is an ordered process used to eliminate unwanted cells, including those deleted during development and those at the end of their life span, necrotic death can be associated with pathological conditions such as viral infection. The ability to capture apoptotic and necrotic cells, and distinguish between these two forms of antigen, may enable DCs to present cell-associated antigens for the maintenance of tolerance (apoptotic cells) and for induction of immune responses (necrotic cells), respectively (Steinman et al., 2000). It has been postulated that not all DC types may possess the capacity to capture cell-associated antigens, suggesting a specialization in DC subset function based on variability in antigen uptake.

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Figure 2 Antigen uptake, processing, and presentation in the MHC II presentation pathway. Antigens are endocytosed by actin-dependent (macropinocytosis and phagocytosis) or clathrindependent (receptor-mediated endocytosis and pinocytosis) mechanisms. The internalized material then moves along the endocytic route, which can be subdivided into three major types of compartments: early endosomes (EE), late endosomes (LE), and lysosomes (Lys). The physicochemical conditions in these compartments are progressively more acidic, reducing, and proteolytic, leading to gradual degradation of the endocytosed antigens. Antigenic peptides are thus released along the entire endocytic route. Newly synthesized MHC II ab dimers are transported into the endocytic route in association with the chaperone Ii, which associates with the MHC II molecules by inserting its CLIP region into the peptide-binding site contained in the ab dimer. The cytosolic tail of Ii carries a sorting motif for delivery of the abIi complexes into EEs directly from the trans-Golgi network or after transient expression on the cell surface. Once the ab–Ii complexes reach the endocytic route, Ii is degraded in a stepwise fashion. The protease asparaginyl endopeptidase (AEP) may be involved in converting full-length Ii into Iip10, which is then degraded by cathepsin (Cat) S to generate CLIP [one of the major intermediate steps (the ab–Iip22 complex) has been removed for simplicity]. The chaperone H-2DM then promotes the exchange of CLIP for antigenic peptides. The conversion of ab–Ii into ab–CLIP has been drawn between brackets in all the endosomal compartments to represent that one or more of the steps involved in this process can occur at several stations of the endocytic route. Therefore the ab–peptide complexes can be generated in EEs, LEs, or Lys, and be transported from all these compartments to the cell surface, although the majority are probably generated in LEs.

Indeed, several groups have reported that only the CD8þ DCs can phagocytose and present cell-associated antigens (Iyoda et al., 2002; Schulz and Reis e Sousa, 2002; Valdez et al., 2002). However, the studies by Thomson’s group (Morelli et al., 2003) and our own unpublished results indicate that CD8
DCs

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 255 are also capable of capturing and presenting cell-associated antigens. Whether these discrepancies are due to differences in the cellular antigens analyzed or the methods of assessment used by the different studies is yet to be established. 4.1.2. Macropinocytosis Macropinocytosis is constitutive in immature DCs, whereas in other cells such as macrophages it must be induced by phorbol esters or growth factors (Swanson and Watts, 1995). It has been estimated that immature DCs can internalize by macropinocytosis the equivalent of their own volume in 60 min (Sallusto et al., 1995). To maintain a steady uptake of such a large volume of extracellular fluid, immature DCs rely on regulated expression of several members of the aquaporin family (de Baey and Lanzavecchia, 2000), membrane channels that facilitate the elimination of excess water across the endosomal membranes (Engel et al., 2000). Engulfment of large portions of extracellular volume followed by release of excess water thus results in highly efficient concentration of solutes in the endosomal compartments of immature DCs. 4.1.3. Receptor-Mediated Endocytosis Receptor-mediated endocytosis is another strategy of antigen capture fully exploited by DCs. DCs express a multitude of receptors that they may use to take up different forms of antigens. These include Fc receptors (den Haan and Bevan, 2002; Fanger et al., 1996; Ravetch and Bolland, 2001; Sallusto and Lanzavecchia, 1994), the immunoglobulin-like molecule ILT3 (Cella et al., 1997a), and members of the C-type lectin family such as the mannose receptor (Sallusto et al., 1995), DEC-205 (Jiang et al., 1995; Mahnke et al., 2000), DCSIGN (van Kooyk and Geijtenbeek, 2003), langerin (Valladeau et al., 2000), and CIRE (Caminschi et al., 2001). Strikingly, the expression pattern of several of these putative antigen receptors varies among DC subsets, suggesting that the DC populations may be specialized at presenting antigens derived from different sets of pathogens (Caminschi et al., 2001; Linehan et al., 1999; Mommaas et al., 1999; Valladeau et al., 2000; Vremec and Shortman, 1997). In addition, some of these receptors can deliver activatory or inhibitory signals, so they may have a dual role as both antigen receptors and modulators of the immune response (Chang et al., 2002; Chieppa et al., 2003; den Haan and Bevan, 2002; Regnault et al., 1999; van Kooyk and Geijtenbeek, 2003). Such receptors could thus be considered ‘‘pathogen recognition receptors’’ (Janeway, 1989) akin to the members of the Toll-like receptor (TLR) family (Medzhitov, 2001). Unfortunately, the putative pathogen ligand(s) for ILT3 and several of the C-type lectins expressed by DCs are still unknown, so it

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remains unclear whether all these molecules have a role in antigen capture in vivo; alternative roles in mediating intercellular interactions or adhesion to extracellular matrix components are also possible (van Kooyk and Geijtenbeek, 2003). Characterizing the role of these receptors and their ligands is important, because they may provide suitable targets for the induction of immunogenic or tolerogenic reactions in vivo (Hawiger et al., 2001; Mahnke et al., 2003). 4.2. Control of Endocytosis in DCs Activation of in vitro-cultured DC leads to a drastic downregulation of phagocytosis and macropinocytosis (Sallusto et al., 1995; Winzler et al., 1997). The groups of Mellman and Watts identified two members of the Rho family of GTPases, cdc42 and rac1, respectively, as key regulators of this process (Garrett et al., 2000; West et al., 2000), but it is still unknown which are the upstream and downstream factors that control the activity of these two GTPases during DC maturation. Endocytosis via some of the antigen receptors is also downregulated during DC maturation because of decreased expression of the receptors (Sallusto et al., 1995; Stoitzner et al., 2003; Valladeau et al., 2000; Winzler et al., 1997). These observations have led to the assumption that mature DCs shut down all forms of endocytosis, but this is incorrect. Pinocytosis still occurs in mature DCs at a comparable rate to immature DCs. This point is illustrated by an approximately equal number of clathrin-coated pinosomes in immature and mature DCs (Garrett et al., 2000; West et al., 2000). In fact, the turnover rate of the bulk of the plasma membrane proteins of immature and mature DCs is comparable (Wilson et al., 2004); since degradation of membrane proteins occurs primarily in the endocytic route, this indicates that the rate of internalization and degradation of plasma membrane components does not substantially decrease on maturation. Furthermore, mature DCs still internalize substantial amounts of soluble proteins, which are most likely endocytosed by micropinocytosis or receptor-mediated endocytosis (Pure et al., 1990; Winzler et al., 1997). Supporting the concept that mature DCs are still proficient at capturing certain antigens, not all the putative antigen receptors are downregulated during DC maturation; for instance, DEC-205 is upregulated, even in DC populations that do not express this lectin in their immature state (Inaba et al., 1994; Vremec and Shortman, 1997). These observations imply that the inability of mature DCs to process and present soluble antigens via the MHC II pathway is not due to impaired uptake. As we describe below, this inability is in fact due to downregulation of MHC II synthesis and therefore a lack of available peptide-receptive molecules. Why then are some antigen receptors still expressed in mature DCs? One reason might be to provide antigens for

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 257 cross-presentation, because mature DCs do not downregulate MHC I synthesis. On the other hand, perhaps those receptors that are not downregulated on maturation are not, after all, dedicated to antigen presentation but rather to adhesion to other cell receptors or extracellular components. This point stresses the need to identify the natural ligands of the putative antigen receptors expressed by DCs. Adding further confusion, it is unclear whether the decrease in phagocytosis and macropinocytosis observed in in vitro–generated DCs is reflected in their in vivo counterparts. Ruedl and Itano have shown that mature skin-derived DCs are still highly endocytic (Itano et al., 2003; Ruedl et al., 2001), an observation confirmed by ourselves (unpublished observations). However, LCs isolated from epidermal sheets and cultured in vitro downregulate phagocytosis (Reis e Sousa et al., 1993). One possible explanation for these discrepancies might be that phagocytosis is regulated independently of the other phenotypic features that define DC maturation, namely the antigenpresenting and T-cell–stimulatory capacities. If this is the case, DCs may downregulate phagocytosis in vivo only if they mature as a response to activatory signals, but not when they mature in the steady state (Itano and Jenkins, 2003; Ruedl et al., 2001). Finally, it is important to note that measuring endocytosis in vivo with tracers is prone to the influence of DC location within the lymphoid organs and peripheral tissues. For instance, tracers inoculated in the skin are inefficiently captured by blood-derived DCs, but the same tracers injected intravenously are preferentially captured by the blood-derived DCs and not the tissue-derived DCs (Itano et al., 2003; Salomon et al., 1998). This probably reflects different pathways followed by the tracers to enter the lymphoid organ (lymph versus blood), which dictates which DC populations will have access to the tracer. In addition, based on a highly organized conduit system, the lymphoid organs impose limits to the size of the particles or molecules that can access different regions (Gretz et al., 1997, 2000; Kaldjian et al., 2001; Nolte et al., 2003). Since the distribution of DC subsets varies (De Smedt et al., 1996; Pulendran et al., 1997), some DC types may be unable to capture certain antigens, not because they lack the capability, but because they do not have access to the antigens (Itano and Jenkins, 2003). 5. Antigen Presentation by MHC II Molecules Formation of MHC II–peptide complexes is an intricate process that entails the orchestration of multiple cellular processes, including protein-sorting mechanisms, proteolytic activities, the intervention of chaperones, etc. Many of these mechanisms have been reported to be regulated in DCs in a manner

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distinct from other APCs. Although the process of MHC II antigen presentation has been reviewed extensively (Sercarz and Maverakis, 2003; Villadangos, 2001; Watts, 1997; Wolf and Ploegh, 1995), in order to discuss the contentious area of how this process is regulated in DCs, it is necessary to recapitulate the major steps involved in this pathway. 5.1. The Endocytic Pathway Recounting the process of MHC II antigen presentation requires a description of the subcellular structures where formation of the MHC II–peptide complexes takes place: the endocytic pathway (Gruenberg, 2001; Mellman, 1996). This pathway is a complex network of tubulovesicular structures, which can be visualized as a major road spanning from the plasma membrane to the lysosomes. This road is intersected by secondary incoming and outgoing tracks (Fig. 2). The incoming tracks represent vesicles originating mostly from the cell surface and the distal components of the Golgi complex, and the exit tracks denote tubules and vesicles that eventually will fuse with the plasma membrane. The contents of the endosomal compartments change gradually along the major track and therefore it is not possible to identify clear boundaries dividing the endocytic pathway, but for the sake of simplicity, three major regions are usually distinguished. First, the early endosomes (EEs), whose limiting membrane and lumen have a similar composition to the plasma membrane and the extracellular medium, respectively; second, the late endosomes (LEs), which already contain many components found only in the endocytic route such as proteases, and are more acidic than the EE; third, the lysosomes, which are considered the final station of the endocytic pathway, are acidic and highly proteolytic. Endocytosed material moves predominantly along the EE–LE–lysosomes axis toward the lysosomes. As a rule, the number of entry and exit tracks along the major axis is most abundant in the EEs and least in the lysosomes, but how any given protein accesses the endocytic route, and how it leaves, is determined by sorting mechanisms that are still poorly understood. For example, the transferrin receptor cycles continuously from the plasma membrane to the EEs and back to the surface (Mellman, 1996); the DC lectin DEC-205 transports antigens bound at the plasma membrane to LEs and lysosomes, releases the antigens, and then returns to the cell surface (Mahnke et al., 2000); and the endosomal proteases are delivered to the endocytic route directly from the Golgi and are largely retained in endocytic compartments (Chapman et al., 1997). In the case of transmembrane proteins, the ‘‘address code’’ that determines how the protein will enter the endocytic route, whether it will leave and how, can be encoded in its cytoplasmic region (Kirchhausen

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 259 et al., 1997). These address codes are probably recognized by cytosolic proteins that regulate protein sorting, but little is known about how the information carried by the transmembrane region is interpreted by the sorting machinery, or how the machinery works (Gruenberg, 2001). It is important to point out two features of the endocytic route that are critical in the context of antigen presentation. First, the sorting mechanisms allow for some degree of ‘‘leakiness,’’ so that, for example, the cathepsins can be both secreted and delivered to endosomal compartments (LennonDumenil et al., 2001; Wolters and Chapman, 2000). Indeed, the MHC II molecules are remarkably flexible and follow multiple entry and exit tracks of the endocytic pathway (see below). The second feature to consider is that the endocytic route is not always ‘‘linear,’’ as otherwise suggested by our simplified description of the relationships between the different compartments. Rather, it is possible that some compartments ‘‘branch off,’’ becoming distinct reservoirs for endocytosed material, or parallel subpathways with different dynamics and composition than those of the main EE–LE–lysosomal track. The significance of such branching off is discussed later in the context of MHC II antigen presentation and MHC I cross-presentation. 5.2. Antigen Degradation Whatever mechanism is used for their internalization (see above), antigens progress along the EE–LE–lysosomal axis, where they are exposed to an increasingly more acidic, reducing, and proteolytic environment (Fig. 2; Bryant et al., 2002; Honey and Rudensky, 2003; Villadangos and Ploegh, 2000). The result is a gradual release of polypeptides along the entire endocytic pathway, a few of which will contain the correct amino acid sequence that permits them to lodge into the peptide-binding sites of the particular MHC II allotypes expressed by the APC (Engelhard, 1994). The objective of the MHC II molecules is to capture the most diverse array of peptides possible in order to display the largest amount of antigenic information to CD4þ T cells (Villadangos, 2001). 5.3. Formation of the MHC II–Peptide Complexes Newly synthesized MHC II ab dimers are cotranslationally translocated into the endoplasmic reticulum (ER). However, the peptides that the MHC II molecules bind are not brought into the ER as is the case for the peptides presented by MHC I; instead, the MHC II molecules need to be transported to the endosomal compartments. To enhance their efficiency as peptide binders, the MHC II molecules have evolved a safety mechanism that prevents

