Advances in Immunology Volume 87

Contents

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Role of the LAT Adaptor in T-Cell Development and Th2 Differentiation Bernard Malissen, Enrique Aguado, and Marie Malissen 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The LAT Signalosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LAT: An Essential Component of the Pre-TCR . . . . . . . . . . . . . . . Role of LAT in gd T-Cell Development . . . . . . . . . . . . . . . . . . . . . Negative Regulatory Role of LAT . . . . . . . . . . . . . . . . . . . . . . . . . . Positive and Negative Selection in LatY136F Mice . . . . . . . . . . . . . . Cooperative Assemblies Within the LAT Signalosome . . . . . . . . . . Redundancy Among LAT Tyrosines. . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of Signal Termination by LAT. . . . . . . . . . . . . . . . . . . Th2-Type Immunity in LatY136F and LatY7/8/9F Mice . . . . . . . . . . . . Is a LAT-Signalosome Pathology Taking Shape? . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 2 5 7 9 13 15 17 18 19 19 22 22

The Integration of Conventional and Unconventional T Cells that Characterizes Cell-Mediated Responses Daniel J. Pennington, David Vermijlen, Emma L. Wise, Sarah L. Clarke, Robert E. Tigelaar, and Adrian C. Hayday Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Integrating Immune Responses in the Public Good . . . . . . . . . . . . 3. Evidence for T-Cell Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

27 28 28 29

c on t e n ts

vi 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Mechanisms of Action of Unconventional T Cells. . . . . . . . . . . . . . Immune Integration via Cytolysis by gd Cells . . . . . . . . . . . . . . . . . Targeting by Cytolytic gd Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunointegration by Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunointegration by Chemokines . . . . . . . . . . . . . . . . . . . . . . . . . Immunointegration by Adhesion and Costimulatory Molecules . . . Additional Clues to Immunoregulation . . . . . . . . . . . . . . . . . . . . . . A Spectrum of Unconventional T Cells . . . . . . . . . . . . . . . . . . . . . . Unconventional T Cells and NK Cells . . . . . . . . . . . . . . . . . . . . . . . A Developmental Program of T-Cell Integration. . . . . . . . . . . . . . . T-Cell Integration: Genetics and Disease . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 34 39 40 43 44 46 47 48 48 50 51

Negative Regulation of Cytokine and TLR Signalings by SOCS and Others Tetsuji Naka, Minoru Fujimoto, Hiroko Tsutsui, and Akihiko Yoshimura 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Cytokine Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Negative Regulation of Cytokine Signaling . . . . . . . . . . . . . . . . . . . 64 Regulation of Cytokine Signaling by SOCS Proteins (Tables 1 and 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 TLR-Mediated Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Signal Transduction Pathways Through TLRs . . . . . . . . . . . . . . . . . 88 Major Biological Events by the TLR-Mediated Cell Activation . . . 92 Pathophysiological Roles for TLR-Mediated Signal Pathways. . . . . 93 Negative Regulation of the TLR Signalings . . . . . . . . . . . . . . . . . . . 96 Regulation of TLR Signaling by SOCS . . . . . . . . . . . . . . . . . . . . . . 101 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Pathogenic T-Cell Clones in Autoimmune Diabetes: More Lessons from the NOD Mouse Kathryn Haskins 1. 2. 3. 4.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenic T-Cell Clones—Effector Function. . . . . . . . . . . . . . . . . Migration of Pathogenic T-Cell Clones . . . . . . . . . . . . . . . . . . . . . . T-Cell Clones in T-Cell Receptor Transgenic (TCR-Tg) Mice . . . .

123 123 126 133 135

c o nt e n t s 5. Antigens for Pathogenic T-Cell Clones . . . . . . . . . . . . . . . . . . . . . . 6. Tracking of Pathogenic T-Cell Clones with MHC Tetramers . . . . . 7. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 140 149 152 154

The Biology of Human Lymphoid Malignancies Revealed by Gene Expression Profiling Louis M. Staudt and Sandeep Dave 1. 2. 3. 4.

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Diagnosis of Lymphoid Malignancies . . . . . . . . . . . . . . . Gene Expression-Based Survival Predictors . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

163 164 165 177 197 198

New Insights into Alternative Mechanisms of Immune Receptor Diversification Gary W. Litman, John P. Cannon, and Jonathan P. Rast 1. 2. 3. 4.

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Variation in Innate Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Mechanisms that Diversify Immune Receptors . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

209 210 211 216 228 231

The Repair of DNA Damages/Modifications During the Maturation of the Immune System: Lessons from Human Primary Immunodeficiency Disorders and Animal Models Patrick Revy, Dietke Buck, Franc¸oise le Deist, and Jean-Pierre de Villartay Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Fundamental Mechanisms of Lymphoid-Specific DNA Cleavage and Repair Mechanisms . . . . . . . . . . . . . . . . . . . . . 3. Human Primary Immunodeficiency Disorders Associated with Defective DNA Repair. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237 238 240 257

c on t e n ts

viii

4. Human Primary Immunodeficiency Disorders Associated with Defective Cell Cycle Control Following DNA Damage . . . . . 5. Defective DNA Repair and Malignancies in the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268 274 278 279

Antibody Class Switch Recombination: Roles for Switch Sequences and Mismatch Repair Proteins Irene M. Min and Erik Selsing 1. 2. 3. 4. 5.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class Switch Recombination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting of the CSR Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . Proteins Involved in CSR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297 297 298 300 305 319 320

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Contents of Recent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 Color Plate Section

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Enrique Aguado (1), Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS-Universite´ de la Me´diterrane´e, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 9, France Dietke Buck (237), De´veloppement Normal et Pathologique du Syste`me Immunitaire, INSERM U429, Hoˆpital Necker, Paris, France John P. Cannon (209), Department of Pediatrics, University of South Florida College of Medicine, USF/ACH Children’s Research Institute, St. Petersburg, Florida 33701 Sarah L. Clarke (27), Peter Gorer Department of Immunobiology, Guy’s King’s St. Thomas’ School of Medicine, King’s College, University of London, London SE1 9RT, United Kingdom Sandeep Dave (163), Metabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892 Franc¸oise le Deist (237), De´veloppement Normal et Pathologique du Syste`me Immunitaire, INSERM U429, Hoˆpital Necker, Paris, France; Assistance Publique–Hoˆpitaux de Paris (AP/HP), Paris, France Minoru Fujimoto (61), Department of Molecular Medicine, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan Kathryn Haskins (123), Department of Immunology and Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center and National Jewish Medical and Research Center, Denver, Colorado, 80206 Adrian C. Hayday (27), Peter Gorer Department of Immunobiology, Guy’s King’s St. Thomas’ School of Medicine, King’s College, University of London, London SE1 9RT, United Kingdom Gary W. Litman (209), Department of Pediatrics, University of South Florida College of Medicine, USF/ACH Children’s Research Institute, St. Petersburg, Florida 33701; All Children’s Hospital, Department of Molecular Genetics, St. Petersburg, Florida 33701; H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612 ix

x

c o n tr i b u t o rs

Bernard Malissen (1), Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS-Universite´ de la Me´ diterrane´ e, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 9, France Marie Malissen (1), Centre d’Immunologie de Marseille-Luminy, INSERMCNRS-Universite´ de la Me´ diterrane´ e, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 9, France Irene M. Min (297), Genetics Program, Tufts University School of Medicine, Boston, Massachusetts 02111 Tetsuji Naka (61), Department of Molecular Medicine, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan Daniel J. Pennington (27), Peter Gorer Department of Immunobiology, Guy’s King’s St. Thomas’ School of Medicine, King’s College, University of London, London SE1 9RT, United Kingdom Jonathan P. Rast (209), Sunnybrook and Women’s College, Health Sciences Centre, Toronto, Ontario, Canada M4N 3M5 Patrick Revy (237), De´ veloppement Normal et Pathologique du Syste`me Immunitaire, INSERM U429, Hoˆ pital Necker, Paris, France Erik Selsing (297), Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111 Louis M. Staudt (163), Metabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892 Robert E. Tigelaar (27), Department of Dermatology, Yale University, New Haven, Connecticut 06511 Hiroko Tsutsui (61), Department of Immunology and Medical Zoology, Hyogo College of Medicine, Hyogo 663-8501, Japan; CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan David Vermijlen (27), Peter Gorer Department of Immunobiology, Guy’s King’s St. Thomas’ School of Medicine, King’s College, University of London, London SE1 9RT, United Kingdom Jean-Pierre de Villartay (237), De´ veloppement Normal et Pathologique du Syste`me Immunitaire, INSERM U429, Hoˆ pital Necker, Paris, France; Assistance Publique–Hoˆ pitaux de Paris (AP/HP), Paris, France Emma L. Wise (27), Peter Gorer Department of Immunobiology, Guy’s King’s St. Thomas’ School of Medicine, King’s College, University of London, London SE1 9RT, United Kingdom Akihiko Yoshimura (61), Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan

Role of the LAT Adaptor in T-Cell Development and Th2 Differentiation Bernard Malissen, Enrique Aguado, and Marie Malissen Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS-Universite´ de la Me´diterrane´e, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 9, France

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

Abstract ........................................................................................................... Introduction ..................................................................................................... The LAT Signalosome ........................................................................................ LAT: An Essential Component of the Pre-TCR ....................................................... Role of LAT in gd T-Cell Development.................................................................. Negative Regulatory Role of LAT ......................................................................... Positive and Negative Selection in LatY136F Mice ..................................................... Cooperative Assemblies Within the LAT Signalosome............................................... Redundancy Among LAT Tyrosines....................................................................... Mechanisms of Signal Termination by LAT............................................................. Th2-Type Immunity in LatY136F and LatY7/8/9F Mice ................................................. Is a LAT-Signalosome Pathology Taking Shape?....................................................... Concluding Remarks .......................................................................................... References .......................................................................................................

1 2 2 5 7 9 13 15 17 18 19 19 22 22

Abstract LAT (linker for activation of T cells) is an integral membrane adaptor protein that constitutes in T cells a major substrate of the ZAP-70 protein tyrosine kinase. LAT coordinates the assembly of a multiprotein signaling complex through phosphotyrosine-based motifs present within its intracytoplasmic segment. The resulting ‘‘LAT signalosome’’ links the TCR to the main intracellular signalling pathways that regulate T-cell development and T-cell function. Early studies using transformed T-cell lines suggested that LAT acts primarily as a positive regulator of T-cell receptor (TCR) signalling. The partial or complete inhibition of T-cell development observed in several mouse lines harboring mutant forms of LAT was congruent with that view. More recently, LAT ‘‘knock-ins’’ harboring point mutations in the four COOH-terminal tyrosine residues, were found to develop lymphoproliferative disorders involving polyclonal T cells that produced high amounts of T helper-type 2 (Th2) cytokines. This unexpected finding revealed that LAT also constitutes a negative regulator of TCR signalling and T-cell homeostasis. Although LAT is also expressed in mast cells, natural killer cells, megakaryocytes, platelets, and early B cells, the present review specifically illustrates the role LAT plays in the development and function of mouse T cells. As discussed, the available data underscore that a novel immunopathology proper to defective LAT signalosome is taking shape.

1 advances in immunology, vol. 87 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)87001-4

2

B ER NA RD MA L I S S EN ET A L .

1. Introduction T cells can be divided into two lineages based on the structure of their T-cell antigen receptor (TCR). In the adult mouse, most T cells express a TCR heterodimer consisting of a and b chains, whereas a minor population expresses an alternative TCR isoform made of g and d chains. The signal transduction cassettes operated by ab- and gd-TCR share many functional components. Among them, the transmembrane adaptor molecule LAT (linker for activation of T cells) is essential in that it coordinates the assembly of a multiprotein signaling complex through phosphotyrosine-based motifs present within its intracytoplasmic segment. The resulting ‘‘LAT signalosome’’ links the TCR to the main intracellular signalling pathways that regulate T-cell development and T-cell function. Early studies using transformed T-cell lines suggested that LAT acts primarily as a positive regulator of TCR signalling. The partial or complete inhibition of T-cell development observed in several mouse lines harboring mutant forms of LAT was congruent with that view. More recently, two distinct LAT ‘‘knock-ins’’ were found to develop lymphoproliferative disorders involving polyclonal T cells that produced high amounts of T helper-type 2 (Th2) cytokines. This unexpected finding revealed that LAT also constitutes a negative regulator of TCR signalling and T-cell homeostasis. Although LAT is also expressed in mast cells, natural killer cells, megakaryocytes, platelets, and early B cells, the present chapter will be limited to illustrate the role LAT plays in the development and the function of mouse T cells. As discussed, the available data underscore the existence of an immunopathology proper to defective LAT signalosome. 2. The LAT Signalosome LAT was identified in 1998 as a 36- to 38-kDa integral membrane adaptor protein that constitutes in T cells a major substrate of the ZAP-70 protein tyrosine kinase (PTK) (Zhang et al., 1998). Adaptor proteins lack both enzymatic and transcriptional activities and act as molecular scaffolds through which multiprotein signaling complexes are transiently assembled via phosphotyrosine-based motifs and/or modular protein-protein interaction domains (e.g., Src homology 2 (SH2)-, Src homology 3 (SH3)-, pleckstrin homology (PH)-domains) (Jordan et al., 2003). Adaptor proteins can be divided into transmembrane adaptor proteins (TRAPs) and cytoplasmic adaptor proteins (Kliche et al., 2004). A subset of TRAPs that includes LAT possesses a juxtamembrane CXXC palmitoylation motif (where C denotes cysteine and X denotes any amino acid). Palmitoylation stabilizes the association of LAT with the plasma membrane and targets it to glycosphingolipid-enriched microdomains (GEMs or lipid

l at a da p t or i n t-c e l l d e ve l o p m e n t

3

rafts). The association of LAT with lipid rafts does not appear, however, essential for its function during T-cell development and T-cell activation (Zhu et al., 2005). The essential role LAT plays in T-cell signalling was first deduced from the analysis of LAT-deficient variants of the Jurkat T-cell line (Finco et al., 1998; Zhang et al., 1999a). Subsequent biochemical studies helped define the binding partners of phosphorylated LAT molecules and showed that in T cells most of the signalling activity of LAT is funnelled through the four COOH-terminal tyrosine residues found at positions 136, 175, 195, and 235 of the mouse LAT sequence (Figs. 1 and 2) (Lin and Weiss, 2001; Paz et al., 2001; Zhang et al., 2000; Zhu et al., 2003). After TCR-induced phosphorylation, these four tyrosines manifest some specialization in the SH2-domain-containing proteins they recruit. For instance, mutation of tyrosine (Y) 136 primarily eliminates binding of phospholipase C-g1 (PLC-g1), whereas the simultaneous mutation of Y175 and Y195, or of Y175, Y195, and Y235 results in loss of binding of the Gads and Grb2/Grap adaptors, respectively (Lin and Weiss, 2001; Paz et al., 2001; Zhang et al., 2000; Zhu et al., 2003). Grb2 comprises a central SH2 domain flanked by two SH3 domains that are constitutively associated with a variety of signalling proteins, including Sos and Cbl. The Grb2-like adaptor Grap is specifically expressed in lymphocytes. Gads resembles Grb2/ Grap and contains an additional proline-rich region between its SH2- and

Figure 1 Schematic representation of the mouse and human LAT molecules. The extracellular (EC), transmembrane (TM), and cytoplasmic (CY) segments are indicated together with the tyrosine (Y) residues found within the cytoplasmic region. Human LAT contains 10 tyrosines of which nine are conserved in mouse LAT. Of these, only the five carboxy-terminal tyrosines appear to be phosphorylated upon TCR engagement (Zhu et al., 2003). The tyrosines conserved in mouse and human LAT have been numbered 1 to 9 by beginning at the membrane proximal tyrosine. Mutant LAT molecules where the three or the four carboxy-terminal tyrosines were mutated to phenylalanine have been denoted LATY7/8/9F (or LAT3YF) and LATY6/7/8/9F (or LAT 4YF), respectively. Molecules with a mutation that replaced tyrosine 136 with a phenylalanine have been denoted LATY136F (or LATY6F).

4

B ER NA RD MA L I S S EN ET A L .

Figure 2 A model of the signalling complexes assembled through LAT in T cells. Raft-associated LAT molecules accumulate in the vicinity of activated TCR and undergo protein tyrosine phosphorylation events. These events are initiated by Src-family PTKs (Lck and Fyn), and proceed through the Syk-family PTK ZAP-70. Once bound to phosphorylated LAT via Gads, SLP-76 is phosphorylated by ZAP-70 and provides a binding site for the SH2-domain of the Tec-family PTK Itk. LAT-bound PLC-g1 becomes activated following phosphorylation by both ZAP-70 and Itk. The activation of PLC-g1 leads to the generation of diacylglycerol (DAG) and inositol triphosphate (IP3). While IP3 triggers Ca2þ fluxes, DAG activates protein kinase C (PCK) and the nucleotide exchange factor RasGRP1, an activator of Ras in T cells. An independent pathway involving the recruitment of Sos trough Grb2 may also connect LAT to the Ras pathway. Phosphorylated SLP-76 also interacts with the cytosolic adaptor protein Nck and with the nucleotide exchange factor Vav. This ternary complex activates the GTPase Rac1, and induces cytoskeletal reorganization. In addition, phosphorylated SLP-76 interacts with the serine-threonine kinase HPK-1, and the adhesion and degranulation promoting adaptor protein (ADAP), thereby altering the function of integrins. LAT is also capable of interacting through Grb2/Gads with the adaptor protein Gab2 and the tyrosine phosphatase SHP-2. The juxtamembrane CXXC motif, which becomes palmitoylated and targets LAT to lipid rafts, is shown by a broken arrow.

COOH-terminal SH3-domains. Gads interacts constitutively with the adaptor SLP-76, thereby recruiting it to LAT, together with its constellation of associated molecules (Vav, Nck, Itk, adhesion and degranulation promoting adaptor protein (ADAP)). SLP-76 contributes to PLC-g1 activation by stabilizing the LAT-PLC-g1 association and by bringing the Tec family PTK Itk in the vicinity of its PLC-g1-substrate (Yablonski et al., 2001). In addition to PLC-g1, another major effector molecule functioning downstream of LAT is the Ras GTPase, whose activation is defective in both Lat- and Slp-76-deficient T cells. In T cells, the functional coupling between LAT and Ras occurs mainly

l at a da p t or i n t-c e l l d e ve l o p m e n t

5

through an SLP-76-PLC-g1-RasGRP1 pathway, and secondarily via a Grb2Sos axis (Fig. 2). 3. LAT: An Essential Component of the Pre-TCR Genetic studies have defined two consecutive developmental checkpoints at which T cells progressing along the ab-lineage undergo programmed cell death if they fail to productively rearrange their TCR genes or express TCR ab heterodimers with inappropriate specificities (Malissen et al., 1999; Von Boehmer et al., 2003). Transition through the earliest checkpoint requires the operation of a molecular sensor known as the pre-TCR complex. Once successfully assembled, this multimolecular complex triggers the transition from the double-negative (DN) CD4 CD8 stage to the double-positive (DP) CD4þCD8þ stage, and ensures that only DN cells with a productive TCR b gene rearrangement are rescued from cell death and become DP cells. At the DP stage, a second molecular sensor assembles and controls the transition to the single-positive (SP) CD4þ CD8 or CD4 CD8þ stages on the basis of the specificity of TCR ab heterodimers. As discussed later, the phenotype of mice deficient in LAT (Lat / ), or having a mutation of the three (LatY7/8/9F) or four (LatY6/7/8/9F ) COOH-terminal tyrosine residues of LAT underscores the positive regulatory role played by LAT during ab T cell development and shows that LAT is essential for the function of the pre-TCR (Nunez-Cruz et al., 2003; Sommers et al., 2001; Zhang et al., 1999b). 3.1. ab T-Cell Development in Lat Mutant Mice