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their expression as empty dimers on the cell surface: they are unstable and have the propensity to aggregate if their peptide-binding cavity is empty (Bonnerot et al., 1994; Ceman et al., 1998; Germain and Rinker, 1993; Zhong et al., 1996). In addition, since the peptide-binding site is highly promiscuous, it can promptly associate with other polypeptides contained in the ER (Busch et al., 1996), a process that would preclude ab dimers from acquiring peptides in the endosomes. To prevent this early inactivation and to stabilize the conformation of the ab dimer, the MHC II molecules are synthesized in an inactive ‘‘proform’’ in which their peptide-binding cleft is occupied by the chaperone invariant chain (Ii) (Fig. 2). The Ii also contains in its cytosolic portion a sorting motif that is recognized in the trans-Golgi network as a signal to haul the ab–Ii complexes out of the secretory pathway and into the EE/LE regions of the endocytic pathway (Bakke and Dobberstein, 1990; Lotteau et al., 1990; Pieters et al., 1993). Once the MHC II–Ii complexes reach the endoctytic compartments, the ab dimers must eliminate Ii to regain their capacity to bind antigenic peptides. This process occurs in several steps. First, several proteases progressively cleave Ii, producing its major degradation intermediates Iip22, Iip10, and finally the CLIP fragment, a stretch of amino acids that occupies the MHC II peptide-binding cleft (reviewed in Bryant et al., 2002; Honey and Rudensky, 2003; Villadangos and Ploegh, 2000). Only one of these proteolytic reactions has been attributed to a particular enzyme: the cleavage of Iip10, which in DCs is accomplished by cathepsin S. The importance of cathepsin S has been demonstrated in vivo by the analysis of knockout mice (Nakagawa et al., 1999; Shi et al., 1999), although the impact of the cathepsin S deficiency on MHC II peptide binding is strongly dependent on the particular MHC II allelic variant (allotype) expressed by the APC (see More Exceptions than Rules, below) (Nakagawa et al., 1999; Riese et al., 2001; Villadangos et al., 1997, 2001). A study by the Watts group suggests that the protease asparaginyl endopeptidase (AEP) may be responsible for the initial cleavage of full-length Ii (Manoury et al., 2003), but the role of this enzyme in vivo awaits confirmation in AEP-deficient animals. The conversion of Iip10 into CLIP has special significance, because it releases the ab dimers from the cytoplasmic portion of Ii, whose endosomal targeting motif acts as a retention signal that prevents the ab dimers associated with Ii, Iip22, or Iip10 from escaping to the cell surface (Amigorena et al., 1995; Brachet et al., 1997; Driessen et al., 1999; Neefjes and Ploegh, 1992; Pierre and Mellman, 1998). Strikingly, interfering with the kinetics of Ii destruction can have profound effects not only on MHC II trafficking, but also on the dynamics of endosomal maturation (Gorvel et al., 1995; Gregers et al., 2003; Nordeng et al., 2002; Pieters et al., 1993; Romagnoli and Germain,

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 261 1994). Thus, in DCs that cannot convert the ab–Iip10 complex into ab–CLIP because of the lack of cathepsin S, the number of endosomal vesicles increases dramatically (Driessen et al., 1999; and our unpublished observations) and trafficking of other components of the endocytic pathway is indirectly affected (Riese et al., 2001). This phenomenon may be due to excessive coating of endosomes expressing Iip10 with the chaperone Hsc70, which associates with the tail of Ii on the cytosolic face of the endosomes (Lagaudriere-Gesbert et al., 2002). Although it is still unclear how Hsc70 influences endosome biogenesis and maturation, the consequences of Iip10 accumulation in cathepsin Sdeficient APCs suggest that the role of Ii may not be solely to control MHC II trafficking and peptide loading but also to act as a master regulator of the endocytic route in APCs. By linking Ii degradation with endosomal trafficking, the APCs may be able to orchestrate the timing of generation of peptidereceptive MHC II dimers with the gradual degradation of endocytosed antigens (Lagaudriere-Gesbert et al., 2002). Once the MHC II–CLIP complex has been generated, the CLIP peptide must be substituted for antigenic peptides. This reaction involves the chaperone H-2DM and (in B cells) its close relative H-2DO, which interact transiently with MHC II–CLIP, destabilizing the complex and facilitating the release of CLIP (reviewed in Alfonso and Karlsson, 2000). This enables antigenic peptides that contain the correct combination of ‘‘anchor residues’’ to associate with the now vacant peptide-binding groove (Engelhard, 1994). The newly formed MHC II–peptide complexes can then be sorted from the endocytic route toward the plasma membrane in transport vesicles (Fig. 2). Since the conversion of Iip10 into CLIP releases the MHC II complex of the Ii cytoplasmic tail, this proteolytic step must be quickly followed by the exchange of CLIP for antigenic peptides. Indeed, in H-2DM–deficient APCs most of the surface MHC II molecules remain associated with CLIP (Fung-Leung et al., 1996; Martin et al., 1996; Miyazaki et al., 1996). H-2DM not only assists in removing CLIP, it also acts as a peptide editor, promoting the exchange of lowaffinity peptides for high-affinity peptides (Jensen et al., 1999; Katz and Sant, 1994; Kropshofer et al., 1996; van Ham et al., 1996). Controlling the abundance or location of H-2DM in the endosomal compartments may thus act as a rheostat regulating the stability of the MHC II–peptide complexes generated by the APCs (Kropshofer et al., 1997b). 5.4. Where Does Peptide Binding Occur? Much of the research performed on MHC II antigen presentation has been directed toward characterizing the compartments of the endocytic route where the MHC II molecules acquire their peptide cargo (Amigorena et al., 1994;

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Kropshofer et al., 1997b; Peters et al., 1991; Qiu et al., 1994; Tulp et al., 1994; West et al., 1994; reviewed in Geuze, 1998; Neefjes, 1999). Today, the accepted view is that just as antigens are degraded gradually along the entire EE–LE–lysosomal track, MHC II molecules become receptive to binding antigenic peptides, or polypeptide precursors, at all the stations of the endosomal pathway (Castellino and Germain, 1995; Driessen et al., 1999; Villadangos et al., 2000). Likewise, MHC II–peptide complexes can exit from all compartments to the plasma membrane (Driessen et al., 1999). This ability enables MHC II molecules to sample peptides from the entire endocytic route, including peptides that may only be present in EEs because they do not survive the harsher conditions of LEs or lysosomes, and also peptides that require thorough degradation of their polypeptide precursors late in the endocytic route (Sercarz and Maverakis, 2003; Villadangos, 2001; Watts, 1997; Wolf and Ploegh, 1995). Importantly, the spatial organization of the mechanisms employed by MHC II molecules to acquire their peptide cargo implies that all the peptides derived from any endocytosed protein, whether endogenous or exogenous, self or foreign, are included together in a single antigenic peptide pool. Therefore, any mechanism controlling antigen presentation that nonspecifically interferes with antigen degradation, or with the formation of peptide-receptive MHC II molecules, should affect the presentation of the entire pool. This concept is important when we later evaluate the hypotheses on how MHC II antigen presentation is regulated in immature and mature DCs. 5.5. More Exceptions than Rules Although our description of the conversion of MHC II–Ii into MHC II– peptide complexes suggests a rigid sequence of events, this process has evolved considerable flexibility. Much of the flexibility is imparted by the effect that MHC polymorphism has on the affinity of the interaction between the peptide-binding cleft and CLIP. This in turn causes variability in the requirements of each allotype for accessory molecules. For instance, I-Ab molecules are highly dependent on association with Ii for conformational stability and eggress out of the ER, but I-Ak can be transported to the cell surface as empty ab dimers in the absence of Ii (Bikoff et al., 1995; Rovere et al., 1998); lack of cathepsin S impairs conversion of MHC II–Iip10 into MHC II–peptide complexes in APCs expressing I-Ab or I-Ad, but Iip10 associates loosely with I-Ak, I-Aq, I-Au, or I-As, enabling these allotypes to acquire peptide cargo even when cathepsin S has been inactivated (Honey et al., 2002; Nakagawa et al., 1999; Riese et al., 2001; Villadangos et al., 1997, 2001); likewise, CLIP can be removed from some allotypes without the assistance of H-2DM (Avva and

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 263 Cresswell, 1994; Bikoff et al., 2001; Brooks et al., 1994; Koonce et al., 2003; Stebbins et al., 1995, 1996; Wolf et al., 1998). Probably these differences allow each MHC II allelic product to acquire its peptide-binding capacity at different positions along the endocytic route, in which the concentration of cathepsin S and H-2DM vary. Since, in normal populations, each individual can express several combinations of I-A and I-E (or HLA-DR and HLA-DP) allotypes, this diversity probably contributes to increase the capacity of each individual to respond to a variety of antigenic challenges (Villadangos, 2001). Another source of plasticity in the MHC II presentation pathway is a degree of ‘‘leakiness’’ in most of the steps described above. Thus, some MHC II–Ii complexes are not delivered directly to the endocytic route but are transiently exported to the plasma membrane and then internalized (Fig. 2; Benaroch et al., 1995; Koch et al., 1991; Saudrais et al., 1998; Wraight et al., 1990). In addition, some peptides can displace the Ii degradation intermediates Iip22 or Iip10 and not just CLIP (Denzin et al., 1996; Kropshofer et al., 1997a; Sanderson et al., 1996; Stebbins et al., 1996). Finally, Ii can be eliminated from a fraction of MHC II–Ii complexes in EEs in a reaction independent of cysteine proteases and H-2DM; a process still poorly understood (Villadangos et al., 2000). All these factors contribute to increase the number of different endocytic compartments where ab dimers can access the peptide currency, thereby enlarging the repertoire of antigenic peptides that APCs can present to CD4þ T cells (Villadangos, 2001). 6. Control of MHC II Antigen Presentation in DCs The studies that have addressed how MHC II antigen presentation is controlled in DCs during their maturation have tried to answer three major questions. First, why do mature DCs express more MHC II molecules on their surface than immature DCs? Second, what causes the striking difference in subcellular distribution of MHC II between immature and mature DCs (Fig. 1C)? Finally, and most importantly, how do the mechanisms that control antigen presentation in DCs translate into the functional antigen-presenting properties of immature and mature DCs. Immature DCs efficiently capture antigens, but they are poor at displaying antigenic peptides at the plasma membrane. In contrast, mature DCs show extended presentation of a ‘‘memory’’ (or a ‘‘snapshot’’) of the antigens they captured at the time of activation, but they are incapable of presenting subsequently encountered antigens. How are these changes regulated? Several mechanisms have been proposed in an attempt to answer the above questions, and in summary they can all be broadly assigned to one of two models (Fig. 3). The first suggests that immature DCs are inefficient in their

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Figure 3 Two models of developmental control of antigen presentation in DCs. In the gain of function model, immature DCs have impaired formation of ab–peptide complexes as a result of poor antigen degradation, inhibition of Cat S, and/or impairment of H-2DM function. Most of the MHC II molecules are thus retained in endosomal compartments and degraded. Activation allows formation of MHC II–peptide complexes that are then transported to the plasma membrane, where they are long-lived. In the interruption model, immature DCs constitutively generate MHC II–peptide complexes and transport them to the cell surface, but then they are endocytosed and degraded. Activation downregulates the rate of MHC II–peptide internalization from the plasma membrane. See Fig. 4 for a more detailed description of the interruption model.

ability to generate MHC II–peptide complexes. On receiving an activation signal, the now ‘‘maturing’’ DCs rapidly gain the capacity to create and present MHC II–peptide complexes. We refer to this model as the ‘‘gain of function’’ model, for it suggests that DCs gain the capacity to process and present antigens during maturation. The second model supports the concept that immature DCs constitutively generate MHC II–peptide complexes and present them transiently on their surface, but the complexes are then rapidly endocytosed and destroyed. According to this model, during maturation, newly generated MHC II–peptide complexes are retained on the plasma membrane, that is, MHC II–peptide turnover is ‘‘interrupted’’ (Fig. 3). Here

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 265 we review the experimental data produced by different groups to analyze MHC II antigen presentation in DCs, assessing whether the results obtained support the gain of function or the interruption model. 6.1. Control of Endosomal Proteolysis in DCs Control of antigen degradation has been proposed as one mechanism used by DCs to regulate MHC II antigen presentation. Low proteolytic activity in the endosomal route of immature DCs could limit the availability of peptide ligands for MHC II molecules, causing impaired antigen presentation. This hypothesis is supported by two studies that found that the overall activity of endosomal cysteine proteases, measured using active site-labeling techniques, increased during maturation in human monocyte–derived DCs (MoDCs) and mouse bone marrow–derived DCs (BMDCs) (Fiebiger et al., 2002; Trombetta et al., 2003). The increase in activity was not simply caused by augmented protease expression in mature DCs because the total amount of the enzymes did not vary significantly on maturation. These studies also showed, using pHsensitive fluorescent endocytic tracers, that the average pH in the endocytic route of mature DCs was more acidic than in the immature DCs, consistent with a low-pH optimum for the majority of endosomal proteases. Therefore, acidification of the endosomal compartments could explain the increase in protease activity in mature DCs. To further support this, the study by Trombetta and colleagues showed that some components of the vacuolar Hþ-ATPase (V-ATPase), the pump responsible for lysosomal acidification, were recruited from the cytosol to the endosomal compartments on DC maturation, suggesting an active mechanism of control of the endosomal pH (Trombetta et al., 2003). Modulating the endosomal pH can also affect the kinetics of endosomal transport: inhibitors of the V-ATPase, such as concanamycin A and B, impair trafficking of MHC II and other components of the endocytic pathway from EEs to LEs and from LEs to lysosomes (Benaroch et al., 1995; van Weert et al., 1995; Villadangos et al., 2000; Yilla et al., 1993). Together these studies suggest that DCs may control endosomal acidification as an elegant mechanism to orchestrate antigen internalization and degradation. On the other hand, considering the importance of endosomal proteolysis in cell metabolism, it might be expected that the activity of the endosomal compartments of immature DCs should still be sufficient to degrade the proteins that are normally eliminated via the endosomal route. This considered, is such ‘‘basal activity’’ sufficient to process antigens in immature DCs? Several studies have suggested that DCs are indeed inefficient at processing antigens until they receive a maturation stimulus. This hypothesis is supported by microscopy and biochemical measurements of the rate of degradation of

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some exogenous proteins. For instance, hen egg lysozyme (HEL) and horseradish peroxidase (HRP) were still detectable in endocytic vesicles of immature BMDCs up to 2 days after their internalization, and their rates of degradation were estimated by Western blot to be less that 25% in 19 h (Trombetta et al., 2003). Activation of the BMDCs led to rapid degradation of the internalized HEL and HRP. These results support the concept that the proteolytic activity in the endosomal compartments of immature BMDCs is low, and this activity is rapidly upregulated on maturation. But are HEL and HRP representative of the bulk of the proteins endocytosed by immature BMDCs? The BMDCs are cultured in medium supplemented with fetal calf serum (FCS), which typically contains approximately 40 mg of protein per milliliter (yielding a concentration of 4 pg/pl in culture medium supplemented with 10% FCS). Since the volume of extracellular medium internalized by one immature DC has been estimated as 3 pl/h (Sallusto et al., 1995), the amount of protein internalized by immature BMDCs in culture can be estimated to be about 12 pg/h. This is likely an underestimate, because it does not consider the amount of protein contained in other cells or cell debris present in the culture, which presumably are also phagocytosed. If the rate of degradation of the bulk of the internalized proteins were that observed for HEL or HRP (roughly 25%/day), immature BMDCs would accumulate over 200 pg of protein per day. Since the protein content of one whole cell has been estimated as 200 pg (see, e.g., Princiotta et al., 2003), this rate of accumulation would obviously be unsustainable. A similar argument can be made for immature DCs constitutively sampling their environment in vivo. Clearly, the half-life of the bulk of the proteins endocytosed by immature BMDCs must be much shorter than that observed for HEL or HRP. Indeed, bovine serum albumin (BSA), which constitutes approximately 60% of the total protein contained in FCS, had a half-life of less than 3 h in immature BMDCs (Trombetta et al., 2003). In fact, immature and mature BMDCs degraded this protein with comparable kinetics (Trombetta et al., 2003). Thus, if BSA were used as a reference model instead of HEL or HRP, it would have to be concluded that the endosomal proteolytic activities of immature and mature DCs are both high and comparable. In an attempt to obtain a more comprehensive assessment of such activity, we examined the turnover rate of plasma membrane proteins, which are mostly degraded in endosomal compartments following internalization. We observed that, with the notorious exception of MHC II molecules (see below), immature and mature DCs degraded most of their plasma membrane proteins with comparable kinetics (Wilson et al., 2004). Moreover, the half-life of the bulk of the plasma membrane proteins was comparable to that of BSA (a few hours) (Wilson et al., 2004). Therefore it can be concluded that the long half-life of HEL and HRP represents an

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 267 exception rather than the rule of protein processing in the endosomal compartments of immature DCs. An explanation for the exceptional resilience of HEL and HRP to endosomal degradation might be their resistance to unfolding, which in turn may be conferred by the disulfide bonds contained in these two proteins. It would be interesting to test whether immature and mature DCs degrade the denatured forms of HEL and HRP with comparable kinetics. If they did, this would suggest that immature DCs are less capable of unfolding native proteins, perhaps due to lower expression of disulfide reductases such as IP30/GILT (Arunachalam et al., 2000; Luster et al., 1988; Maric et al., 1994). An alternative mechanism that may explain the long half-life of some endocytosed proteins in immature DCs could be the existence of ‘‘storage’’ compartments with low proteolytic activity (Lutz et al., 1997). Such compartments would represent ‘‘cul-de-sacs’’ branching off the main endocytic track, where a small fraction of the endocytosed material could be stored for later processing, while the remaining protein mass progresses to the lysosomes and is degraded. Some proteins such as HEL or HRP may perhaps be preferentially sorted from the major endocytic pathway into these compartments, thus explaining their unusual long life in immature DCs. Activation signals could induce recruitment of proteases into the storage compartments. This hypothesis is supported by a study by Driessen and colleagues, which showed that DC activation induces protease recruitment from lysosomes into late endosomes (Lautwein et al., 2002). However, the existence of these storage compartments remains to be verified by other independent studies. To complicate this picture further, a report by Lennon-Dumenil and collaborators found that when DCs receive a maturation signal, they downregulate the proteolytic activity of the endosomal compartments encountered by the endocytosed antigens (Lennon-Dumenil et al., 2002). This conclusion is in apparent contradiction with the studies mentioned above and suggests that mature DCs degrade endocytosed antigens slower than immature DCs. However, the analysis by Lennon-Dumenil and colleagues used an active protease probe linked to latex beads to directly assess the proteolytic activity of the compartments traversed by an endocytic tracer, whereas the former studies measured protease activity in cell lysates or disrupted subcellular fractions. In addition, this study measured the changes in endosomal proteolysis at early stages of the maturation process. Therefore, it is possible that although the overall proteolytic activity of the endosomal route increases in mature DCs, these gross changes are not uniformly distributed in all the endocytic compartments. We favor the concept that immature DCs do not accumulate endocytosed proteins, because these would constitute a liability when the DCs encounter a pathogen. If immature DCs were constantly accumulating endocytosed

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proteins, their endosomal compartments would be ‘‘saturated’’ with self-proteins. As a result, a small amount of foreign antigen captured in the presence of an activatory signal would need to compete with a pool of preendocytosed selfproteins for binding to the available MHC II molecules. A more plausible scenario is that immature DCs quickly dispose of the proteins they endocytose in the steady state, so that a foreign antigen captured in the context of an immune challenge can achieve a high relative representation on MHC II molecules, with minimum interference from self-proteins. In this situation, a reduction in proteolytic activity in the compartments containing the foreign antigen would provide a more controlled environment for antigenic processing, favoring the preferential loading of newly synthesized MHC II molecules with foreign peptides (see below). In conclusion, while the proteolytic activity of the endocytic route undergoes some changes during DC maturation, which are likely to affect the processing of certain antigens, endosomal proteases contained in immature DCs have adequate activity to process antigens in the steady state. The question that remains is whether the resulting antigenic peptides generated in immature DCs are loaded and presented by MHC II molecules. 6.2. Formation of MHC II–Peptide Complexes in DCs Indeed, whether immature and mature DCs regulate antigen presentation at the level of formation of MHC II–peptide complexes has been a contentious issue. Some reports conclude that immature DCs are inefficient at generating peptide-receptive MHC II molecules and therefore cannot present antigens (Inaba et al., 2000; Kleijmeer et al., 2001; Pierre and Mellman, 1998; Turley et al., 2000), whereas others reach the opposite conclusion (Cella et al., 1997b; Colledge et al., 2002; El-Sukkari et al., 2003; Pierre et al., 1997; Veeraswamy et al., 2003; Villadangos et al., 2001; Wilson et al., 2004). Determining whether immature DCs can present antigens is central to understanding their putative role in maintaining tolerance to self-antigens, a role that would obviously require them to present those antigens. In this section we describe experimental approaches that have been employed to analyze the formation of MHC II–peptide complexes in DCs and the conclusions from such studies. 6.2.1. Is Ii Degradation a Regulatory Mechanism in DC Antigen Presentation? The first study that suggested the generation of MHC II–peptide complexes in DCs was developmentally regulated reported a difference in degradation of Iip10 between immature and mature mouse BMDCs (Pierre and Mellman,

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 269 1998). Immature DCs contained more Iip10 in their endocytic compartments than mature DCs, suggesting that immature DCs were inefficient at degrading Iip10. Since Iip10 cleavage is a prerequisite step in MHC II–peptide complex formation (see above and Fig. 2), deficient Iip10 processing would impair antigen presentation by immature DCs. Moreover, because the MHC II–Iip10 complexes carry the endosomal retention signal contained in the cytosolic portion of Ii, the complexes would accumulate in endosomal compartments, consistent with the observations of microscopy analysis of MHC II distribution in immature DCs (Fig. 1). This study also reported the presence of the cysteine protease inhibitor cystatin C (Abrahamson, 1994) in the endosomal compartments of immature DCs and suggested that cystatin C prevented Iip10 degradation by blocking the activity of cathepsin S (Fig. 3). Cystatin C disappeared from the endosomal route of mature DCs, which led to the hypothesis that on DC activation cathepsin S would become active and degrade Iip10, allowing the production of MHC II–peptide complexes (Pierre and Mellman, 1998). These observations supported an attractive gain of function model to explain how DCs regulate antigen presentation, with cystatin C acting as the key switch. However, this study had a potential caveat: measurement of Iip10 content was performed by Western blot analysis of whole cells or cell fractions, not from immunoprecipitated MHC II molecules. Therefore, the relative amounts of MHC II molecules that were associated with Iip10 in immature and mature DCs were not determined. This was relevant because DCs downregulate MHC II synthesis on maturation (see below; and Cella et al., 1997b; Kampgen et al., 1991; Pure et al., 1990; Rescigno et al., 1998; Villadangos et al., 2001; Wilson et al., 2004). Therefore, immature DCs are at any time synthesizing and processing more MHC II molecules than mature DCs (a 3-fold difference in BMDCs). This implies that immature DCs also contain a larger number of processing intermediates (including MHC II–Iip10 complexes) than mature DCs, even though they are degrading Iip10 normally. In fact, we have shown by Western blot that most of the MHC II molecules contained at any time within immature BMDCs, D1DCs, or lymphoid organ DCs are associated with peptides, not with Iip10 (El-Sukkari et al., 2003; Villadangos et al., 2001; Wilson et al., 2004). Therefore, the accumulation of MHC II molecules in the endosomal compartments of immature DCs (Fig. 1) cannot be attributed to their association with Iip10. The concept that the developmental control of antigen presentation is regulated in DCs independently of the rate of Iip10 degradation was also suggested by reports examining ‘‘cathepsin S–independent’’ MHC II allotypes (see above). These allotypes (which include I-Ak, I-Aq, I-Au, and I-As) can convert MHC II–Iip10 into MHC II–peptide even in cathepsin S–deficient

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DCs (Honey et al., 2002; Nakagawa et al., 1999; Riese et al., 2001; Villadangos et al., 1997, 2001). If MHC II antigen presentation were regulated in DCs by controlling cathepsin S activity, the cathepsin S–independent allotypes would be oblivious to this control mechanism. However, it was found that they behave identically to the ‘‘cathepsin S–dependent’’ allotypes (Villadangos et al., 2001). Analysis of DCs from Ii-deficient mice (Rovere et al., 1998) and cathepsin S–deficient mice (Driessen et al., 2001; Nakagawa et al., 1999; Shi et al., 1999) also suggested that the mechanism of control of MHC II expression was not based on the kinetics of Ii degradation. More recently, we were able to directly assess the role of cystatin C in controlling DC antigen presentation by studying cystatin C knockout mice: we observed no alteration in control of MHC II expression and subcellular distribution in immature cystatin C-null BMDCs, D1DCs, or spleen DCs (ElSukkari et al., 2003). Interestingly, we detected a striking difference in the levels of cystatin C expression among DC populations: CD8þ DCs synthesize much larger quantities of cystatin C than do CD8
DCs, and they accumulate cystatin C in MHC IIþ endosomal compartments. This was surprising because cystatin C is considered a ubiquitously expressed protein and its gene lacks any obvious regulatory elements (Abrahamson, 1994; Abrahamson et al., 1990; Huh et al., 1995). The differential pattern of expression of cystatin C among closely related DC subsets suggests the existence of hitherto unknown mechanisms of expression of this protease inhibitor. Nevertheless, the expression of cystatin C in CD8þ DCs did not affect the MHC II presentation of several antigens (El-Sukkari et al., 2003), so it is unclear whether cystatin C plays any specific antigen presentation function in this DC subset. 6.2.2. Is H-2DM a Regulatory Molecule in DC Antigen Presentation? The chaperone H-2DM has also been suggested as a key regulatory element controlling MHC II antigen presentation in DCs. Analysis of the subcellular localization of MHC II and H-2DM in immature D1DCs revealed that MHC II was localized to the internal vesicles of multivesicular bodies, whereas H2DM localized to the limiting membrane of the bodies (Kleijmeer et al., 2001). The physical separation of H-2DM from MHC II might prevent the conversion of MHC II–CLIP into MHC II–peptide and impair antigen presentation. On maturation, the internal vesicles fuse with the outer membrane of the multivesicular bodies, which would enable H-2DM to exert its chaperone activity on MHC II–CLIP and allow formation of MHC II–peptide complexes (Kleijmeer et al., 2001). As in the reports that suggested cystatin C represented the rate-limiting step in the production of peptide-receptive MHC II molecules, this study favored the gain of function model of control of antigen

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 271 presentation (Fig. 3). However, limitations were also evident: first, it did not explain how it would operate on MHC II allotypes that do not require H-2DM to convert MHC II–CLIP into MHC II–peptide (Albert et al., 1996; Avva and Cresswell, 1994; Bikoff et al., 2001; Koonce et al., 2003; Stebbins et al., 1995; Wolf et al., 1998); second, for the I-Ab allotype, lack of H-2DM activity leads to accumulation of I-Ab–CLIP complexes, but this study did not observe such complexes in immature DCs; third, in H-2DM-null mice MHC II–CLIP complexes are transported normally to the plasma membrane (Fung-Leung et al., 1996; Martin et al., 1996; Miyazaki et al., 1996), so even if the MHC II molecules of immature DCs failed to eject CLIP, it is unclear how this would result in their poor expression at the plasma membrane. We suggest that the segregation of H-2DM from MHC II observed in the multivesicular bodies of immature DCs may occur after the MHC II molecules have acquired their peptide cargo. This segregation, and its control during maturation, may reflect differential sorting events that control MHC II trafficking in immature and mature DCs (see below) (van Lith et al., 2001). 6.2.3. Is MHC II–Peptide Complex Formation Regulated in DCs? Techniques that directly assess the formation of individual MHC II–peptide combinations have been used by several laboratories to examine how DCs control MHC II peptide loading. These include using monoclonal antibodies (mAbs) specific for individual MHC–peptide complexes and antigen presentation assays T-cell hybridomas or naive T cells. In a series of studies, immature BMDCs were incubated with HEL in the absence or the presence of an activation stimulus (LPS). Formation of a complex between I-Ak and an HEL-derived peptide was then assessed, employing an mAb or T cells specific for this complex. Strikingly, the I-Ak–HELpep complexes could not be detected by microscopy until the BMDCs were activated, suggesting that immature BMDCs did not generate peptide-receptive I-Ak molecules (Inaba et al., 2000; Turley et al., 2000). However, the complex could be detected by Western blot analysis of cell lysates of both immature and mature DCs (Trombetta et al., 2003). Moreover, fluorescence-activated cell sorting (FACS) analysis revealed that the proportion of surface I-Ak molecules that were associated with the HEL peptide in immature and mature DCs was comparable (Inaba et al., 2000; Veeraswamy et al., 2003). Finally, probably the most compelling evidence that I-Ak–HELpep complexes were formed by immature BMDCs incubated with HEL was that these cells stimulated HEL-specific T cells, demonstrating that they did present the complex (Inaba et al., 2000; Veeraswamy et al., 2003). Therefore, even though immature DCs appear to be relatively inefficient at processing HEL (see above), this deficiency certainly