/

, LatY7/8/9F, and LatY6/7/8/9F

Compared to wild-type thymi, adult thymi from Lat / , LatY7/8/9F, and LatY6/7/ 8/9F mice were hypocellular. They showed a complete absence of DP and SP cells, and contained TCR-b gene rearrangements that were as extensive and diverse as those found in wild-type DN cells. Analysis of the DN compartment found in Lat / , LatY7/8/9F, and LatY6/7/8/9F thymi for the expression of CD44 and CD25 showed a strict developmental blockade at the CD44 CD25þ to CD44 CD25 transition. This phenotype resembles the one found in thymi of mutant mice unable to assemble a functional pre-TCR (Malissen et al., 1999; Von Boehmer et al., 2003), and indicates that mutation of the three COOHterminal tyrosines of LAT suffices to prevent pre-TCR function. CD25þCD44 DN thymocytes from Lat / , LatY7/8/9F and LatY6/7/8/9F mice express lower levels of pre-TCR complexes than CD25þ CD44 DN thymocytes from wild-type mice (Nunez-Cruz et al., 2003; Sommers et al., 2001). This suggests that LAT normally control the steady-state levels of

6

B ER NA RD MA L I S S EN ET A L .

pre-TCR complexes at the surface of DN cells. Signal transduction through the pre-TCR, and through the clonotype independent complexes (CICs) that are expressed at the surface of wild-type and of Rag-deficient CD25þCD44 DN cells, respectively, can be triggered by injection of anti-CD3 monoclonal antibodies (Wiest et al., 1995). CICs consist of calnexin and of either CD3-g or CD3-d polypeptide pairs. Antibody-mediated cross-linking of the pre-TCR and CIC complexes found on Lat / and LatY6/7/ 8 /9F DN cells did not induce their proliferation and maturation to the DP stage (NunezCruz et al., 2003; Sommers et al., 2001). In contrast, treatment of LatY7/8/9F mice with anti-CD3 monoclonal antibodies induced the development of rare DP cells. This suggests that under the supra-physiological stimulation conditions provided by anti-CD3 treatment, LAT Y7/8/9F molecules can still manifest residual signalling potential in DN cells, an attribute exploited by some developing gd T cells (see Section 4). Consistent with the view that LATY7/8/9F molecules fail to recruit the SLP-76 adaptor, treatment of Slp-76 / mice with anti-CD3 monoclonal antibodies induced the development of a few DP cells with a magnitude similar to that observed in LatY7/8/9F mice (Pivniouk et al., 1998). 3.2. ab T-Cell Development in LatY136F Mutant Mice To address the importance of LAT Y136 in vivo and analyze the consequence of selectively eliminating binding of PLC-g1, knock-in mice with a mutation that replaced Y136 with phenylalanine (Y136F) were independently derived by two groups (Aguado et al., 2002; Sommers et al., 2002). Thymi from mice homozygous for this mutation, LatY136F, contained approximately tenfold fewer cells than wild-type thymi and showed reduced numbers of DP and SP thymocytes. Analysis of the DN compartment found in LatY136F thymi further demonstrated that the LatY136F mutation constitutes a hypomorphic (partial loss of function) mutation of the pre-TCR checkpoint. After reaching a peak in mutant newborn mice, DP cells decreased and were almost undetectable in mutant mice older than 7 weeks. Coincident with this progressive DP erosion, a population of CD4 T cells started to dominate the thymus. The phenotype of these CD4 T cells (CD44high, CD62Llow, CD69þ, and CD24 ) was distinct from that expected for genuine CD4 SP thymocytes. These CD4 T cells corresponded to abnormal CD4 T cells that, after expanding in the periphery of LatY136F mice, infiltrated the thymus among other organs (see Section 5). The IL-5 and IL-13 cytokines they produced in the thymic parenchyma were responsible for tissue-fibrosis and tissue-eosinophilia, and resulted in the subsequent erosion of the DP cell compartment. Consistent with that view, when the development of the infiltrating CD4 T cells was prevented by breeding the LatY136F mutation on a genetic background deprived of MHC class II molecules (see later

l at a da p t or i n t-c e l l d e ve l o p m e n t

7

discussion), the small complement of DP thymocytes characteristics of young LatY136F mice remained stable over time (Aguado et al., 2002). Prior to their infiltration by peripheral CD4 T cells, LatY136F thymi contain very small numbers of CD4 and CD8 SP thymocytes, suggesting that the LatY136F mutation also affects the DP to SP transition (Aguado et al., 2002). The absence of CD8 and CD4 T cells in LatY136F mice rendered deficient for both MHC class I and MHC class II molecules indicates that the development of these rare SP cells requires a selective process involving MHC class I and MHC class II, respectively (Aguado et al., 2002). Therefore, the LatY136F mutation negatively affects the two checkpoints that punctuate intrathymic ab T-cell development and globally results in a severe but partial impairment of ab T-cell development. 3.3. ab T-Cell Development in Lat Expressing a NTAL/LAB Adaptor

/

Mutant Mice Ectopically

A transmembrane adaptor protein called NTAL (for non–T-cell activation linker (Brdicka et al., 2002)), or LAB (for linker of activation of B cells (Janssen et al., 2003)) has been recently identified as the product of the Wbscr5 gene. NTAL is structurally similar to LAT. It possesses a short extracellular domain, a transmembrane region, two palmitoylated membrane proximal cysteine residues, and a long cytoplasmic tail with several tyrosine residues that are conserved between mouse and human. In B cells and mast cells, NTAL is rapidly tyrosine phosphorylated following ligation of immunoreceptors. Despite a remarkable conservation of the exon-intron organization of the Ntal and Lat genes and of the NTAL and LAT structural domains, suggesting that these two adaptors originate from the duplication of an ancestral gene (Brdicka et al., 2002), important differences exist, however, in the intracytoplasmic partners capable of binding to LAT or to NTAL. Five of the nine NTAL tyrosines are potential binding sites for Grb2. However, none of the nine tyrosines is in a consensus binding-motif for PLC-g1. As a consequence, NTAL does not bind to PLC-g, and thus resembles LATY136F mutant molecules. Indeed, when ectopically expressed in developing T cells of LAT-deficient mice, NTAL functionally behaved like LATY136F mutant molecules (Janssen et al., 2004). 4. Role of LAT in gd T-Cell Development As described in Section 3, T cells progressing along the ab-lineage encounter two consecutive developmental checkpoints. Likewise, thymocytes committed to the gd-lineage encounter a single checkpoint at the penultimate DN stage (CD44 CD25þ), which counterbalances the stochastic nature of the

8

B ER NA RD MA L I S S EN ET A L .

concurrent TCR gand TCR d rearrangements. This checkpoint allows only cells _ to rapidly differentiate into CD44 CD25þCD4 CD8 gd expressing a gd TCR _ T cells (Ferrero et al., 2001; Passoni et al., 1997; Wilson et al., 1999). Although gd TCR firing is mandatory for the differentiation of CD44 CD25þ DN cells into_ gd T cells, it does not induce their concomitant proliferation as the pre-TCR does in developing DN T cells (Passoni et al., 1997). Although the LatY136F mutation resulted in a severe but partial impairment of ab T-cell development, it did not detectably affect the development of gd T cells (Aguado et al., 2002; Sommers et al., 2002). gd T cells are also found in the thymus of young Lat / , LatY7/8/9F and LatY6/7/8/9F mice (Nunez-Cruz et al., 2003). However, the total numbers of gd T cells found in these mutant mice are reduced relative to wild-type thymi, and the levels of TCR they expressed at their surface reduced in comparison to wild-type gd thymocytes. Therefore, some thymic gd T cells had the capacity to emerge in the absence of LAT, or in the presence of LATY7/8/9F or LATY6/7/8/9F mutant molecules. However, the progeny of these thymic gd T cells can only be found in the spleen and lymph nodes of LatY7/8/9F mice where they expand and give rise, as described later, to a lymphoproliferative disorder (Nunez-Cruz et al., 2003). The lack of gd T cells in the spleen and lymph nodes of both Lat / and LatY6/7/8/9F mice may reflect their defective development, defective peripheral homeostasis, or a combination of both. The gd T cells found in LatY7/8/9F thymi had a phenotype distinct from that expected for wild-type gd thymocytes (Nunez-Cruz et al., 2003). First, they expressed low levels of CD5 molecules. CD5 is a negative regulator of TCR signalling, the expression of which increases during T-cell development in a manner proportional to the intensity of pre-TCR and TCR signalling (Azzam et al., 1998). Second, whereas only a small fraction of wild-type gd thymocytes scored as CD25þ, close to 90% of gd cells found in LatY7/8/9F thymi expressed the CD25 molecule. Therefore, the blunted signalling potential of LATY7/8/9F molecules likely impeded the development of most gd T cell precursors at the time of their transit to the CD44 CD25 stage. Consistent with this view, the few CD25 gd thymocytes that succeeded to cross this checkpoint in LatY7/8/9F mice expressed a higher level of TCR gd (a hallmark of gd T-cell maturation) than their CD25þ gdþ precursors. Because it does not prevent the assembly and surface expression of gd TCR, the LATY7/8/9F mutation contrasts with null mutations involving the CD3 subunits (Malissen et al., 1999), and provides a unique opportunity to visualize a transitory CD25þ TCR gdþ population. Although it permitted an inefficient development of some gdT cells normally present in the blood, lymph nodes, and spleen (see later discussion), the LatY7/8/9F mutation completely ablated the development of the gd T cells that normally reside in the epidermis and in the gut epithelium. Therefore, the

l at a da p t or i n t-c e l l d e ve l o p m e n t

9

development of the various gdT cell subsets differs in their dependence on LAT signalling, and it is plausible that the ‘‘crippled’’ LATY7/8/9F molecules only allowed the selective maturation of those few gd cells that express TCR with a high affinity for self-ligands. Consistent with the observation that the LatY7/8/9F mutation affects the docking of a larger number of binding partners than the LatY136F mutation (Fig. 2), the overall in vivo effects of the former are more severe than those of the latter. LatY136F constitutes a hypomorph mutation for ab T-cell development and had no detectable effect on gdT–cell development, whereas LatY7/8/9F is a hypomorph mutation for the development of some gd T-cell subsets and a null mutation for the development of ab T cells and of most gd Tcell subsets. The differential effects of these two mutations on the development of the ab and gd lineages might be explained by the fact that lower levels of TCR signalling are needed for the development of gd T cells than for ab T-cell development. Alternatively, ab and gd T cells may differ in their ability to adapt to the effects of these two mutations through ‘‘rewiring’’ of the signalling network operating downstream of LAT. 5. Negative Regulatory Role of LAT 5.1. ab T Cells in the Periphery of LatY136F Mice Given the scarcity of SP thymocytes found in LatY136F newborn mice, one would expect very few SP cells in secondary lymphoid organs. However, T cells are readily found in the spleen and lymph nodes of LatY136F mice (Aguado et al., 2002; Sommers et al., 2002). These are primarily an expanding population of CD4 T cells. As a result, spleen and lymph node enlarge, such that by 7 weeks of age, spleen cellularity is approximately five times greater than that of wild-type mice. These CD4 cells have a CD25 , CD44high, CD62Llow, CD69þ phenotype resembling activated-memory T cells and express low levels of TCR on their surface, an attribute that may in part account for their inability to proliferate in response to TCR stimulation in vitro (see later discussion). The progressive accumulation of CD4 T cells in the periphery of LatY136F mice is probably due to their extended survival and increased proliferation. Paradoxically, CD4 peripheral T cells from LatY136F mice are largely refractory to direct TCR stimulation in vitro. Upon treatment with anti-TCR or anti-TCR plus anti-CD28 antibodies, they proliferate rather poorly and do not increase their levels of CD69 or CD25. CD4 T cells freshly isolated from LatY136F mice expressed sufficient IL-4 and IL-10 transcripts to allow their detection even without ex vivo restimulation (Aguado et al., 2002). Upon activation by phorbol 12-myristate 13-acetate

10

B ER NA RD MA L I S S EN ET A L .