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does not translate to their inability to load and present to T cells I-Ak molecules loaded with HEL-derived peptides. Indeed, a conclusion of these studies is that since most antigens are probably more sensitive to proteolysis than HEL (see above), immature DCs will present most antigens even more efficiently than HEL. Perhaps the failure to detect the I-Ak–HELpep complex by microscopy was due to low sensitivity of the antibody used. In a similar study, Colledge and colleagues tracked formation of a complex between I-Ek and a pigeon cytochrome c (PCC)–derived peptide in immature BMDCs, showing that the complex could be detected by microscopy in endosomal compartments just 5 min after incubation of the cells with intact PCC (Colledge et al., 2002). The immature BMDCs then rapidly transported the I-Ek–PCCpep complex to the cell surface. This demonstrated that immature BMDCs can eficiently process PCC, load their I-Ek dimers with the resulting antigenic peptides, and then present these complexes on the plasma membrane. Importantly, all the studies summarized above checked that neither HEL nor PCC induced BMDC maturation, discarding the possibility that contaminating LPS or other inflammatory compounds triggered activation of the immature BMDCs. Together these studies support a scenario in which immature BMDCs constitutively load their MHC II molecules with peptides derived from endocytosed antigens and transport the resulting complexes to the cell surface. Is this also the case for DCs generated in vivo? To answer this question, the group of Unanue (Veeraswamy et al., 2003) and ourselves (Wilson et al., 2004) used transgenic mice expressing HEL or OVA as models of self-proteins, respectively, to determine whether the immature lymphoid organ DCs presented antigens in the steady state. Both studies used mAbs and/or antigenspecific T cells to assess formation of I-Ak–HELpep or I-Ab-OVApep complexes. The conclusions of these two studies were that in vivo immature DCs constitutively load their MHC II molecules with peptides derived from self-proteins and present these complexes to T cells. Using mAbs and T cells to assess MHC II–peptide loading restricts the scope of studies to the few complexes for which mAbs or T cells have been generated. A complementary approach consists of using biochemical methods to assess the composition of the MHC II molecules. This approach takes advantage of the unique resistance of the MHC II–peptide complexes to denaturation in SDS at room temperature (Germain and Hendrix, 1991; Springer et al., 1977). Even though the ab–peptide trimers do not contain interchain disulfide bonds, they run in SDS–PAGE as a complex with an Mr of approximately 50 kDa. This property is lost if the complexes are first denatured at high temperature (‘‘boiling’’). Used in Western blot or combined with metabolic radiolabeling, this biochemical approach represents a powerful

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 273 tool to measure the kinetics, intermediate steps, and efficiency of the conversion of MHC II–Ii into MHC II–peptide in DCs and other APCs. For example, cathepsin S-deficient DCs show impaired formation of MHC II– peptide SDS-stable complexes and accumulate MHC II–Iip10 complexes instead; this deficiency can be easily visualized by Western blot or pulse– chase analysis of MHC II immunoprecipitates (Nakagawa et al., 1999; Shi et al., 1999; Villadangos et al., 2000). Likewise, the MHC II–CLIP complexes that accumulate in H-2DM–deficient APCs can be distinguished from the bulk of normal MHC II–peptide complexes by their distinct mobility in SDS– PAGE (Fung-Leung et al., 1996; Martin et al., 1996; Miyazaki et al., 1996; Villadangos et al., 2000). Studies that have employed this biochemical approach to compare MHC II peptide loading in immature and mature mouse BMDCs (El-Sukkari et al., 2003; Pierre et al., 1997; Veeraswamy et al., 2003), D1DCs (Rescigno et al., 1998; Villadangos et al., 2001), or lymphoid organ DCs (El-Sukkari et al., 2003; Wilson et al., 2004), and in human monocytederived DCs (Cella et al., 1997a; Saudrais et al., 1998), have reported unimpaired generation of MHC II–peptide complexes in immature DCs. One study also indicates that immature DCs express a relatively large number of ‘‘empty’’ molecules (ab dimers devoid of antigen peptides), which can be recognized with an mAB (Santambrogio et al., 1999). In any case, this population of empty molecules represented only a fraction of the total (20% approximately, L. Santambrogio, personal communication), with the majority of MHC II molecules associated with regular peptides. Therefore it can be concluded that immature DCs from both mouse and human, either generated in vitro or in vivo, constitutively load their MHC II molecules with antigenic peptides. Hence, the mechanisms that control MHC II antigen presentation in DCs must operate post-MHC II–peptide complex formation. 6.3. Control of MHC II–Peptide Trafficking in DCs Although the bulk of the MHC II molecules contained at any time in both immature and mature DC are loaded with peptides, the fate and localization of these complexes clearly differ between the two maturational stages: MHC II molecules are short-lived and accumulate in endosomal compartments in immature DCs but are long-lived on the plasma membrane of mature DCs. What mechanism is responsible for these fundamental differences? A straightforward explanation would be that only a few MHC II–peptide complexes can escape to the cell surface in immature DCs, the rest being retained intracellularly and then degraded in lysosomes; on maturation, these complexes would be permitted to traffic to the cell surface. However, an alternative possibility is that the MHC II–peptide complexes are in fact

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transiently expressed on the surface of the immature DCs and then quickly internalized, delivered to lysosomes, and degraded. Quantitation of the number of MHC II–peptide complexes that are transported to the cell surface in immature DCs, measured by pulse–chase followed by cell surface biotinylation and immunoprecipitation, favor the second mechanism (Baron et al., 2001; Cella et al., 1997a; Villadangos et al., 2001). Therefore, most of the MHC II molecules that accumulate in the endosomal compartments of immature DCs are not really ‘‘retained,’’ but rather are in the process of acquiring peptide cargo to progress to the cell surface or else are moving along the endocytic route after being internalized from the cell surface (Figs. 3 and 4). An appropriate metaphor would be a playing videotape (see below), where the MHC II molecules are represented by the tape, the portion of the tape accessible to the head of the VCR the ‘‘cell surface,’’ and the interior of the cassette the ‘‘endocytic route’’: for an outside observer, most of the tape never appears to leave the interior of the cassette, but over time the entire length of tape will be transiently exposed on the surface. What happens when immature DCs receive an activation signal? The disappearance of MHC II molecules from internal compartments, and their accumulation on the plasma membrane, suggest a direct transfer of MHC II molecules from endosomes to the DC surface. However, it has long been recognized that the increase in MHC II surface expression that accompanies LC maturation requires protein synthesis (Pure et al., 1990; Shimada et al., 1987; Witmer-Pack et al., 1988). In accordance with this, we have described that brefeldin A (BFA, an inhibitor of protein export out of the ER) and cycloheximide (CHX, a protein synthesis inhibitor) block the increase in MHC II surface expression in maturing D1DCs or spleen DCs without impairing the degradation of the preexisting intracellular MHC II–peptide pool (Villadangos et al., 2001; Wilson et al., 2004). Similar effects have been observed with actinomycin D (a transcription inhibitor) (D. Vremec and K. Shortman, personal communication). Therefore, the majority of the MHC II–peptide complexes that accumulate on the surface of maturing DCs originate from de novo synthesis; the preexisting MHC II–peptide complexes that were localized in the endosomal compartments when the immature DCs received the activation signal contribute little, and most are degraded instead. This conclusion makes sense because removing CLIP from newly synthesized MHC II–CLIP is kinetically more favorable than displacing peptides from preloaded MHC II–peptide complexes (Kropshofer et al., 1999). Therefore a DC maturing in response to an endocytosed pathogen will present more efficiently pathogen-derived peptides by using newly synthesized MHC II molecules.

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Figure 4 DCs regulate MHC II antigen presentation by controlling the rates of MHC II synthesis and MHC II–peptide endocytosis. The MHC II molecules of immature DCs (top left) constitutively generate MHC II–peptide complexes, which are transiently expressed on the cell surface, internalized, and then degraded. This causes the apparent accumulation of MHC II molecules in the endosomal compartments of immature DCs (see Fig. 1C). Therefore, immature DCs can constantly present self-antigens (open triangles) to T cells. On activation (top right), MHC II synthesis is transiently upregulated and turnover of surface MHC II–peptide complexes is downregulated. This causes an accumulation of newly generated MHC II–peptide complexes on the plasma membrane. Some of these complexes are loaded with peptides derived from antigens captured at the site and time of activation (solid triangles). MHC II synthesis is subsequently downregulated (bottom left) to prevent the substitution of the MHC II–peptide complexes that reached the plasma membrane early during maturation. This process enables mature DCs (bottom right) to present a long-lived ‘‘snapshot’’ of the antigenic material captured when they became activated, even if the foreign antigens have been exhausted by proteolytic processing. The MHC II–peptide complexes generated on activation may traffic to the cell surface associated to membrane microdomains.

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A fascinating series of studies by the groups led by Kleijmeer (Barois et al., 2002; Kleijmeer et al., 2001), Ploegh (Bertho et al., 2003; Boes et al., 2002, 2003), and Mellman (Chow et al., 2002) have shown that maturation signals trigger dramatic changes in the morphology of the endocytic compartments of DCs (reviewed in Boes et al., 2004). While the compartments containing MHC II molecules in immature DCs appear vesicular, long tubular structures are generated on DC maturation, providing exit ‘‘tracks’’ to the cell surface for the MHC II–peptide complexes. Strikingly, in the absence of T cells these structures extend randomly toward the plasma membrane of the activated DCs, but in their presence the tubules reach out toward the DC–T-cell interface in an antigen-specific manner (Bertho et al., 2003; Boes et al., 2002, 2003). Although the implication of these observations in vivo are still unclear, they suggest that T cells signal the maturing DCs to polarize transport of the newly generated MHC II–peptide complexes toward the site of DC–T cell contact. Moreover, T-cell–costimulatory molecules are delivered to the cell surface in close proximity with the MHC II–peptide complexes (Turley et al., 2000). These mechanisms have obvious advantages for forming the immunological synapse (Huppa and Davis, 2003). Why do the MHC II–peptide complexes accumulate on the surface of the maturing DCs? We support the view that this is due to reduced internalization of MHC II–peptide complexes during DC maturation (Fig. 4). Indeed, we believe this is the key step that controls MHC II expression and antigen presentation in DCs. In immature DCs, surface MHC II–peptide complexes are rapidly internalized and then degraded, but on maturation they are retained on the plasma membrane as a long-lived ‘‘snapshot’’ of antigenic information (Baron et al., 2001; Cella et al., 1997b; Villadangos et al., 2001; Wilson et al., 2004). This is not a passive consequence of overall reduction of macropinocytosis and phagocytosis in the mature DCs, because the turnover of most plasma membrane proteins, including MHC I, proceeds at comparable rates in immature and mature DCs (Delamarre et al., 2003; Wilson et al., 2004). This is also the case for the rate of pinocytosis, which shows little change on DC maturation (see above) (Garrett et al., 2000; West et al., 2000). Therefore, MHC II–peptide complexes are selectively excluded from endocytosis and subsequent degradation on DC maturation. This conclusion implies the existence of a specific mechanism of sorting of MHC II–peptide complexes actively regulated during DC maturation. Where and how do these mechanisms operate? We can speculate on three possibilities. The first might be control of internalization of MHC II–peptide complexes from the plasma membrane, a process that would be downregulated on DC maturation. The second possibility is that the complexes are internalized by default in both the immature and the mature DCs, but in the mature DCs most of the complexes

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 277 are recycled back from EEs to the cell surface instead of proceeding toward LEs and lysosomes, thus following a cycle similar to that of the transferrin receptor (Mellman, 1996). It is well established that surface MHC II molecules can undergo such recycling (Pinet et al., 1995; Ramachandra and Harding, 2000; Reid and Watts, 1990), and indeed the kinetics of internalization of MHC II in immature DCs suggest that a fraction of the endocytosed MHC II–peptide complexes return to the surface (Cella et al., 1997a; Villadangos et al., 2001). Therefore it is plausible that DC maturation upregulates this recycling mechanism, causing the MHC II–peptide complexes of mature DCs to constantly cycle between the plasma membrane and the EEs (Maxfield and McGraw, 2004). A third mechanism of control could be based on recruitment of MHC II–peptide complexes to membrane microdomains: Roche’s group described accumulation of MHC II–peptide complexes in lipid rafts on B cells (Anderson et al., 2000), whereas Kropshofer and colleagues observed a similar phenomenon in DC, but involving tetraspan rich microdomains, not lipid rafts (Kropshofer et al., 2002). This mechanism is not incompatible with the previous two; indeed, recruitment to membrane microdomains might be the factor that modulates internalization and/or recycling of the MHC II–peptide complexes in DCs. If this were the case, then the sorting signals controlling MHC II trafficking may not be contained in the MHC II molecules themselves but in other, closely associated molecules within the membrane microdomains. In any case, the exact nature of these signals and the sorting machinery associated with them have yet to be characterized. It is known that the cytosolic portion of the MHC II b chain contains sorting motifs (for a review see Bakke and Nordeng, 1999), but whether these have a role in control of MHC II trafficking in DCs is unknown. 6.4. Control of MHC II Synthesis in DCs If DC maturation results in ‘‘freezing’’ the MHC II–peptide complexes on the plasma membrane (or their entry in a cycle between the plasma membrane and the EEs), the net addition of newly synthesized MHC II–peptide complexes explains the increase in surface expression of MHC II that follows DC activation. However, this process must stop when the number of MHC II molecules reaches a certain level. DCs accomplish this by coordinating the reduction in the rate of surface MHC II turnover with a reduction in the rate of MHC II synthesis (Fig. 4). MHC II synthesis increases transiently shortly after DC activation, but then decreases gradually (Cella et al., 1997a; Rescigno et al., 1998; Wilson et al., 2004). In DCs generated in vitro, MHC II synthesis is reduced by approximately 70% 24–48 h postactivation (Cella et al., 1997a; Rescigno et al., 1998; Villadangos, 2001). This reduction is much more