(PMA) and ionomycin, IL-5, IL-13, and IFN-g transcripts were additionally detected, and close to 80% of the CD4 T cells expressed high levels of intracytoplasmic IL-4. Thus, over the first weeks of life and in the absence of deliberate antigenic stimuli, the CD4 ab T cells that expanded in LatY136F mice deployed a Th2-like effector program. Secondary lymphoid organs of 6-week old LatY136F mice contained 7 to 10 times more B cells than their wild-type counterparts. Most of the B cells found in those enlarged secondary lymphoid organs were highly activated and contained antibody-producing cells (Aguado et al., 2002; Sommers et al., 2002). Serum IgG1 and IgE concentrations were elevated approximately 200 times and up to 10,000 times, respectively, compared to wild-type mice. In contrast, the levels of the other Ig isotypes did not differ significantly from those of wild-type serum. In support of the idea of polyclonal hypergammaglobulinemia G1 and E, the concentrations of both kappa and lambda light chains were both augmented in the serum of LatY136F mice. IgE and IgG1 antibody concentrations reached a plateau at 5 weeks of age, with values exceeding those reported for mice deprived of NFATc2 and NFATc3 transcription factors (Ranger et al., 1998). Given that isotype switching to IgE and IgG1 depends on IL-4 and IL-13, the overproduction of IgE and IgG1 noted in LatY136F mice is probably secondary to the presence of an abnormally high frequency of Th2 effectors. Analysis of lymph nodes from LatY136F mice older than 4 weeks also showed the presence of high levels of eosinophils, probably resulting from the IL-5 produced by the expanding CD4 cells. Important lymphocytic infiltrations were also observed in multiple organs, including lungs, kidneys, and liver. Even when preserved from the noxious effect of the CD4 T cells, as occurs in a LatY136F mice crossed onto an MHC class II-deficient background, the CD8 T cells that are found in the periphery of LatY136F mice neither gave rise to a lymphoproliferative disorder, nor adopted a Tc2-type phenotype (Aguado et al., 2002). However, akin to CD4 cells, they were refractory to direct TCR stimulation in vitro, exhibited a memory-activated phenotype, and their TCR levels were downregulated to the same extent as on CD4 T cells. The reason why the lymphoproliferative disorder induced by the LatY136F mutation remains limited to the CD4 T cells and spares the CD8 T cells as well as the gd T cells remains to be determined. Finally, it should be noted that LAT mutant molecules containing only the three COOH-terminal tyrosines functioned similarly to LATY136F mutant molecules. Therefore, the five NH2-terminal tyrosines of LAT are dispensable for the manifestation of the immunopathology triggered by the selective elimination of Y136 (Zhu et al., 2003).

l at a da p t or i n t-c e l l d e ve l o p m e n t

11

5.2. In What Cell Type(s) Does the LatY136F Mutation Operate? Considering that LAT was recently found expressed in early B cells (Oya et al., 2003; Su and Jumaa, 2003), the hypergammaglobulinemia E and G1 observed in LatY136F mutant mice may have resulted from the fact that this mutation not only forces developing CD4þ T cells to adopt a Th2 fate, but also directly affects B cell development and makes the resulting mature B cells particularly receptive to the action of the LatY136F CD4 T cells. When purified CD4 T cells from LatY136F mice were adoptively transferred into hosts that are both T-cell deficient and B-cell proficient (as a result of the Cd3-D5/D5 mutation, (Malissen et al., 1995)), they expanded over time and converted most host B cells into IgE- and IgG1-producing cells (Y. Wang and M. Malissen, unpublished data). Therefore, the LatY136F mutation acts primarily at the level of CD4 T cells, and the development of the hypergammaglobulinemia E and G1 does not require the expression of LATY136F molecules within B cells. 5.3. Residual TCR Signaling Responses in LatY136F CD4 T Cells Biochemical analyses of LatY136F CD4 T cells stimulated with anti-CD3 and anti-CD4 antibodies showed that proximal activation events that do not depend on LAT phosphorylation, such as tyrosine phosphorylation of CD3 subunits and of ZAP-70, were unaffected (Sommers et al., 2002). As expected for a mutation selectively eliminating PLC-g1 binding, TCR cross-linking also failed to induce calcium mobilization and to activate the calcineurindependent transcription factors NF-AT c1 and c2. Tyrosine phosphorylation of SLP-76, which binds to LAT tyrosines other than Y136, was unimpaired in response to TCR engagement. This constitutes an unexpected finding considering that, after TCR cross-linking, the overall phosphorylation of LAT was dramatically reduced in LatY136F CD4 T cells. Surprisingly, ERK activation in response to CD3 plus CD4 cross-linking was also normal or slightly reduced in CD4 T cells and DP thymocytes purified from LatY136F mice. Therefore, despite the markedly reduced levels of tyrosine phosphorylation displayed by LATY136F molecules, they may still trigger a weak activation of PLC-g1 and activate RasGRP1 via a ‘‘trickle through’’ mechanism (Hartgroves et al., 2003; Rosette et al., 2001; Werlen et al., 2003). Alternatively, LATY136F molecules may still recruit enough Grb2-Sos complex to activate the Ras-ERK pathway. Rather than specifically ‘‘unplugging’’ a discrete signalling pathway from the TCR transduction cassette and triggering partial and uncoordinated signals, the LatY136F mutation may thus result in a quantitative reduction of generic signals, more than in qualitative changes in the LAT signalosome output.

12

B ER NA RD MA L I S S EN ET A L .

5.4. gd T Cells in the Periphery of LatY7/8/9F Mice More than 90% of LatY7/8/9F mice older than 20 weeks exhibit splenomegaly and lymphadenopathy (Nunez-Cruz et al., 2003). Given the small size of the gd T-cell population found in young LatY7/8/9F thymi (see Section 4), the presence of large numbers of gd T cells in the spleen and lymph nodes of older LatY7/8/9F mice suggests that the progeny of these cells have expanded in the periphery. Consistent with that view, peripheral gd T cells were found to increase steadily over time, and by 20 weeks of age, gave rise to up to 500  106 gd T cells per spleen. A lymphoproliferative disorder of this magnitude has never been documented for gd T cells developing in the mere absence of ab T cells (e.g., in Tcrb-deficient mice (Mombaerts et al., 1992), or in mice carrying a homozygous deletion of the TCR b transcriptional enhancer (Eb / mice) (Leduc et al., 2000)). Therefore, the disorder that develops in the ab T-cell–less LatY7/8/9F mice cannot be accounted for by the lack of an extrinsic negative regulatory loop normally provided by ab T cells. The gd T cells expanding in the spleen and lymph nodes of LatY7/8/9F mice had a CD25 CD44highCD62Llow CD69þ phenotype closely resembling activated-memory T cells. As previously described in the case of the CD4 ab T cells that develop and expand in LatY136F mice, a protracted expression of CD5 also occurred in the case of the gd T cells developing in LatY7/8/9F mice, with high levels of CD5 only being reached in the periphery (Aguado et al., 2002; Sommers et al., 2002). CD5 expression is thought to be proportional to the signalling potential of the TCR (Azzam et al., 1998). Therefore, the reduced CD5 expression found on the few thymocytes that develop in both LatY136F and LatY7/8/9F mice may allow them to adapt to the lowered signalling potential of the mutated LAT molecules. Conversely, the high levels of CD5 found on their peripheral progeny may correspond to an attempt to desensitize unrestrained, chronic TCR signalling (see later discussion). The subsets of gd T cells found in LatY7/8/9F spleen differ from the ones normally found in the spleen of wild-type mice. Vg1-bearing cells represented 70–90% of all gd T cells found in the spleen and lymph nodes of 6-week-old LatY7/8/9F mice and accounted for most of the gd T cells found in mice older than 20 weeks. The Vg1-bearing cells expressed TCRs composed of Vd5 (20– 60%), Vd6 (3–12%), and of as yet unidentified Vd chains. Although Vg1- and Vg4-bearing cells constitute major gd T cell populations of wild-type spleen and lymph nodes of mice (Pereira et al., 1995), Vg4-bearing cells were clearly not expanded and even underrepresented in LatY7/8/9F mice. The oligoclonal and nonmalignant nature of the gd T cell populations found in LatY7/8/9F mice was established by analysis of the distribution of complementary-determining