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dramatic in LCs or splenic DCs activated in vitro, in which MHC II expression is virtually shut off in 18 h (Kampgen et al., 1991; Pure et al., 1990; Wilson et al., 2004). This downregulation of MHC II synthesis reflects a normal program of DC maturation in vivo, as it is observed in splenic DCs from mice injected with inflammatory compounds (Villadangos et al., 2001; Wilson et al., 2004), and in the tissue-derived DCs isolated from LNs, which acquire a mature phenotype in the steady state (see above) (Wilson et al., 2004). In contrast to MHC II, the rate of MHC I synthesis increases on DC maturation (Cella et al., 1997a; Delamarre et al., 2003; Rescigno et al., 1998; Villadangos, 2001; Wilson et al., 2004), which is consistent with the continued turnover of surface MHC I observed in mature DCs (Wilson et al., 2004). The surface expression level of MHC II is thus regulated in DCs by a finely coordinated regulation of the rates of MHC II synthesis and MHC II–peptide internalization and degradation (Fig. 4). 6.5. Corollary: The Cell Biological Basis of ‘‘Antigenic Memory’’ We conclude that the experimental evidence gathered by different laboratories in studies of mouse and human DCs obtained from in vitro or in vivo sources support the interruption model of control of MHC II antigen presentation in DCs (Figs. 3 and 4). Using the metaphor described above, MHC II molecules represent the tape of a videocassette. In immature DCs, the MHC II molecules are constantly ‘‘unwound’’ (synthesized) from the left ‘‘roll.’’ The MHC II molecules then bind peptides derived from both endogenous and exogenous self-proteins, which are constantly endocytosed and processed by the immature DCs. The resulting MHC II–peptide complexes are transiently exposed on the plasma membrane for recognition by the ‘‘VCR head’’ (the T cells), but the complexes are then internalized and degraded (‘‘wound’’ into the videocassette roll at the right) (Fig. 4). For immature DCs in uninflamed peripheral tissues, this constant presentation of peptide-loaded MHC II molecules may be irrelevant, because these DCs are unlikely to encounter naive T cells. But for the immature DCs that reside in the lymphoid organs, this process would enable DCs to provide T cells with a constant and updated supply of information about the antigens that reach the lymphoid organs, whether those antigens get there by themselves or are brought by other incoming cells (Belz et al., 2002; Inaba et al., 1998; Pooley et al., 2001; Salomon et al., 1998; Scheinecker et al., 2002; Zhong et al., 1997). If immature DCs capture even a small amount of foreign antigen in the presence of maturation signals, ‘‘winding’’ of the ‘‘tape’’ stops and MHC II molecules start to accumulate on the ‘‘videocassette surface.’’ Many MHC II molecules will still carry self-peptides, but some will present peptides derived

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 279 from the foreign antigen. As the MHC II–peptide complexes accumulate on the surface of the maturing DCs, MHC II synthesis slows down and eventually stops (Fig. 4). The net result of these synchronized changes is the characteristic increase in the level of surface MHC II that accompanies DC maturation. More importantly, by the time the DCs reach full maturity, even if the antigen captured when DC activation took place is no longer available and all the endocytosed antigen ‘‘meal’’ has been processed, the mature DCs will have accumulated a sufficient number of MHC II molecules loaded with foreign peptides for scanning by naive T cells. These complexes will be exposed on the DC surface for a long time (‘‘antigenic memory’’), thus increasing the chances of encountering a pathogen-specific T-cell. In this scenario, the changes undergone by DCs during the initial period of maturation are the most important for efficient antigen presentation. MHC II synthesis is initially upregulated (Cella et al., 1997a; Rescigno et al., 1998; Wilson et al., 2004), whereas the proteolytic environment encountered by recently endocytosed antigen may be partially downregulated (LennonDumenil et al., 2002). Together these two events would facilitate a steady accumulation of MHC II molecules loaded with peptides derived from antigens captured at the time of activation. The egress of these molecules to the cell surface may initially follow random directions and is accompanied by expression of T-cell–costimulatory molecules (Turley et al., 2000). But as soon as an antigen-specific T cell detects its cognate MHC II–peptide complex, the T-cell signals the DC to polarize the transport of the MHC II–peptide complexes toward the DC–T-cell interface, thus promoting the formation of the immunological synapse (Bertho et al., 2003; Boes et al., 2002, 2003). In conclusion, the major checkpoints that control antigen presentation in DCs and the mechanisms responsible for their antigenic memory are the rate of MHC II synthesis and the rate of turnover of surface MHC II–peptide complexes. We think that this interruption model of MHC II antigen presentation is consistent with most of the experimental evidence obtained by the different groups engaged in studies of control of MHC II antigen presentation in DCs and can also explain the results that have been interpreted as favoring the gain of function model. These conclusions have several implications for studies of DC subtype characterization. Surface MHC II levels are often used as a marker of DC maturity. For this reason, immature blood-derived DCs, which express more MHC II than, for instance, immature BMDCs, are often referred to as ‘‘semimature’’ or ‘‘intermediate’’ DCs. However, to contradict this assumption the turnover of surface MHC II is much higher in the immature blood-derived DCs than in immature BMDCs, D1DCs, or MoDCs (Cella et al., 1997a; Villadangos et al., 2001; Wilson et al., 2004). Likewise, activated lymphoid

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organ DCs downregulate MHC II synthesis faster and more profoundly than their in vitro–grown counterparts (Cella et al., 1997a; Rescigno et al., 1998; Villadangos et al., 2001; Wilson et al., 2004). Therefore, the expression level of MHC II is misleading as a marker of the antigen-presenting properties of different DC types. It is the behavior of the MHC II molecules, and the immunological manifestation of such behavior (capacity to process and present newly encountered antigens and antigenic memory), that distinguish DC maturity. Our conclusions also have implications for DC-based immunotherapy. Namely, the success of a DC-based vaccine may depend more on the duration of presentation of MHC II–peptide complexes than on the total amount of complexes generated during preparation of the vaccine (Schuler et al., 2003). Therefore the mechanisms that control MHC II–peptide turnover represent potential targets for drugs that could prolong the half-life of the MHC II– peptide complexes. Conversely, drugs that increased the turnover of selfMHC II–peptide combinations on mature DCs could interfere with autoimmune responses. Indeed, one of the effects of the immunosuppresive cytokine interleukin 10 (IL-10) is to counteract the changes induced by DC maturation, promoting MHC II–peptide turnover (Koppelman et al., 1997) and regulating endosomal proteolysis (Fiebiger et al., 2001). In addition, some studies suggest that to induce optimal T-cell activation, MHC II–peptide complexes must be generated within the antigen presentation pathway, whereas incubating DCs with synthetic peptides leads to suboptimal recognition by T cells, even if a similar number of MHC II–peptide complexes is thus generated on the DC surface (Bertho et al., 2003). This might be because only in the first instance do the MHC II–peptide complexes localize in membrane microdomains that favor extended MHC II–peptide presentation and T-cell activation (Anderson et al., 2000; Kropshofer et al., 2002). 7. DCs and Cross-Presentation 7.1. When Cross-Presentation Is Cross-Presentation? Most cell types in the body express MHC I molecules that are loaded with peptides generated in the cytosol and then translocated into the ER by the TAP transporter (Fig. 5). These peptides are derived from proteins degraded in the cytosol mostly by the proteasome (Yewdell et al., 2003). Such proteins include any endogenously produced polypeptide, because even noncytosolic proteins such as secreted or membrane proteins, which are cotranslationally translocated into the ER, can be processed in the cytosol as defective

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Figure 5 Mechanisms of MHC class I ‘‘direct presentation’’ and ‘‘cross-presentation.’’ In the direct presentation pathway (left), MHC I molecules acquire peptide cargo in the endoplasmic reticulum (ER). The antigenic peptides are derived from polypeptides degraded in the cytosol mostly by the proteasome. The MHC I–peptide complexes shuttle to the cell surface by the default secretory pathway. Cross-presentation entails the formation of a hybrid phagosome–ER hybrid compartment (ergosome), which contains endocytosed exogenous antigens, MHC I molecules, and the components of the MHC I–peptide loading complex, including the peptide transporter TAP. Exogenous polypeptides are transported to the cytosol, perhaps via the Sec61 complex, and are degraded in the proximity of the ergosome by the proteasome. The exogenous peptides are then transported back into the ergosome by TAP for loading onto MHC I molecules. The resulting MHC I–peptide complexes are transported to the cell surface without crossing the Golgi complex.

ribosomal products (DRiPs) (Reits et al., 2000; Schubert et al., 2000; Yewdell et al., 1996, 2003). Exogenous proteins can also be presented by MHC I molecules, a process that has been termed cross-presentation (Bevan, 1976). This term is sometimes used to refer to presentation of cell-associated antigens, even via the MHC II pathway. However, we suggest that the word crosspresentation should be used to refer only to presentation of exogenous antigens via MHC I molecules, independently of whether those antigens are soluble, bound to serum Ig as immunocomplexes, cell associated, etc.

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Although the existence or practical applications of the cross-presentation pathway are not contested, its significance in maintaining tolerance (crosstolerance) and inducing immune responses (cross-priming) in vivo is highly controversial (Melief, 2003; Zinkernagel, 2002). In this section we focus on advances in the delineation of the molecular mechanisms involved in crosspresentation. For a more thorough discussion on the role of cross-presentation in the immune system we refer the reader to other reviews (Heath and Carbone, 2001; Heath et al., 2004). Cross-presentation may occur by different mechanisms. Some proteins can traverse the plasma membrane and access the cytosol, where they are processed as endogenous proteins (Jeannin et al., 2000; Kim et al., 1997). Heat shock proteins may be capable of transporting small bound peptides into cells (Srivastava, 2002), although the exact nature of this phenomenon remains controversial (Nicchitta, 2003). Endocytosed exogenous proteins, processed in the endocytic route, can generate antigenic peptides suitable for binding to MHC I molecules recycled from the plasma membrane (Svensson et al., 1997). This mechanism is probably independent of the TAP transporter and the components of the MHC I peptide-loading complex and thus is usually considered a distinct form of cross-presentation, sometimes referred to as the ‘‘alternative pathway’’ (Campbell et al., 2000; Chen and Jondal, 2004; Gromme et al., 1999; Song and Harding, 1996). Finally, endocytosed antigens can be actively transferred to the cytosol by a specialized mechanism and access the classic MHC I presentation pathway. In this review we focus on this latter mechanism of cross-presentation because of its particular significance in DCs. We refer the reader to an excellent previous review by Yewdell and colleagues in this series (Yewdell et al., 1999), which discusses in detail the different mechanisms of cross-presentation mentioned above. 7.2. A Novel Compartment for Cross-Presentation The first direct evidence to suggest a mechanism of transport from the endosomes to the cytosol in cross-presentation was reported by Norbury and colleagues, first using macrophages (Norbury et al., 1995) and then BMDCs (Norbury et al., 1997). These studies tracked the transport of horseradish peroxidase (HRP) from endosomal compartments into the cytosol. Rodriguez et al., published a similar study tracking the transport of HRP and OVA in D1DCs (Rodriguez et al., 1999). This study also demonstrated that OVA taken up as immunocomplexes was endocytosed and then transferred into the cytosol, whereas the Ig portion of the immunocomplex remained in the endosomal compartments. This indicated that transfer to the cytosol was not simply by physical disruption of the endocytic vesicle (Reis e Sousa and Germain, 1995)

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 283 but rather by a selective mechanism of transport. Indeed, the putative channel that mediated endosome to cytosol transport could accommodate molecules with an Mr of up to 50 to 500 kDa, as indicated by analysis of transfer of dextrans of variable size (Rodriguez et al., 1999). A seminal study by the group of Desjardins provided evidence for the generation in macrophages of endosomal compartments that contained endoplasmic reticulum (ER) components (Garin et al., 2001). These compartments result from fusion of ER membranes with phagosomes (Desjardins, 2003). Building on this work, three groups have independently described a similar compartment in macrophages and DCs, which may provide the physical structure sufficient for cross-presentation (Ackerman et al., 2003; Guermonprez et al., 2003; Houde et al., 2003). We refer to these ER–phagosomal hybrid compartments as ergosomes (Fig. 5). The ergosomes contain endocytosed antigens, newly synthesized MHC class I molecules, and the multimolecular machinery required for efficient formation of MHC I–peptide complexes, including the TAP transporter (Purcell, 2000). The study by Cresswell’s group showed that macropinosomes can also fuse with ER membranes, at least in DCs (Ackerman et al., 2003). This indicates that the range of antigens that can access ergosomes is probably not restricted to those that are phagocytosed, but also includes soluble antigens taken up by macropinocytosis and perhaps receptormediated endocytosis. This suggestion is consistent with the observation that DCs can cross-present a wide range of antigens including cell-associated, bacterial, immunocomplexed, and soluble proteins (Heath et al., 2004). It has been hypothesized that the antigens contained in the ergosome are transferred into the cytosol, where they are processed by the proteasome. The resulting peptides are then translocated back into the ergosome via TAP to generate MHC I–peptide complexes, which are then transported to the plasma membrane (Fig. 5) (Ackerman et al., 2003; Guermonprez et al., 2003; Houde et al., 2003). Interestingly, a feature of the complexes generated by the crosspresentation mechanism is that they do not appear to traffic through the Golgi, and so their carbohydrates do not undergo the modifications that would make them resistant to treatment with the enzyme endoglycosidase H (Ackerman et al., 2003). This endoglycosidase H sensitivity may allow the distinction between MHC I–peptide complexes generated via cross-presentation from those generated via the classic pathway. 7.3. Where Is the Exit, Please? Perhaps the major ‘‘black box’’ in this model of cross-presentation is the transfer of antigens into the cytosol. It has been suggested that this transport may be mediated by the Sec61 complex, a component of the ER that is also present in