l at a da p t or i n t-c e l l d e ve l o p m e n t

13

region 3 lengths of Vg-Jg junctions and was corroborated by histological analyses. Upon activation by PMA and ionomycin, freshly isolated LatY7/8/9F gd T cells expressed IL-4, IL-5, IL-10, and IL-13 transcripts in amounts comparable to those found in CD4 ab T cells freshly isolated from LatY136F mice (Nunez-Cruz et al., 2003). Therefore, the T-cell–lymphoproliferative disorders that characterize LatY7/8/9F and LatY136F mice are each associated with a Th2 polarization, the magnitude of which is only achieved in wild-type mice after prolonged antigenic stimulation in the presence of IL-4. Moreover, the gd T cells that expand in LatY7/8/9F mice resembled the CD4 T cells that expand in LatY136F mice in that they also failed to proliferate in response to CD3 cross-linking in vitro (Nunez-Cruz et al., 2003). The spleen and the lymph nodes of LatY7/8/9F mice older than 20 weeks contained 3 to 5 times as many B cells as their wild-type counterparts. They showed a hyperactivated phenotype, and some expressed a phenotype typical of antibody-producing cells (Nunez-Cruz et al., 2003). Serum IgG1 and IgE concentrations were elevated about 500 and 1000 times, respectively, compared with wild-type mice. Therefore, the B-cell disorder that afflicts LatY7/8/9F mice resembles the one found in LatY136F mice and is probably contingent on the presence of an abnormally high frequency of gd T cells displaying a Th2-like effector phenotype. The splenomegaly and lymphadenopathy that develop in LatY7/8/9F mice is therefore mostly accounted for by expansion of cells belonging to the gd T-cell and B-cell lineages. In contrast to the situation observed in the thymus and lymph nodes of LatY136F mice, no sign of tissue eosinophilia was detected in LatY7/8/9F mice. 6. Positive and Negative Selection in LatY136F Mice To explain the presence in LatY136F mutant mice of a lymphoproliferative disorder and of autoantibodies against double stranded DNA and nucleoproteins, it has been hypothesized that this mutation results in a failure to completely eliminate self-reactive T cells by negative selection (Sommers et al., 2002). Autoreactive T cells could then escape to the periphery, where they expand and become causative of the autoimmune syndrome. According to this hypothesis, antibodies against DNA and nuclear antigens result from T-B cooperation events involving T cells that specifically react against self-peptide-MHC class II complexes expressed by B cells. In LatY136F mice, the inefficient selective process that allows a few DP T cells to reach the CD4 and CD8 SP stages requires the presence of MHC class II and MHC class I molecules, respectively (Aguado et al., 2002). To assess

14

B ER NA RD MA L I S S EN ET A L .

whether this MHC-dependent selection process is associated with impaired negative selection, a TCR ab transgene specific for MHC class II molecules and originally selected in the context of LAT-sufficient mice was introduced into mice with a LatY136F  Rag-1 / background. Despite the presence of signs of attempted selection, supporting the view that the TCR-LATY136F signalling axis was not completely dead and transmitted some signals upon encounter with intrathymic self peptide-MHC complexes, the LatY136F mutation prevented both negative and positive selection, and the TCRþ DP cells remained essentially in a state of non-selection (Y. Wang and M. Malissen, unpublished data). Therefore, an MHC class II-restricted ab TCR originally calibrated in a LAT-proficient context triggered neither positive nor negative selection when forced to cooperate with LAT molecules that had a crippled signalling ability. Interestingly, the previously described experiment results in a mismatch between a TCR ab transgene originally selected in the context of signallingproficient LAT molecules and crippled LATY136F molecules and constitutes a symmetric situation to the one previously observed using T-cell hybridomas derived from LatY136F CD4 T cells (Aguado et al., 2002). Most of these hybridomas unexpectedly reacted with antigen-presenting cells expressing syngeneic MHC class II molecules, whereas none of the control hybridomas derived in parallel from wild-type CD4 T cells showed autoreactivity. The TCR ab heterodimers expressed on the T-cell hybridomas derived from LatY136F CD4 T cells have been originally calibrated in a LatY136F signalling-deficient context. By introducing them into T-cell hybridomas, these TCR were artificially forced to cooperate with the wild-type LAT molecules contributed by the BW5147 fusion partner. It is likely that this mismatch resulted in an increase in TCR signalling output, accounting for the reactivity toward self-MHC class II molecules. Therefore these data cannot be used in support of the view that the LatY136F mutation results in a failure to eliminate self-reactive T cells by negative selection (see Lin et al., 1997 for an analogous reasoning on the autoreactivity manifested by some T cells, the TCR signalling output was experimentally enhanced after their intrathymic calibration phase). It still remains possible that the LatY136F mutation commensurably altered the sensitivity of DP thymocytes to both positive and negative selection. Accordingly, we would like to suggest that the SP cells that develop in LatY136F mice are appropriately calibrated in the context of the crippled LAT molecules. The low intensity signals expected to emanate from the TCRLATY136F axis support the selection of only those DP cells expressing TCR whose affinity for self is shifted toward higher values than in a normal, LATproficient background. Based on this alternative hypothesis, which does not invoke any intrinsic defect in negative selection, the presence of autoantibodies

l at a da p t or i n t-c e l l d e ve l o p m e n t

15

in LatY136F mice might reflect the fact that the LatY136F CD4 effector T cells have acquired the ability to help B cells in a TCR-independent, ‘‘quasimitogenic’’ mode, thereby inducing a massive polyclonal B cell activation that is accompanied by the production of autoantibodies among other antibodies. 7. Cooperative Assemblies Within the LAT Signalosome In recent years, attention has focused on the fact that many proteins are devoid of a-helices or of b-sheets (Dafforn and Smith, 2004). Preliminary structural data suggest that the LAT intracytoplasmic tail may also constitute an intrinsically unstructured protein (D. Housset and B. Malissen, unpublished data). LAT may thus consist of a long flexible cytoplasmic segment that is attached to lipid rafts and constitutes a kind of ‘‘protein fishing line’’ containing several low-affinity, phosphotyrosine-based docking motifs. Owing to its putative unstructured nature, the cytoplasmic segment of LAT would have a greater capture radius than a compact, folded protein with restricted conformational flexibility. As depicted in Figure 3, some combinatorial diversity may occur at the level of the LAT scaffold and produce distinct LAT signalosomes. Moreover, because some of the partners binding to phosphorylated LAT may compete for overlapping docking sites, distinct LAT signalosomes might sequentially assemble, or alternatively there might exist at a given moment more than one LAT signalosome, opening the possibility of a dynamic interplay between them (Bunnell et al., 2002)(Fig. 3A and B). Determination of the specificity and thermodynamic parameters of the individual binary interactions that are involved in the formation of the LATPLC-g1-Gads-SLP-76 complex (Fig. 2) showed that these interactions are relatively weak (in the micromolar range) and display a limited binding specificity (Cho et al., 2004; Houtman et al., 2004). Therefore, the selective assembly of the LAT-Gads-SLP-76-PLC-g1 complex minimally requires the coincident phosphorylation of tyrosines at positions 136, 175, and 195, and is governed by cooperative interactions (Lin and Weiss, 2001;Yablonski et al., 2001). As emphasized by two studies (Hartgroves et al., 2003; Zhu et al., 2003), this structural cooperativity results in some important functional cooperativity: SLP-76 not only stabilizing the association between PLC-g1 and LAT but also helping the activation of bound PLC-g1 molecules by recruiting the Tec kinase Itk (Yablonski et al., 2001). Genetic data further suggest that the LATGads-SLP-76-PLC-g1 complex must nucleate on the same LAT molecule to mediate its function. In mice with a LatY7/8/9F  LatY136F genotype, LAT molecules with a complementary set of phosphotyrosine-based docking sites likely colocalize into lipid rafts and have the possibility to cooperate in trans to restore a wild-type phenotype. However, LatY7/8/9F  LatY136F mice develop a

16

B ER NA RD MA L I S S EN ET A L .

Figure 3 The LAT fishing line: competing complexes, combinatorial diversity and cis-trans interactions. Panels A and B outline the various discrete complexes that can assemble on the LAT scaffold. Most of the signalling activity of LAT appears funnelled through the four COOHterminal tyrosine residues (numbered 6 to 9). Threonine at position 155 of human LAT is shown. Once phosphorylated by ERK, this residue diminishes the docking of PLC-g1 on phosphotyrosine 132 (136 in mouse, see numbering key on the right hand side). Starting with a LAT mutant where all tyrosines were mutated into phenylalanine, Zhu and colleagues (Zhu et al., 2003) performed a systematic tyrosine ‘‘add-back’’ approach, that allowed them to document the minimal number of tyrosine residues needed to reconstitute interaction with Grb2, Gads, and PLC-g1. Once expressed

l at a da p t or i n t-c e l l d e ve l o p m e n t

17

lymphoproliferative disorder, the features and magnitude of which were similar to that unfolding in LatY136F homozygous mice (Nunez-Cruz et al., 2003). Therefore, consistent with previous results obtained in Jurkat cells (Lin and Weiss, 2001), and provided that LATY136F and LATY7/8/9F molecules are properly phosphorylated following TCR engagement (see later discussion), it appears that the reconstitution of a full constellation of LAT phosphotyrosine residues in trans does not permit coordinated pre-TCR and TCR signalling (Fig. 3C). The highly conserved spacing existing between the phosphotyrosinebased binding motifs found in human and rodent forms of LAT further suggests that these motifs may need some stereospecific arrangement to preside to the formation of LAT-Gads-SLP-76-PLC-g1 ‘‘cis-complex’’ on a given LAT scaffold. In Jurkat T cells, the phosphorylation of Y136 of LAT appeared dependent on the prior phosphorylation of the three COOH-terminal tyrosines (Zhu et al., 2003). Consistent with that observation, the LATY7/8/9F and LATY6/7/8/9F mutant polypeptides were indistinguishable in terms of their interaction with Grb2 and PLC-g1 (Tanimura et al., 2003). In case such a processive tyrosine phosphorylation mechanism extends to in vivo conditions, the lack of phosphorylation of Y136 in LATY7/8/9F mutant polypeptides may thus provide a concurrent and rather trivial explanation to the genetic data indicating that LATY136F and LATY7/8/9F molecules do not have the possibility to cooperate in trans. Note that it is presently difficult to reconcile the existence of processive tyrosine phosphorylation events with the view that the LAT intracytoplasmic tail constitutes an intrinsically unstructured protein. Moreover, the distinct phenotype of the LatY7/8/9F and LatY6/7/8/9F mice clearly reveal that the corresponding LAT mutant molecules likely differ in their in vivo signalling proficiency (see Section 3). 8. Redundancy Among LAT Tyrosines Using a Pax5 / pro-B cell-based experimental system intended to speed up the study of T-cell differentiation and function, single mutation of tyrosines 175, 195, or 235 of LAT were analyzed and found to have no effect on in a LAT-deficient Jurkat cell line, LAT mutants with one single Grb2-consensus binding site (at position 7, 8, and 9) were phosphorylated upon TCR cross-linking but failed to associate with Grb2. In contrast, LAT mutants with any two of tyrosines 7, 8 or 9 were capable of associating with Grb2. This finding that is unexpected on the basis of the structural data available on Grb2, strongly suggests, as shown in panel B, that two Grb2 molecules need to bind cooperatively to phosphorylated LAT to form a stable complex. Panel C shows that reconstitution of a full constellation of LAT tyrosine residues in trans does not permit assembly of a LAT-Gads-SLP-76-PLC-g1 complex. The juxtamembrane CXXC motif, which becomes palmitoylated and targets LAT to lipid rafts, is shown by a broken arrow.