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ergosomes (Guermonprez et al., 2003; Houde et al., 2003). The Sec61 complex is a heterotrimer that forms the channel that allows newly synthesized secretory or membrane proteins to cross from the cytosol into the ER (Rapoport et al., 1996). This channel can also be used to transfer misfolded polypeptides from the ER to the cytosol for proteasomal degradation, a process termed ‘‘dislocation’’ or ‘‘retrotranslocation’’ (Brodsky and McCracken, 1999; Wiertz et al., 1996). The ergosome-to-cytosol transport of exogenous antigens required for cross-presentation would thus be reminiscent of the process of dislocation (Fig. 5). An involvement of Sec61 in this process is suggested by the observation that the cholera toxin A1 subunit, a protein known to use Sec61 as a gate to cross from within the ER to the cytosol (Schmitz et al., 2000), can be exported from ergosomes (Houde et al., 2003). However, this observation does not prove that the cross-presented antigens are in fact translocated via Sec61, only that Sec61 maintains its integrity in the ergosome. The major problem with the hypothesis that Sec61 mediates transport of exogenous antigens to the cytosol is the size of its channel. The group of Rapoport has provided the closest picture yet of Sec61 by reporting the crystal structure of SecY, the archaeal homolog of Sec61 (Van den Berg et al., 2004). This study reveals that the diameter of the channel contained in a single SecY complex, through which proteins would have to cross the ER membrane, is only 5–8 A˚ , enough to accommodate only polypeptides in extended conformation or, at most, a disulfide-linked loop. This contrasts with the studies by the Watts and Amigorena groups, which have shown that the ergosome-to-cytosol transport mechanism can accommodate dextrans with an Mr over 50 kDa and globular proteins such as enzymatically active HRP (Norbury et al., 1995, 1997; Rodriguez et al., 1999). These observations would suggest that the transport of antigens from ergosomes to the cytosol is not carried out by Sec61 but by a different transporter. On the other hand, the study by Rapoport and co-workers does not discard the possibility that several juxtaposed Sec61 complexes could create a much larger pore (Van den Berg et al., 2004). Indeed, electron microscopy studies of solubilized Sec61 revealed ringlike structures with an open pore of 20 A˚ (Hanein et al., 1996), whereas indirect measurements of the pore size, using fluorescent probes, raise this figure to at least 40 A˚ —sufficient to accommodate an Fab fragment, for instance (Hamman et al., 1997). Furthermore, previous studies of dislocation of MHC I heavy chains suggested that Sec61 can mediate the transport of glycosylated polypeptides (Wiertz et al., 1996). Based on the crystal structure reported by Rapoport and colleagues, oligomerization of SecY to form large pores appears unlikely, but the authors did not discard this possibility (Van den Berg et al., 2004). Further elucidation of this question awaits resolution of the three-dimensional structure of the mammalian Sec61 complex. In conclusion, Sec61 remains an attractive candidate as the channel

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 285 responsible for ergosomal export, but until direct evidence for its involvement is obtained, a verdict for its implication in cross-presentation might be one more case of ‘‘guilt by association.’’ 7.4. Keeping the Team Together Another intriguing question is whether cross-presentation requires the presence of TAP, MHC I, and the peptide-loading machinery within the ergosome, and a close association between ergosomes and proteasomes. It would be reasonable to assume that the only critical step for cross-presentation would be antigen transport into the cytosol. Once there, the exogenous antigens should be processed as any endogenous component. However, antigen transfer to the cytosol may not necessarily be followed by its presentation via MHC I. It has been shown that the products of proteosomal degradation are short lived in the cytosol, and are quickly eliminated unless they are transported by TAP into the ER, and then protected by lodging into the MHC I peptide-binding cavity (Reits et al., 2003). Therefore, failure to recruit to the ergosome any of the components of the MHC I antigen presentation machinery could prevent cross-presentation from occurring. Indeed, a report from the Mellman group has shown that both DCs and CD11c
cells (probably macrophages) growing in bone marrow cultures could transfer soluble OVA to the cytosol, but only the BMDCs loaded their MHC I molecules with OVA-derived peptides (Delamarre et al., 2003). Since the CD11c
cells could present endogenous proteins, indicating their MHC I peptide-loading machinery was operative, this report suggested that the failure of the CD11c
cells to cross-present OVA was due to a deficiency downstream of translocation of OVA to the cytosol. This could be a lack of proteasomes, TAP, MHC I, or other components of the peptide-loading complex in the ergosomes of CD11c
cells. A report from the Jefferies group has described a conserved tyrosine-based sorting motif in the cytosolic portion of the MHC I heavy chain that is required for crosspresentation but not for endogenous antigen presentation (Lizee et al., 2003). This study supports the notion that the recruitment of MHC I molecules to ergosomes may depend on an active sorting mechanism that recognizes this tyrosine motif; such a mechanism could be operative in BMDCs but not in CD11c
cells. 7.5. Is Cross-Presentation Regulated During DC Maturation? Similar to MHC II presentation, MHC I cross-presentation may be developmentally regulated in DCs. Signals mediated by Fc receptors (den Haan and Bevan, 2002), inflammatory compounds (Datta et al., 2003; Gil-Torregrosa

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et al., 2004), CD4þ T cells (Machy et al., 2002), and disruption of intercellular contacts (Delamarre et al., 2003) have been reported to induce crosspresentation. We stress that we are referring to enhancement of formation of MHC I–peptide complexes by cross-presentation and not to enhancement of T-cell activation by cross-priming; the second is a direct consequence of increased T-cell stimulatory capacity in activated DCs, which may or may not be accompained by increased efficiency of loading of MHC I molecules with exogenously derived peptides. The reports above suggest that certain components of the cross-presentation machinery are upregulated on DC maturation, but the mechanisms underlying this regulation are still poorly characterized. One obvious mechanism that may contribute to enhancement of crosspresentation (and direct presentation) is increased synthesis of MHC I (Cella et al., 1997a; Rescigno et al., 1998; Villadangos et al., 2001; Wilson et al., 2004) and of the components of the peptide-loading machinery (Gil-Torregrosa et al., 2004). However, since not all activatory signals that enhance MHC I synthesis upregulate cross-presentation (Datta et al., 2003; Delamarre et al., 2003), some more specific mechanisms must exist. Pierre’s group has reported that activated DCs concentrate endogenous ubiquitinated proteins and DRiPs in cytosolic aggregates (DALIs) (Lelouard et al., 2002). Formation of DALIs might focus the DC proteasomal activity on exogenous proteins translocated from ergosomes to the cytosol. Another mechanism that might be regulated is the efficiency of formation of ergosomes or, as mentioned above, the recruitment of MHC I molecules or other components of the peptide-loading machinery to these compartments. Evidence for this, however, is still lacking. A report from the Amigorena group has also shown that mature DCs downregulate uptake and delivery of antigen to the cross-presentation pathway (Gil-Torregrosa et al., 2004). As described before for the MHC II pathway, transient upregulation of cross-presentation followed by its downregulation may allow the mature DCs to focus their cross-presenting activity on antigens captured at the time of activation. But providing a ‘‘memory’’ of exogenous antigens via cross-presentation would require the DCs to extend the half-life of the MHC I–peptide complexes generated by this pathway (Ludewig et al., 2001). If maturing DCs loaded their MHC I molecules with exogenous antigens captured in a peripheral tissue but the complexes were rapidly degraded, the cross-presented antigens could be eliminated before or shortly after the DCs reached the lymphoid organs. However, the half-life of surface MHC I has been reported to increase only by 2- to 3-fold (Ackerman and Cresswell, 2003), or not at all (Cella et al., 1997a; Delamarre et al., 2003; Wilson et al., 2004) in mature DCs. This is consistent with the increase in MHC I synthesis observed in mature DCs (Cella et al., 1997a; Rescigno et al., 1998; Villadangos et al., 2001; Wilson et al., 2004). Indeed, it makes sense for mature DCs to keep

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 287 presenting endogenous antigens via MHC I molecules to allow detection of viruses that might infect the DCs themselves. So how can mature DCs maintain a memory of their cross-presented antigens? We can provide three hypotheses. The first is that the epitopes known to be cross-presented may be particularly stable. It is known that the half-life and immunogenicity of different MHC I–peptide complexes is determined by their stability, which in turn depends on the affinity of the MHC I–peptide interaction (Chen et al., 1994; Sette et al., 1994). This affinity can vary broadly among MHC I–peptide combinations. Perhaps only those peptides that interact with MHC I molecules with high affinity can be cross-presented long enough to induce cross-priming or cross-tolerance. If this were the case, it would not be necessary to invoke a specific mechanism of enhancement of the half-life of these complexes in mature DCs. The second hypothesis is that a fraction of the ergosomes may be relatively stable and not fuse with lysosomes. As discussed for MHC II presentation, DCs may deliver some antigens into ‘‘storage’’ compartments with a lower proteolytic activity. Similarly, some of the ergosomes may ‘‘branch off’’ the main endocytic track and store some of the exogenous antigens for sustained cross-presentation. The third hypothesis is that the MHC I–peptide complexes generated via the cross-presentation pathway may be ‘‘tagged’’ in the ergosomes, so that once they are deposited on the cell surface, their endocytosis and turnover rates are much slower than those of their counterparts generated via the classic pathway. Such tagging might consist of their association with membrane microdomains, posttranslational modifications such as phosphorylation, or the expression of carbohydrates not modified by transport through the Golgi complex. 8. Conclusions and Future Directions Biochemical and cell biological studies have revealed the molecular basis of some of the unique antigen presentation capabilities shown by DCs. In general, components of the antigen presentation machinery expressed in DCs are not different from those of other antigen-presenting cells. However, this machinery has been adapted in DCs to fulfill two distinct functions linked to the maturational state of the DCs. In the steady state, immature DCs constitutively express on their surface MHC class II molecules loaded with self-peptide ligands. By virtue of cross-presentation, some DC types can also cross-present on their MHC I molecules peptides derived from proteins collected from the environment (Heath et al., 2004). This allows immature DCs to provide naive T cells with MHC–peptide ligands for maintenance of homeostasis and a constant supply of information about the peripheral self for maintenance of tolerance. In contrast, DCs activated by the presence of pathogens or tissue damage

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undergo dramatic changes that result in their presentation of a snapshot of their antigenic environment at the time and site of activation. This is accomplished by interrupting the processes of MHC II presentation and MHC I crosspresentation once the activated DC has accumulated a substantial amount of MHC–peptide complexes on its surface. As has been summarized here, the checkpoints that control these developmental changes for the MHC II and the MHC I pathways are different. The studies carried out on the MHC II pathway indicate that the regulatory mechanisms operate at the level of MHC II synthesis and MHC II–peptide turnover. However, the signals that control the trafficking and turnover of the peptide-loaded MHC II molecules remain unknown, as does the sorting machinery involved in interpreting those signals. Is the turnover regulated at the level of internalization from the plasma membrane or at the level of recycling from early endosomes back to the cell surface? Does the association with lipid rafts play a direct role in controlling MHC II trafficking? How does the formation of tubular tracks for the delivery of MHC II–peptide complexes fit with the dynamics of DC trafficking and maturation and with establishment of the immunological synapse? The development of sophisticated experimental systems that allow direct visualization of the DC–T-cell interaction in the lymphoid organs may provide answers to some of these questions. In the case of MHC I cross-presentation, the mechanisms of regulation are much less defined; these may involve the generation or ergosomes, the recruitment of proteasomes to the vicinity of these compartments, or the delivery of the components of the MHC I peptide-loading machinery to the ergosomes. The mechanisms involved in formation of the ergosomes themselves, and the minimum requirements that make them antigen presentation–competent compartments need to be defined. Is Sec61 the channel used by exogenous antigens to access the cytosol? Are the peptides generated by the crosspresentation pathway identical to those generated in the direct pathway? Does the formation of MHC I–peptide complexes in the ergosome necessitate all the components of the peptide-loading machinery involved in the endoplasmic reticulum? Answering these questions will provide us not only with a better understanding of the functions played by DCs in tolerance and immunity, they may also point to novel targets for the improvement of DC-based vaccines and other immunotherapeutic strategies. Acknowledgments We thank our colleague Bill Heath at the Walter and Eliza Hall Institute for critically reading the manuscript and for suggestion of the term ‘‘ergosome.’’ Nicholas S. Wilson is supported by a Melbourne University Research Scholarship and by a Student Project Grant from the Cooperative

c on t r o l o f a n t i g en p r e s e n tat i o n i n d e nd r i t i c ce l l s 289 Research Centre for Vaccine Technology. Jose A. Villadangos is funded by a Scholarship of the Leukemia and Lymphoma Society and grants of the Human Frontiers Science Program Organization, the Anti-Cancer Council of Australia, and the Australian National Health and Medical Research Council.

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Index

A ACE, see Angiotensin-converting enzyme Activation-induced cytidine deaminase Apobec-1 homology and function, 79–80 discovery and isolation, 76–77 expression regulation, 77 mutation studies, 85 replication protein A interactions, 78 single-stranded DNA substrates, 78 S region targeting, 78 substrate formation, 80–82 ADA deficiency, see Adenosine deaminase deficiency Adenosine deaminase deficiency adenosine signaling and immune development and function, 17–20 CD26–enzyme interactions, 5–6 20 -deoxyadenosine accumulation and toxicity S-adenosylhomocysteine hydroxylase inhibition, 12, 16–17 apoptosis derangements, 13–14 lymphoid tissues, 12 ribonucleotide reductase inhibition, 13 enzyme function, 2 gene mutations, 3–4 gene therapy prospects, 4–5, 35 hairy cell leukemia treatment implications, 20–21 knockout mouse models adenosine receptor expression eosinophils, 26–27 mast cells, 26 pulmonary signaling, 27 generation, 7 immune system effects B cell maturation effects, 11–12 T cell development, 9–11, 34

neurological defects, 32–33 phenotypes, 7, 9 respiratory effects, 21, 32 trophoblast cell essentiality and gene delivery for fetal rescue, 8–9 phenotypic and metabolic disturbances, 6–7 pulmonary consequences of adenosine elevation adenosine amplification model in lung disease, 29–31 enzyme therapy reversal of abnormalities, 23, 25 inflammation, 23 interleukin-13 mediation of lung injury, 28–29 lung disease implications, 21–22, 27–29 severe combined immunodeficiency disease, 2–4, 31 treatment, 4–5, 35 S-Adenosylhomocysteine hydroxylase, inhibition in adenosine deaminase deficiency, 12, 16–17 AID, see Activation-induced cytidine deaminase Allergy, contact activation role, 191–192 Alzheimer’s disease, contact activation pathway in pathophysiology, 194–195 b-Amyloid, bradykinin formation cascade component interactions, 194–195 Angiotensin-converting enzyme blood pressure regulation, 192 bradykinin inactivation, 184–185 Antigen presentation, see also Dendritic cell systems, 251–252 terminology, 251 Anti-phospholipid antibodies, fetal loss syndrome role, 152–153

307

308 a1-Antitrypsin, contact activation inhibition, 183 Apobec-1, activation-induced cytidine deaminase homology and function, 79–80 Apoptosis, adenosine deaminase deficiency derangements, 13–14 Artemis class switch recombination role, 90–91 DNA double-strand break joining mediation in V(D)J recombination, 60 Asthma, adenosine signaling, 21–22, 31 ATM, class switch recombination role, 91 B B cell activation and class switch recombination regulation, 72 adenosine deaminase deficiency effects on maturation, 11–12 terminal deoxynucleotidyltransferase splice variants in development, 120–121, 123, 127–129 B cell receptor, V(D)J recombination genomic organization in mouse, 47–50 overview, 44–46 recombinase-activating genes, see Recombinase-activating genes regulated accessibility of gene segments chromatin modification, 67–68 histone H2AX role, 68–69 overview, 64–65 transcriptional regulation, 65–67 Bcl-2, cytokine induction and natural killer cell survival promotion, 223–224 Bradykinin cardiovascular disease and blood pressure control implications, 193–194 contact activation allergy role, 191–192 Alzheimer’s disease implications, 194–195 blood cell binding of mediators, 173–175 factor XII binding to human umbilical vein endothelial cells, 169, 172 high molecular weight kininogen binding to human umbilical vein endothelial cells, 166–169 inflammatory disease derangements, 194 inhibition, 181–184 kinin cascade activation cell surface binding, 175–177

I ND EX

factor XII-independent activation of prekallikrein–high molecular weight kininogen complex, 177–180 factor XII role, 180–181 pathway, 161–166 inactivation, 184–185 plasma pathway of generation, 160–161 C C1 inhibitor contact activation inhibition, 181–183 deficiency and disease pathogenesis, 188–191 CD26, adenosine deaminase interactions, 5–6 Chronic obstructive pulmonary disease, adenosine signaling, 21–22 Class switch recombination diseases hyper-IgM syndromes, 93–94 X-linked hypohydrotic ectodermal dysplasia, 95 heavy chain constant region gene organization, 71–72 mechanism activation-induced cytidine deaminase role, see also Activation-induced cytidine deaminase cytidine deamination, 82 DNA double-strand break induction, 85–86 mutation studies, 85 substrate formation, 80–82 Artemis role, 90–91 ATM role, 91 base excision repair, 82 DNA deamination model, 82–84 DNA double-strand break repair, 90–92 DNA-PKcs role, 88–91 53BP1 role, 91–92 histone H2AX modifications, 88, 92 Ku role, 90 mismatch repair, 89, 93 NBS1 role, 92–93 promoter/enhancer interactions, 88 S region deletions, 86–87 mutations, 87 recombination and synapsis, 87–89 transcription, 80–82

I ND EX

overview, 44–46, 69–71 regulation B cell activation, 72 germline heavy chain constant region transcripts, 72–74 regional specificity in regulation, 74–76 S region sequence mutations, 76 V(D)J recombination comparison, 95–97 CLIP peptide, major histocompatibility complex II–peptide complex formation, 260–261, 274 Clq receptor high molecular weight kininogen and factor XII binding on human umbilical vein endothelial cells, 166–169, 172 human umbilical vein endothelial cell membrane interactions, 172–173 Complement C1 activation, 187–188 fetal loss syndrome mediation, 151–153 ischemia–reperfusion injury mediation overview, 139 renal ischemia–reperfusion injury, 147–151 COPD, see Chronic obstructive pulmonary disease Crry, fetal loss syndrome role, 152–153 CSR, see Class switch recombination Cytidine deaminase, see Activation-induced cytidine deaminase Cytokeratin 1 high molecular weight kininogen binding on human umbilical vein endothelial cells, 168–169 human umbilical vein endothelial cell membrane interactions, 172–173 D DC, see Dendritic cell Dendritic cell antigen capture macropinocytosis, 255 overview, 252–253 phagocytosis, 253–255 receptor-mediated endocytosis, 255–256 regulation, 256–257 antigen presentation degradation of antigen, 259 endocytic pathway, 258–259

309 major histocompatibility complex II–peptide complex formation, 259–261 plasticity, 262–263 subcellular localization of peptide binding, 261–262 cross-presentation antigen transfer to cytosol, 283–285 compartmentalization, 282–283 overview, 280–282 peptide-loading machinery, 285 regulation during maturation, 285–287 culture from precursors, 250–251 immunity and tolerance interactions, 241–242 interleukin-15 production and natural killer cell modulation, 230 major histocompatibility complex II antigen presentation control antigenic memory biological basis, 278–280 endosomal proteolysis regulation, 265–268 major histocompatibility complex II molecule synthesis, 277–278 major histocompatibility complex II–peptide complex formation regulation H-2DM regulation, 270–271 Ii degradation, 268–270 techniques for study, 271–273 models, 263–265 trafficking control, 273–274, 276–277 maturation, 243 prospects for study, 287–288 subtypes blood-derived cells, 247–250 mouse versus human systems, 250 overview, 243, 245 tissue-derived cells, 245–247 DNA ligase 4, DNA double-strand break joining mediation in V(D)J recombination, 60 DNA mismatch repair, class switch recombination, 89, 93 DNA-PKcs class switch recombination role, 88–91 DNA double-strand break joining mediation in V(D)J recombination, 59–60 terminal deoxynucleotidyltransferase interactions, 126

310 E Elf-1, terminal deoxynucleotidyltransferase expression regulation, 119 Eosinophils, adenosine receptor expression, 26–27 F Factor XII activation, 164–165, 180 b-amyloid interactions, 194–195 blood cell binding, 173–175 blood pressure regulation, 192 bradykinin formation role, 160, 162, 164–165 human umbilical vein endothelial cell binding, 169, 172 inflammatory disease derangements, 194 inhibition of contact activation, 181 intrinsic fibrinolytic cascade, 185–187 leukocyte interactions, 188 plasma protease interactions, 187–188 prekallikrein–high molecular weight kininogen complex activation, 180–181 Fetal loss syndrome, innate autoimmunity, 151–153 53BP1, class switch recombination role, 91–92 Flt3 ligand, synergy with interleukin-15 in natural killer cell development, 217 G Gene therapy, adenosine deaminase deficiency, 4–5, 35 H H-2DM, major histocompatibility complex II–peptide complex formation regulation in dendritic cells, 270–271 Heat shock protein 90, factor XII-independent activation of prekallikrein–high molecular weight kininogen complex, 178–180 High molecular weight kininogen (HK) b-amyloid interactions, 194–195 blood cell binding, 173–175 bradykinin formation role, 160, 162–165 domains, 162–163 factor XII activation, 164–165 human umbilical vein endothelial cell binding, 166–169 HK, see High molecular weight kininogen

I ND EX

Histone H2AX class switch recombination modifications, 88, 92 regulation in V(D)J recombination, 68–69 HK, see High molecular weight kininogen HSP90, see Heat shock protein 90 Hyper-IgM syndromes class switch recombination defects, 93–94 I IL-2, see Interleukin-2 IL-13, see Interleukin-13 IL-15, see Interleukin-15 Innate immune system components, 138 autoimmune injury, see Fetal loss syndrome; Ischemia–reperfusion injury Interleukin-2 interleukin-15 comparison cell distribution, 213–214 cis versus trans signaling, 214–215 signaling, 212–213 in vitro versus in vivo similarities, 212 natural killer cell function and homeostasis role apoptosis, 225–226 CD56bright cell subset cytokine production, 228 expansion with low-dose interleukin-2 therapy mechanisms, 221–222 features, 220–221 maturation in secondary lymphoid organs with low-dose interleukin-2 therapy, 228–229 rationale for expansion with low-dose interleukin-2 therapy, 221 cytokine production modulation, 227–228 cytolytic activity enhancement, 226–227 development studies cultured cell versus peripheral blood cell studies, 217–218 historical perspective, 215–216 mechanisms, 219–220 overview, 209–210, 231–232 proliferation role, 224–225 survival promotion mechanisms, 223–224 receptors, 121 signal transduction cascades, 211–212 Interleukin-13, lung injury in transgenic mouse and adenosine role, 28–29

311

I ND EX

Interleukin-15 interleukin-12 comparison cell distribution, 213–214 cis versus trans signaling, 214–215 receptor a-chain signaling, 212–213 in vitro versus in vivo similarities, 212 natural killer cell function and homeostasis role apoptosis, 225–226 cytokine production modulation, 227–228 cytolytic activity enhancement, 226–227, 230 development studies cultured cell versus peripheral blood cell studies, 217–218 endogenous cytokine pools, 218 hematopoietic precursor differentiation induction, 216–217 mechanisms, 219 synergy with c-Kit ligand and Flt3 ligand, 217 immune cell sources for modulation, 230 overview, 209–210, 231–232 proliferation role, 224–225 survival promotion Bcl-2 independent mechanisms, 223–224 Bcl-2 induction, 223 cultured cell and animal studies, 222–223 receptors, 121 signal transduction cascades, 211–212 Ischemia–reperfusion injury acute inflammatory response, 138–139 antibody mediation ischemia-related antigens, 145, 147 knockout mouse studies, 140 monoclonal immunoglobulin M initiation, 143–145 specificity of natural immunoglobulin M, 140–141, 143 complement mediation overview, 139 renal ischemia–reperfusion injury, 147–151 K Kallikrein bradykinin formation role, 160–162

contact activation inhibition, 181 factor XII-independent activation of prekallikrein–high molecular weight kininogen complex, 177–180 inflammatory disease derangements, 194 intrinsic fibrinolytic cascade, 185–187 leukocyte interactions, 188 prekallikrein complex, 162 Kidney, ischemia–reperfusion injury and complement mediation, 147–151 Kininogen, see High molecular weight kininogen c-Kit ligand synergy with interleukin-15 in natural killer cell development, 217 natural killer cell CD56bright subset expression, 220 Ku class switch recombination role, 90 DNA double-strand break joining mediation in V(D)J recombination, 59 terminal deoxynucleotidyltransferase interactions, 126 L Leukemia hairy cell leukemia treatment, 20–21 terminal deoxynucleotidyltransferase expression and activity, 131–133 M a2-Macroglobulin, contact activation inhibition, 182–183 Major histocompatibility complex class II antigen presentation, see Dendritic cell Mast cells, adenosine receptor expression, 26 N Natural killer cell interleukin-2 role in function and homeostasis apoptosis, 225–226 CD56bright cell subset cytokine production, 228 expansion with low-dose interleukin-2 therapy mechanisms, 221–222

312 Natural killer cell (continued) features, 220–221 maturation in secondary lymphoid organs with low-dose interleukin-2 therapy, 228–229 rationale for expansion with low-dose interleukin-2 therapy, 221 cytokine production modulation, 227–228 cytolytic activity enhancement, 226–227 development studies cultured cell versus peripheral blood cell studies, 217–218 historical perspective, 215–216 mechanisms, 219–220 overview, 209–210, 231–232 proliferation role, 224–225 survival promotion mechanisms, 223–224 interleukin-15 role in function and homeostasis apoptosis, 225–226 cytokine production modulation, 227–228 cytolytic activity enhancement, 226–227, 230 development studies cultured cell versus peripheral blood cell studies, 217–218 endogenous cytokine pools, 218 hematopoietic precursor differentiation induction, 216–217 mechanisms, 219 synergy with c-Kit ligand and Flt3 ligand, 217 immune cell sources for modulation, 230 overview, 209–210, 231–232 proliferation role, 224–225 survival promotion Bcl-2-independent mechanisms, 223–224 Bcl-2 induction, 223 cultured cell and animal studies, 222–223 NBS1, class switch recombination role, 92–93 P PCNA, see Proliferating cell nuclear antigen

I ND EX

Proliferating cell nuclear antigen, terminal deoxynucleotidyltransferase interactions, 127 R RAGs, see Recombinase-activating genes Recombinase-activating genes DNA double-strand break joining mediators in V(D)J recombination Artemis, 60 DNA ligase 4, 60 DNAPKcs, 59–60 Ku, 59 pathways, 58–59 XRCC4, 60 expression regulation in V(D)J recombination allelic exclusion and feedback regulation, 61–64 downregulation effects, 64 lymphoid-specific expression, 61 gene structure, 50–51 genetic translocation induction, 51 severe combined immunodeficiency disease defects, 51 V(D)J recombination initiation cleavage reaction biochemistry, 54–56 coding and signal joint formation, 57–58 postcleavage complex, 56–57 precleavage complex assembly, 53–54 target sequence recognition, 51–53 Reperfusion injury, see Ischemia–reperfusion injury Replication protein A, activation-induced cytidine deaminase interactions, 78 Ribonucleotide reductase, inhibition in adenosine deaminase deficiency, 13 RPA, see Replication protein A S SAH hydrolase, see S-Adenosylhomocysteine hydroxylase SCID, see Severe combined immunodeficiency disease Severe combined immunodeficiency disease adenosine deaminase deficiency, 2–4, 31 recombinase-activating gene deficiency, 51 X-linked form, 5