18

B ER NA RD MA L I S S EN ET A L .

thymocyte development and on the emergence of mature T-cell lymphocytes (Ardouin et al., 2005). Therefore, tyrosine 136 appears unique among the four COOH-terminal LAT tyrosine residues, in that, when mutated into a phenylalanine, it is the only one that triggers a Th2-type lymphoproliferative disorder. The distinct effect of these mutations reflects the unique ability of tyrosine 136 to recruit PLC-g1, and the redundant role played by tyrosines 175, 195, and 235 in the recruitment of the Gads and of the Grb2/Grap adaptor molecules (Fig. 3). In contrast, the simultaneous mutation of tyrosines 175 and 195 resulted in a partial restoration of thymocyte development and in a LatY136Flike phenotype (Ardouin et al., 2005). 9. Mechanisms of Signal Termination by LAT Once phosphorylated, it is likely that wild-type LAT molecules trigger first the signalling cascade and activate negative regulatory loop only at later time points. Accordingly, the delayed activation of the negative loop allows the effects of the positive signals to become manifest only transiently. Once phosphorylated, crippled LAT molecules, as found in LatY136F and LatY7/8/9F mice, appear still capable of delivering low-intensity activating signals that suffice to trigger the development of a few ab and gd T cells, respectively. However, these signals may fall below a threshold required to trigger the negative regulatory loop expected to deactivate the LAT signalosome. Consistent with its expected negative feedback function, phosphorylated LAT has been shown to recruit inhibitory effectors. For instance, the docking of Gab2 to phosphorylated LAT occurs through Gads/Grb2 and results in the recruitment of inhibitory molecules such as the SHP-2 protein tyrosine phosphatase (Yamasaki et al., 2003). The possibility to recruit inhibitory molecules is congruent with the recessive nature of the LatY136F mutation: in heterozygous mice, the chronic signals expected to be delivered by the LATY136F molecules are likely blunted by dominant, negative signals originating from wild-type LAT molecules that colocalize to GEMs. Following TCR engagement, LAT is also phosphorylated on serine and threonine residues. It has been proposed that when ERK phosphorylates the threonine found at position 155 of human LAT, its affinity for PLC-g1 decreases, and this results in the dampening of TCR signalling (Matsuda et al., 2004). Reconstitution of LATdeficient Jurkat cells with a T155A mutant of LAT increased calcium mobilization and also resulted in a more robust ERK2 phosphorylation following TCR stimulation. Therefore, as previously documented for the regulation of the Lck PTK (Stefanova et al., 2003), the Ras-ERK pathway may also attenuate LAT signalling. Since LAT T155 is not conserved in the other species that have been analyzed so far, it remains to be demonstrated whether this putative

l at a da p t or i n t-c e l l d e ve l o p m e n t

19

negative feedback loop operates in species other than humans. Finally, it remains also to be determined whether intercellular mechanisms, resulting for instance from the presence of abnormal CD4þ CD25þ regulatory T cells, contribute to the pathology that develops in LatY136F mutant mice. 10. Th2-Type Immunity in LatY136F and LatY7/8/9F Mice Although the differentiation of naive CD4 T cells into polarized populations of Th1 or Th2 daughter cells appears predominantly determined by the cytokine milieu, the strength of TCR signalling also influences the terminal differentiation of CD4 T cells (Lanzavecchia and Sallusto, 2002). In general, high antigen doses induce Th1 cell development whereas low antigen doses favor Th2 cell development (Boonstra et al., 2003; Jorritsma et al., 2003; Rogers and Croft, 2000). In wild-type peripheral T cells, TCR encounters with self-peptide/ MHC complexes generate tonic signals that are required for the survival and priming of peripheral T cells (Stefanova et al., 2002). Provided such tonic signals are normally kept in check through a LAT-based negative regulatory loop, the LatY136F mutation may allow peripheral T cells to escape from this negative regulatory loop, thereby enhancing the magnitude of these tonic signals and/or prolonging their action. In LatY136F mice, the conversion of most naive CD4 T cells into Th2 effectors may thus result from their chronic stimulation through the TCR and in the mere absence of extrinsic inflammatory stimuli such as IL-12 and IFN-g (Grogan and Locksley, 2002; Ho and Glimcher, 2002; Murphy and Reiner, 2002). A similar line of reasoning might probably be applied to the gd T cell subsets that expand in the periphery of LatY7/8/9F mice and also adopt a Th2-like effector program. Although TCR signalling is central to T cell development and to the activation of naive T cells, effector and memory T cells have the potential to use receptors other than the TCR to secrete cytokines (review in Vivier and Malissen, 2005). It remains thus possible that the Th2-type effector T cells expanding in the periphery of LatY136F and LatY7/8/9F mice have reached a terminal differentiation stage where their physiology is no more subjected to the control of the TCR. 11. Is a LAT-Signalosome Pathology Taking Shape? In the absence of any intentional immunization, the LatY136F and LatY7/8/9F mutations lead in two distinct T-cell lineages to the unfolding of a remarkably similar developmental and differentiation program (Aguado et al., 2002; Nunez-Cruz et al., 2003; Sommers et al., 2002). The recurrent features observed in these two models are depicted in Figures 4 and 5 and can be summarized as follows:

20

B ER NA RD MA L I S S EN ET A L .

Figure 4 A scheme of T-cell development and of the immunopathological events that unfold in LatY136F mice. The left part of the figure outlines the events occurring in the thymus of young mutant mice. For ab and gd T-cell development, a line that narrows when development is impaired depicts the course of development. Also shown is the position of two developmental checkpoints (TCR-b dependent selection and TCR-ab dependent selection) encountered by developing ab T cells. The right part of the figure outlines the events that occur in the periphery, following the expansion of CD4 T cells that display a Th2 effector phenotype.

Figure 5 A scheme of T-cell development and of the immunopathological events that unfold in LatY7/8/9F mice. The left part of the figure outlines the events occurring in the thymus of young mutant mice. For ab and gd T-cell development, a line that narrows when development is impaired depicts the course of development. Also shown is the position of one developmental checkpoint (TCR-b dependent selection) encountered by developing ab T cells. The LatY7/8/9F mutation ablated the development of a population of gd cells that resides in the epidermis and is referred to as dendritic epithelial T cells (DETC), and of the CD8aa TCR gdþ intraepithelial lymphocytes (IEL) present in the gut. The right part of the figure outlines the events that occur in the periphery, following expansion of gd T cells displaying a Th2-type effector phenotype.

l at a da p t or i n t-c e l l d e ve l o p m e n t

21

1. Both mutations are recessive: they are only detectable after breeding mutant mice to homozygosity or to mice carrying null allele of the Lat gene. 2. Both mutations result in a severe but incomplete impairment of ab (LatY136F) or gd (LatY7/8/9F) T-cell development. 3. The few T cells that reach the periphery of these mutant mice give rise to polyclonal lymphoproliferative disorders involving either ab (LatY136F ) or gd (LatY7/8/9F ) T cells. 4. The ab and gd T cells expanding in the periphery of LatY136F and LatY7/8/ 9F mice, respectively, had a CD25 CD44high CD62LlowCD69þ phenotype closely resembling activated-memory T cells. 5. Paradoxically, the T cells expanding in the periphery of LatY136F and LatY7/8/9F mice are largely refractory to direct TCR stimulation in vitro. 6. The T cells expanding in the periphery of LatY136F and LatY7/8/9F mice express low levels of TCR on their surface, an attribute that only partially account for their inability to proliferate in response to TCR stimulation in vitro. 7. In the absence of deliberate antigenic stimuli, the populations of CD4 ab and of gd T cells that expand in LatY136F and LatY7/8/9F mice, respectively, deploy a Th2-like effector program and trigger Th2-type disorder characterized by hypergammaglobulinemia E and G1 (LatY136F and LatY7/8/9F mice), and tissue eosinophilia (LatY136F mice). 8. Autoantibodies against DNA and nuclear antigens are present at least in the serum of LatY136F mice. 9. Despite prominent lymphocytic infiltrations in the thymus, lung, liver, and kidney, homozygous LatY136F and LatY7/8/9F mice showed no chronic intestinal inflammation or tumor formation. Mice mutated for genes encoding molecules belonging to the LAT signalosome or lying proximal to it (e.g., c-Cbl and RasGRP1, (Chiang et al., 2004; Priatel et al., 2002)) showed a phenotype that largely recapitulates that of LatY136F and LatY7/8/9F mutant mice (see previous discussion). Although a careful side-by-side comparison of these various mutant mice remains to be done in the same laboratory, these data strongly suggest the existence of an immunopathology proper to the LAT signalosome. We propose to coin this novel pathology as ‘‘LAT-signalling pathology’’ (LSP). LSP differs from other T-cell lymphoproliferative disorders due to defects in CD152 (CTLA-4)function or in CD95-CD95L (Fas-FasL) interactions. Interestingly, LSP is reminiscent of the conditions manifested by some patients suffering from idiopathic hypereosinophilic syndrome (Roufosse et al., 1999; Renner et al., 2004), raising the possibility that some of the afflicted patients may harbor similar mutations in their LAT signalosome.

22

B ER NA RD MA L I S S EN ET A L .