I ND EX

SHM, see Somatic hypermutation Somatic hypermutation mechanism activation-induced cytidine deaminase role, see also Activation-induced cytidine deaminase cytidine deamination, 82 DNA double-strand break induction, 85–86 mutation studies, 85 substrate formation, 80–82 Artemis role, 90–91 ATM role, 91 base excision repair, 82 DNA deamination model, 82–84 DNA double-strand break repair, 90–92 DNA-PKcs role, 88–91 53BP1 role, 91–92 histone H2AX modifications, 88, 92 Ku role, 90 mismatch repair, 89, 93 NBS1 role, 92–93 promoter/enhancer interactions, 88 S region deletions, 86–87 mutations, 76, 87 recombination and synapsis, 87–89 transcription, 80–82 overview, 69–71 regional specificity in regulation, 74–76 V(D)J recombination comparison, 95–97 S region, see Class switch recombination; Somatic hypermutation T T cell adenosine deaminase deficiency effects on development, 9–11, 34 adenosine receptors, 19–20 terminal deoxynucleotidyltransferase splice variants in development, 120–121, 123, 127–129 T cell receptor, V(D)J recombination genomic organization in mouse, 47–50 overview, 44–46 recombinase-activating genes, see Recombinase-activating genes regulated accessibility of gene segments chromatin modification, 67–68

313 histone H2AX role, 68–69 overview, 64–65 transcriptional regulation, 65–67 TdIF1, terminal deoxynucleotidyltransferase interactions, 127 TdT, see Terminal deoxynucleotidyltransferase Terminal deoxynucleotidyltransferase DNA polymerase family X polymerases, 115 domains, 124–126 leukemia expression and activity, 131–133 phylogenetic analysis, 115, 117 prospects for study, 133 protein–protein interactions, 126–127 splice variants junctional diversity role in V(D)J recombination, 123–124 knockout and transgenic mouse studies of repertoire development, 127–130 lymphocyte development, 120–121, 123 sequences, 122 TdTL1, 119 TdTL2, 119 TdTS, 119 substrate specificity, 130 tissue distribution, 115 transcriptional regulation of expression, 117, 119 U u-PAR, see Urokinase plasminogen activator receptor Urokinase plasminogen activator receptor high molecular weight kininogen and factor XII binding on human umbilical vein endothelial cells, 168–169, 172 human umbilical vein endothelial cell membrane interactions, 172–173 V V(D)J recombination antigen receptor gene rearrangement genomic organization in mouse, 47–50 recombinase-activating genes, see Recombinase-activating genes regulated accessibility of gene segments chromatin modification, 67–68 histone H2AX role, 68–69 overview, 64–65

314 V(D)J recombination (continued) transcriptional regulation, 65–67 class switch recombination comparison, 95–97 junctional diversity, 114–115, 123–124 overview, 44–46, 114

I ND EX

X X-linked hypohydrotic ectodermal dysplasia, class switch recombination defects, 95 XRCC4, DNA double-strand break joining mediation in V(D)J recombination, 60

Contents of Recent Volumes

Ju¨rgen Hess, Ulrich Schaible, Ba¨rbel Raupach, and Stefan H. E. Kaufmann

Volume 74 Biochemical Basis of Antigen-Specific Suppressor T Cell Factors: Controversies and Possible Answers Kimishice Isihzaka, Yasuyuki Ishii, Tatsumi Nakano, and Katsuji Sugik

The Cytoskeleton in Lymphocyte Signaling A. Bauch, F. W. Alt, G. R. Crabtree, and S. B. Snapper

The Role of Complement in B Cell Activation and Tolerance Michael C. Carroll

TGF- Signaling by Smad Proteins Kohei Miyazono, Peter ten Dijke, and Carl-Henrik Heldin

Receptor Editing in B Cells David Nemazee

MHC Class II-Restricted Antigen Processing and Presentation Jean Pieters

Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection Pius Loetscher, Bernhard Moser, and Marco Bacciolini

T-Cell Receptor Crossreactivity and Autoimmune Disease Harvey Cantor

Escape of Human Solid Tumors from T-Cell Recognition: Molecular Mechanisms and Functional Significance Francesco M. Marincola, Elizabeth M. Jaffee, Daniel J. Hicklin, and Soldano Ferrone

Strategies for Immunotherapy of Cancer Cornelis J. M. Meliey, Rene E. M. Toes, Jan Paul Medema, Sjoerd H. van der Burg, Ferry Ossendorp, and Rienk Offringa

The Host Response to Leishmania Infection Werner Solbacii and Tamas Laskay

Tyrosine Kinase Activation in the Decision between Growth, Differentiation, and Death Responses Initiated from the B Cell Antigen Receptor Robert C. Hsueh and Richard H. Scheuermann

Index

Volume 75 Exploiting the Immune System: Toward New Vaccines against Intracellular Bacteria

The 30 IgH Regulatory Region: A Complex Structure in a Search for a Function

315

316 Ahmed Amine Khamlichi, Eric Pinaud, Catherine Decourt, Christine Chauveau, and Michel Cogne´

co n t e nt s o f re c e nt vo l um es Human Basophils: Mediator Release and Cytokine Production John T. Schroeder, Donald W. MacGlashan, Jr., and Lawrence M. Lichtenstein

Index

Volume 76 MIC Genes: From Genetics tok Biology Seiamak Bahram CD40-Mediated Regulation of Immune Responses by TRAF-Dependent and TRAF-Independent Signaling Mechanisms Amrif C. Grammer and Peter E. Lipsky Cell Death Control in Lymphocytes Kim Newton and Andreas Strassen Systemic Lupus Erythematosus, Complement Deficiency, and Apoptosis M. C. Pickering, M. Botto, P. R. Taylor, P. J. Lachmann, and M. J. Walport Signal Transduction by the High-Affinity Immunoglobulin E Receptor FceRI: Coupling Form to Function Monica J. S. Nadler, Sharon A. Matthews, Helen Tuhner, and Jean-Pierre Kinet Index

Btk and BLNK in B Cell Development Satoshi Tsukada, Yoshihiro Baba, and Dai Watanabe Diversity and Regulatory Functions of Mammalian Secretory Phospholipase A2s Makoto Murakami and Ichiro Kudo The Antiviral Activity of Antibodies in Vitro and in Vivo Paul W. H. I. Parren and Dennis R. Burton Mouse Models of Allergic Airway Disease Clare M. Lloyd, Jose-Angel Gonzalo, Anthony J. Coyle, and Jose-Carlos Gutierrez-Ramos Selected Comparison of Immune and Nervous System Development Jerold Chun Index

Volume 78 Toll-like Receptors and Innate Immunity Shizuo Akira

Volume 77 The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction S. Celeste Posey Morley and Barbara E. Bierer Raft Membrane Domains and Immunoreceptor Functions Thomas Harder

Chemokines in Immunity Osamu Yoshie, Toshio Imai, and Hisayuki Nomiyama Attractions and Migrations of Lymphoid Cells in the Organization of Humoral Immune Responses Christoph Schaniel, Antonius G. Rolink, and Fritz Melchers

317

c o nt e n t s of re c e n t vo l u m es Factors and Forces Controlling V(D)J Recombination David G. T. Hesslein and David G. Schatz T Cell Effector Subsets: Extending the Th1/Th2 Paradigm Tatyana Chtanova and Charles R. Mackay MHC-Restricted T Cell Responses against Posttranslationally Modified Peptide Antigens Ingelise Bjerring Kastrup, Mads Hald Andersen, Tim Elliot, and John S. Haurum Gastrointestinal Eosinophils in Health and Disease Marc E. Rothenberg, Anil Mishra, Eric B. Brandt, and Simon P. Hogan Index

Volume 79 Neutralizing Antiviral Antibody Responses Rolf M. Zinkernagel, Alain Lamarre, Adrian Ciurea, Lukas Hunziker, Adrian F. Ochsenbein, Kathy D. McCoy, Thomas Fehr, Martin F. Bachmann, Ulrich Kalinke, and Hans Hengartner Regulation of Interleukin-12 Production in Antigen-Presenting Cells Xiaojing Ma and Giorgio Trinchieri

Regulation of Antibacterial and Antifungal Innate Immunity in Fruitflies and Humans Michael J. Williams Functional Heavy-Chain Antibodies in Camelidae Viet Khong Nguyen, Aline Desmyter, and Serge Muyldermans Uterine Natural Killer Cells in the Pregnant Uterus Chau-Ching Liu and John Ding-E Young Index

Volume 80 Protein Degradation and the Generation of MHC Class I-Presented Peptides Kenneth L. Rock, Ian A. York, Tomo Saric, and Alfred L. Goldberg Proteoanalysis and Antigen Presentation by MHC Class II Molecules Paula Wolf Bryant, Ana-Maria Lennon-Dume´ nil, Edda Fiebiger, Ce´ cile Lagaudrie´ re-Gesbert, and Hidde L. Ploegh Cytokine Memore of T Helper Lymphocytes Max Lo¨ hning, Anne Richter, and Andreas Radbruch

Mechanisms of Signaling by the Hematopoietic-Specific Adaptor Proteins, SLP-76 and LAT and Their B Cell Counterpart, BLNK/SLP-65 Deborah Yablonski and Arthur Weiss

Ig Gene Hypermutation: A Mechanism is Due Jean-Claude Weill, Barbara Bertocci, Ahmad Faili, Said Aoufouchi, Ste´ phane Frey, Annie De Smet, Se´ bastian Storck, Auriel Dahan, Fre´ de´ ric Delbos, Sandra Weller, Eric Flatter, and Claude-Agne´ s Reynaud

Xenotransplantation David H. Sachs, Megan Sykes, Simon C. Robson, and David K. C. Cooper

Generalization of Single Immunological Experiences by Idiotypically Mediated Clonal Connections Hilmar Lemke and Hans Lange

318

co n t e nt s o f re c e nt vo l um es

The Aging of the Immune System B. Grubeck-Loebenstein and G. Wick

Volume 82

Index

Transcriptional Regulation in Neutrophils: Teaching Old Cells New Tricks Patrick P. McDonald

Volume 81 Regulation of the Immune Response by the Interaction of Chemokines and Proteases Jo Van Damme and Sofie Struyf Molecular Mechanisms of Host-Pathogen Interaction: Entry and Survival of Mycobacteria in Macrophages Jean Pieters and John Gatfield B Lymphoid Neoplasms of Mice: Characteristics of Naturally Occurring and Engineered Diseasse and Relationships to Human disorders Herbert Morse et al. Prions and the Immune System: A Journey Through Gut Spleen, and Nerves Adriano Aguzzi Roles of the Semaphorin Family in Immune Regulation H. Kikutani and A. Kumanogoh HLA-G Molecules: from Maternal-Fetal Tolerance to Tissue Acceptance Edgardo Carosella et al. The Zebrafish as a Model Organism to Study Development of the Immune System Nick Trede et al.

Tumor Vaccines Freda K. Stevenson, Jason Rice, and Delin Zhu Immunotherapy of Allergic Disease R. Valenta, T. Ball, M. Focke, B. Linhart, N. Mothes, V. Niederberger, S. Spitzauer, I. Swoboda, S.Vrtala, K. Westritschnic, and D. Kraft Interactions of Immunoglobulins Outside the Antigen-Combining Site Roald Nezlin and Victor Ghetie The Roles of Antibodies in Mouse Models of Rheumatoid Arthritis, and Relevance to Human Disease Paul A. Monach, Christophe Benoist, and Diane Mathis MUC1 Immunology: From Discovery to Clinical Applications Anda M. Vlad, Jessica C. Kettel, Nehad M. Alajez, Casey A. Carlos, and Olivera J. Finn Human Models of Inherited Immunoglobulin Class Switch Recombination and Somatic Hypermutation Defects (Hyper-IgM Syndromes) Anne Durandy, Patrick Revy, and Alain Fischer

Control of Autoimmunity by Naturally Arising Regulatory CD4þ T Cells S. Sakaguchi

The Biological Role of the C1 Inhibitor in Regulation of Vascular Permeability and Modulation of Inflammation Alvin E. Davis, III, Shenghe Cai, and Dongxu Liu

Index

Index

319

c o nt e n t s of re c e n t vo l u m es

Volume 83

Volume 84

Lineage Commitment and Developmental Plasticity in Early Lymphoid Progenitor Subsets David Traver and Koichi Akashi

Interactions Between NK Cells and B Lymphocytes Dorothy Yuan

The CD4/CD8 Lineage Choice: New Insights into Epigenetic Regulation during T Cell Development Ichiro Taniuchi, Wilfried Ellmeier, and Dan R. Littman CD4/CD8 Coreceptors in Thymocyte Development, Selection, and Lineage Commitment: Analysis of the CD4/CD8 Lineage Decision Alfred Singer and Remy Bosselut Development and Function of T Helper 1 Cells Anne O’Garra and Douglas Robinson Th2 Cells: Orchestrating Barrier Immunity Daniel B. Stetson, David Voehringer, Jane L. Grogan, Min Xu, R. Lee Reinhardt, Stefanie Scheu, Ben L. Kelly, and Richard M. Locksley Generation, Maintenance, and Function of Memory T Cells Patrick R. Burkett, Rima Koka, Marcia Chien, David L. Boone, and Averil Ma

Multitasking of Helix-Loop-Helix Proteins in Lymphopoiesis Xiao-Hong Sun Customized Antigens for Desensitizing Allergic Patients Fa´ tima Ferreira, Michael Wallner, and Josef Thalhamer Immune Response Against Dying Tumor Cells Laurence Zitvogel, Noelia Casares, Marie O. Pe´ quignot, Nathalie Chaput, Mathew L. Albert, and Guido Kroemer HMGB1 in the Immunology of Sepsis (Not Septic Shock) and Arthritis Christopher J. Czura, Huan Yang, Carol Ann Amella, and Kevin J. Tracey Selection of the T-Cell Repertoire: Receptor-Controlled Checkpoints in T-Cell Development Harald Von Boehmer The Pathogenesis of Diabetes in the NOD Mouse Michelle Solomon and Nora Sarvetnick

CD8þ Effector Cells Pierre A. Henkart and Marta Catalfamo

Index

An Integrated Model of Immunoregulation Mediated by Regulatory T Cell Subsets Hong Jiang and Leonard Chess

Volume 85

Index

Cumulative Subject Index Volumes 66–82