12. Concluding Remarks LAT functions as a central platform for recruitment of multiprotein complexes that are responsible for both TCR signal activation and attenuation. In contrast to null mutants, partial loss-of-function mutations of LAT revealed the complex equilibrium that exists within the LAT signalosome between positive and negative regulators. Based on available biochemical data, it is presently difficult to understand why the disruption of a single phosphotyrosine-based docking site, and of a subset of phosphotyrosine-based docking sites, results in the LatY136F and LatY7/8/9F phenotypes, respectively. How the signals originating from two distinctly mutated LAT signalosomes lead, in two distinct T cell lineages and in the absence of any intended immunization, to the unfolding of a conspicuously similar developmental and terminal differentiation program constitutes another puzzling issue that remains to be elucidated. Acknowledgments We thank Pierre Golstein, Lee Leserman, Hans-Acha Orbea, Dominique Housset, Sho Yamasaki, as well as the members of the Malissen’s laboratory for discussion. Supported in part by CNRS, INSERM, and a specific grant from ARC/ARECA.

References Aguado, E., Richelme, S., Nunez-Cruz, S., Miazek, A., Mura, A.-M., Richelme, M., Guo, X.-J., Sainty, D., He, H.-T., Malissen, B., and Malissen, M. (2002). Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science 296, 2036–2040. Ardouin, L., Rolink, A. G., Mura, A.-M., Gommeaux, J., Melchers, F., Busslinger, M., Malissen, M., and Malissen, B. (2005). Eur. J. Immunol. In press. Azzam, H. S., Grinberg, A., Lui, K., Shen, H., Shores, E. W., and Love, P. E. (1998). CD5 expression is developmentally regulated by T-cell receptor (TCR) signals and TCR avidity. J. Exp. Med. 188, 2301–2311. Boonstra, A., Asselin-Paturel, C., Gilliet, M., Crain, C., Trinchieri, G., Liu, Y. J., and O’Garra, A. (2003). Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: Dependency on antigen dose and differential toll-like receptor ligation. J. Exp. Med. 197, 101–109. Brdicka, T., Imrich, M., Angelisova, P., Brdickova, N., Horvath, O., Spicka, J., Hilgert, I., Luskova, P., Draber, P., Novak, P., Engels, N., Wienands, J., Simeoni, L., Osterreicher, J., Aguado, E., Malissen, M., Schraven, B., and Horejsi, V. (2002). Non-T-cell activation linker (NTAL): A transmembrane adaptor protein involved in immunoreceptor signaling. J. Exp. Med. 196, 1617–1626. Bunnell, S. C., Hong, D. I., Kardon, J. R., Yamazaki, T., McGlade, C. J., Barr, V. A., and Samelson, L. E. (2002). T-cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275. Chiang, Y. J., Sommers, C. L., Jordan, M. S., Gu, H., Samelson, L. E., Koretzky, G. A., and Hodes, R. J. (2004). Inactivation of c-Cbl reverses neonatal lethality and T cell developmental arrest of SLP-76-deficient mice. J. Exp. Med. 200, 25–34.

l at a da p t or i n t-c e l l d e ve l o p m e n t

23

Cho, S., Velikovsky, C. A., Swaminathan, C. P., Houtman, J. C., Samelson, L. E., and Mariuzza, R. A. (2004). Structural basis for differential recognition of tyrosine-phosphorylated sites in the linker for activation of T cells (LAT) by the adaptor Gads. EMBO J. 23, 1441–1451. Dafforn, T. R., and Smith, C. J. (2004). Natively unfolded domains in endocytosis: Hooks, lines and linkers. EMBO Rep. 5, 1046–1052. Ferrero, I., Wilson, A., Beermann, F., Held, W., and MacDonald, H. R. (2001). T-cell receptor specificity is critical for the development of epidermal gammadelta T cells. J. Exp. Med. 194, 1473–1483. Finco, T. S., Kadlecek, T., Zhang, W., Samelson, L. E., and Weiss, A. (1998). LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway. Immunity 9, 617–626. Grogan, J. L., and Locksley, R. M. (2002). T helper cell differentiation: On again, off again. Curr. Opin. Immunol. 14, 366–372. Hartgroves, L. C., Lin, J., Langen, H., Zech, T., Weiss, A., and Harder, T. (2003). Synergistic assembly of linker for activation of T cells signaling protein complexes in T-cell plasma membrane domains. J. Biol. Chem. 278, 20389–20394. Ho, I. C., and Glimcher, L. H. (2002). Transcription: Tantalizing times for T cells. Cell 109, S109–S120. Houtman, J. C., Higashimoto, Y., Dimasi, N., Cho, S., Yamaguchi, H., Bowden, B., Regan, C., Malchiodi, E. L., Mariuzza, R., Schuck, P., Appella, E., and Samelson, L. E. (2004). Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences. Biochemistry 43, 4170–4178. Janssen, E., Zhu, M., Craven, B., and Zhang, W. (2004). Linker for activation of B cells: A functional equivalent of a mutant linker for activation of T cells deficient in phospholipase C-gamma1 binding. J. Immunol. 172, 6810–6819. Janssen, E., Zhu, M., Zhang, W., and Koonpaew, S. (2003). LAB: A new membrane-associated adaptor molecule in B cell activation. Nat. Immunol. 4, 117–123. Jordan, M. S., Singer, A. L., and Koretzky, G. A. (2003). Adaptors as central mediators of signal transduction in immune cells. Nat. Immunol. 4, 110–116. Jorritsma, P. J., Brogdon, J. L., and Bottomly, K. (2003). Role of TCR-induced extracellular signalregulated kinase activation in the regulation of early IL-4 expression in naive CD4þ T cells. J. Immunol. 170, 2427–2434. Kliche, S., Lindquist, J. A., and Schraven, B. (2004). Transmembrane adapters: Structure, biochemistry and biology. Semin. Immunol. 16, 367–377. Lanzavecchia, A., and Sallusto, F. (2002). Progressive differentiation and selection of the fittest in the immune response. Nat. Rev. Immunol. 2, 982–987. Leduc, I., Hempel, W. M., Mathieu, N., Verthuy, C., Bouvier, G., Watrin, F., and Ferrier, P. (2000). T-cell development in TCR beta enhancer-deleted mice: implications for alpha beta T-cell lineage commitment and differentiation. J. Immunol. 165, 1364–1373. Lin, J., and Weiss, A. (2001). Identification of the minimal tyrosine residues required for linker for activation of T cell function. J. Biol. Chem. 276, 29588–29595. Lin, S. Y., Ardouin, L., Gillet, A., Malissen, M., and Malissen, B. (1997). The single positive T cells found in CD3-zeta/eta-/- mice overtly react with self-major histocompatibility complex molecules upon restoration of normal surface density of T-cell receptor-CD3 complex. J. Exp. Med. 185, 707–715. Malissen, B., Ardouin, L., Lin, S. Y., Gillet, A., and Malissen, M. (1999). Function of the CD3 subunits of the pre-TCR and TCR complexes during T-cell development. Adv. Immunol. 72, 103–148. Malissen, M., Gillet, A., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E., and Malissen, B. (1995). Altered T-cell development in mice with a targeted mutation of the CD3-epsilon gene. EMBO J. 14, 4641–4653.

24

B ER NA RD MA L I S S EN ET A L .

Matsuda, S., Miwa, Y., Hirata, Y., Minowa, A., Tanaka, J., Nishida, E., and Koyasu, S. (2004). Negative feedback loop in T-cell activation through MAPK-catalyzed threonine phosphorylation of LAT. EMBO J. 23, 2577–2588. Mombaerts, P., Clarke, A. R., Rudnicki, M. A., Iacomini, J., Itohara, S., Lafaille, J. J., Wang, L., Ichikawa, Y., Jaenisch, R., Hooper, M. L., and Tonegawa, S. (1992). Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages. Nature 360, 225–231. Murphy, K. M., and Reiner, S. L. (2002). The lineage decisions of helper T cells. Nat. Rev. Immunol. 2, 933–944. Nunez-Cruz, S., Aguado, E., Richelme, S., Chetaille, B., Mura, A. M., Richelme, M., Pouyet, L., Jouvin-Marche, E., Xerri, L., Malissen, B., and Malissen, M. (2003). LAT regulates gammadelta T-cell homeostasis and differentiation. Nat. Immunol. 4, 999–1008. Oya, K., Wang, J., Watanabe, Y., Koga, R., and Watanabe, T. (2003). Appearance of the LAT protein at an early stage of B-cell development and its possible role. Immunology 109, 351–359. Passoni, L., Hoffman, E. S., Kim, S., Crompton, T., Pao, W., Dong, M. Q., Owen, M. J., and Hayday, A. C. (1997). Intrathymic delta selection events in gammadelta cell development. Immunity 7, 83–95. Paz, P. E., Wang, S., Clarke, H., Lu, X., Stokoe, D., and Abo, A. (2001). Mapping the ZAP-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells. Biochem. J. 356, 461–471. Pereira, P., Gerber, D., Huang, S. Y., and Tonegawa, S. (1995). Ontogenic development and tissue distribution of V gamma 1-expressing gamma/delta T lymphocytes in normal mice. J. Exp. Med. 182, 1921–1930. Pivniouk, V., Tsitsikov, E., Swinton, P., Rathbun, G., Alt, F. W., and Geha, R. S. (1998). Impaired viability and profound block in thymocyte development in mice lacking the adaptor protein SLP76. Cell 94, 229–238. Priatel, J. J., Teh, S. J., Dower, N. A., Stone, J. C., and Teh, H. S. (2002). RasGRP1 transduces lowgrade TCR signals which are critical for T-cell development, homeostasis, and differentiation. Immunity 17, 617–627. Ranger, A. M., Oukka, M., Rengarajan, J., and Glimcher, L. H. (1998). Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development. Immunity 9, 627–635. Renner, E. D., Puck, J. M., Holland, S. M., Schmitt, M., Weiss, M., Frosch, M., Bergmann, M., Davis, J., Belohradsky, B. H., and Grimbacher, B. (2004). Autosomal recessive hyperimmunoglobin E syndrome: A distinct disease entity. J. Pediatr. 144, 93–99. Rogers, P. R., and Croft, M. (2000). CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen. J. Immunol. 164, 2955–2963. Rosette, C., Werlen, G., Daniels, M. A., Holman, P. O., Alam, S. M., Travers, P. J., Gascoigne, N. R., Palmer, E., and Jameson, S. C. (2001). The impact of duration versus extent of TCR occupancy on T-cell activation: A revision of the kinetic proofreading model. Immunity 15, 59–70. Roufosse, F., Schandene, L., Sibille, C., Kennes, B., Efira, A., Cogan, E., and Goldman, M. (1999). T-cell receptor-independent activation of clonal Th2 cells associated with chronic hypereosinophilia. Blood 94, 994–1002. Sommers, C. L., Menon, R. K., Grinberg, A., Zhang, W., Samelson, L. E., and Love, P. E. (2001). Knock-in mutation of the distal four tyrosines of linker for activation of T cells blocks murine T cell development. J. Exp. Med. 194, 135–142. Sommers, C. L., Park, C. S., Lee, J., Feng, C., Fuller, C. L., Grinberg, A., Hildebrand, J. A., Lacana, E., Menon, R. K., Shores, E. W., Samelson, L. E., and Love, P. E. (2002). A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 296, 2040–2043.

l at a da p t or i n t-c e l l d e ve l o p m e n t

25

Stefanova, I., Dorfman, J. R., and Germain, R. N. (2002). Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes. Nature 420, 429–434. Stefanova, I., Hemmer, B., Vergelli, M., Martin, R., Biddison, W. E., and Germain, R. N. (2003). TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways. Nat. Immunol. 4, 248–254. Su, Y. W., and Jumaa, H. (2003). LAT links the pre-BCR to calcium signaling. Immunity 19, 295–305. Tanimura, N., Nagafuku, M., Minaki, Y., Umeda, Y., Hayashi, F., Sakakura, J., Kato, A., Liddicoat, D. R., Ogata, M., Hamaoka, T., and Kosugi, A. (2003). Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T-cell activation. J. Cell Biol. 160, 125–135. Vivier, E., and Malissen, B. (2005). Innate and adaptive immunity: specificities and signaling hierarchies revisited. Nat. Immunol. 6, 17–21. Von Boehmer, H., Aifantis, I., Gounari, F., Azogui, O., Haughn, L., Apostolou, I., Jaeckel, E., Grassi, F., and Klein, L. (2003). Thymic selection revisited: How essential is it? Immunol. Rev. 191, 62–78. Werlen, G., Hausmann, B., Naeher, D., and Palmer, E. (2003). Signaling life and death in the thymus: timing is everything. Science 299, 1859–1863. Wiest, D. L., Burgess, W. H., McKean, D., Kearse, K. P., and Singer, A. (1995). The molecular chaperone calnexin is expressed on the surface of immature thymocytes in association with clonotype-independent CD3 complexes. EMBO J. 14, 3425–3433. Wilson, A., Capone, M., and MacDonald, H. R. (1999). Unexpectedly late expression of intracellular CD3epsilon and TCR gammadelta proteins during adult thymus development. Int. Immunol. 11, 1641–1650. Yablonski, D., Kadlecek, T., and Weiss, A. (2001). Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-gamma1 and NFAT. Mol. Cell. Biol. 21, 4208–4218. Yamasaki, S., Nishida, K., Sakuma, M., Berry, D., McGlade, C. J., Hirano, T., and Saito, T. (2003). Gads/Grb2-mediated association with LAT is critical for the inhibitory function of Gab2 in T cells. Mol. Cell. Biol. 23, 2515–2529. Zhang, W., Irvin, B. J., Trible, R. P., Abraham, R. T., and Samelson, L. E. (1999a). Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11, 943–950. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P., and Samelson, L. E. (1998). LAT: the ZAP70 tyrosine kinase substrate that links T-cell receptor to cellular activation. Cell 92, 83–92. Zhang, W., Sommers, C. L., Burshtyn, D. N., Stebbins, C. C., DeJarnette, J. B., Trible, R. P., Grinberg, A., Tsay, H. C., Jacobs, H. M., Kessler, C. M., Long, E. O., Love, P. E., and Samelson, L. E. (1999b). Essential role of LAT in T-cell development. Immunity 10, 323–332. Zhang, W., Trible, R. P., Zhu, M., Liu, S. K., McGlade, C. J., and Samelson, L. E. (2000). Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T-cell antigen receptor-mediated signaling. J. Biol. Chem. 275, 23355–23361. Zhu, M., Janssen, E., and Zhang, W. (2003). Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development. J. Immunol. 170, 325–333. Zhu, M., Shen, S., Liu, Y., Granillo, O., and Zhang, W. (2005). Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development. J. Immunol. 174, 31–35.

The Integration of Conventional and Unconventional T Cells that Characterizes Cell-Mediated Responses Daniel J. Pennington,* David Vermijlen,* Emma L. Wise,* Sarah L. Clarke,* Robert E. Tigelaar,{ and Adrian C. Hayday * *Peter Gorer Department of Immunobiology, Guy’s King’s St Thomas’ School of Medicine, King’s College, University of London, London SE1 9RT, United Kingdom { Department of Dermatology, Yale University, New Haven, Connecticut 06511

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Abstract ........................................................................................................... Introduction ..................................................................................................... Integrating Immune Responses in the Public Good.................................................. Evidence for T-cell Integration............................................................................. Mechanisms of Action of Unconventional T Cells .................................................... Immune Integration via Cytolysis by gd Cells.......................................................... Targeting by Cytolytic gd Cells ............................................................................. Immunointegration by Cytokines .......................................................................... Immunointegration by Chemokines....................................................................... Immunointegration by Adhesion and Costimulatory Molecules................................... Additional Clues to Immunoregulation .................................................................. A Spectrum of Unconventional T Cells .................................................................. Unconventional T Cells and NK Cells ................................................................... A Developmental Program of T-Cell Integration...................................................... T-Cell Integration: Genetics and Disease ............................................................... References .......................................................................................................

27 28 28 29 33 34 39 40 43 44 46 47 48 49 50 52

Abstract This review builds on evidence that cell-mediated immune responses to bacteria, viruses, parasites, and tumors are an integration of conventional and unconventional T-cell activities. Whereas conventional T cells provide clonal antigen-specific responses, unconventional T cells profoundly regulate conventional T cells, often suppressing their activities such that immunopathology is limited. By extrapolation, immunopathologies and inflammatory diseases may reflect defects in regulation by unconventional T cells. To explore the function of unconventional T cells, several extensive gene expression analyses have been undertaken. These studies are reviewed in some detail, with emphasis on the mechanisms by which unconventional T cells may exert their regulatory functions. Highlighting the fundamental nature of T-cell integration, we also review emerging data that the development of conventional and unconventional T cells is also highly integrated.

27 advances in immunology, vol. 87 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)87002-6

28

d a ni el j . p e n ni ng t o n

E T AL .

1. Introduction This review will consider the interactions between conventional and unconventional T cells that appear to underpin cell-mediated immune responses to infectious agents, to tumors, and to themselves. Although such integration has yet to be assimilated in mainstream coverage of cell-mediated immunity, the first parts of this review consider the many sets of data that attest to it. We then consider the ongoing attempts to identify the underlying mechanisms of T-cell integration. On the assumption that each type of T cell must be making a distinct contribution, there has been an intensive gene-profiling of unconventional T cells, which is considered here in some depth. We then consider the newly emerging evidence that conventional and unconventional T-cell integration commences in the thymus. Collectively, the data suggest that T-cell integration is an important, developmentally programmed phenomenon, and that it is inappropriate to continue to depict cell-mediated immunity as being mounted solely by conventional T cells. Nonetheless, the functional importance of T-cell integration seems to vary in different strains of mice, and we conclude the review by considering how the identification of genes that regulate an individual’s dependence on T-cell integration may be germane to diseases such as psoriasis that reflect a local, genetically determined dysregulation of T-cell activity in the tissues. 2. Integrating Immune Responses in the Public Good The general public has two major expectations of immunological research. On one hand, people expect prophylactic and even therapeutic vaccines for specific pathogens and tumors, without exacerbating responses to essential commensals and allergens. On the other hand, people expect medicines that will ameliorate graft rejection and autoimmune diseases (such as Type I diabetes, rheumatoid arthritis, and multiple sclerosis), without limiting the potency of the immune system against tumors or other autologous dysregulation. The key to delivering these advances is the adaptive immune system, as only this has the specificity required to selectively affect particular immune responses and not others. Hence, even in cases where somewhat generic agents have shown promising clinical efficacy (e.g., the use of tolerogenic anti-CD3 in Type I diabetes [Herold and Taylor, 2003]) there is an ongoing search for more specific tools (e.g., antigen-specific tolerogenic vaccines). The almost infinite diversity of B cells and ab T cells conferred by somatic gene rearrangement (Tonegawa, 1983), coupled with the deposition of fine spe-

i n t e gr ati on o f t c e l ls

29

cificities in the memory pool means that essentially any challenge can be approached. Consistent with this view, generating antigen-specific lymphocytes is rarely problematic. Be it HIV1 or breast cancer, antigen-specific lymphocytes can often be detected; yet they are often ineffective, the challenge being to achieve desired functional outcomes. Among several complexities encountered, antigen-specific lymphocytes may be (i) anergised by immunoevasion mechanisms or by other lymphocytes (Feuerer et al., 2001); (ii) active but unable to function in the appropriate anatomical site (Klavinskis et al., 1999); (iii) so active as to be catastrophically pathologic (Roberts et al., 1996). These and related considerations indicate the limitations of restricting research to the generation of conventional, antigen-specific B cells and T cells. Instead, the prognosis for immunotherapies will be improved by recognizing that many physiological immune responses are an integration of multiple cellular activities, particularly within the tissues where the immune response is directed. We hypothesize that an enhanced understanding of such activities will ultimately confer predictive power in our attempts to exploit conventional antigenspecific lymphocytes in clinically useful ways. This review focuses on progress in understanding unconventional T cells that regulate and augment the activities of conventional, MHC restricted CD4þ and CD8þ ab T cells. It is increasingly clear that there are multiple subtypes of unconventional T cells. However, this review focuses on gd cells which are the prototypic unconventional T cells, and from which much information of general importance can be learned. 3. Evidence for T-cell Integration The discovery of the ab TCR receptor and its recognition of peptides presented by MHC was followed by the intensive study of conventional T cells. Among the myriad insights gained was the finding that full activation of T cells requires co-stimulation of CD28 via CD80 and CD86, on antigenpresenting cells (Leung and Linsley, 1994), and that the same two molecules will likewise limit T-cell activation by their engagement of CTLA4 (Walunas et al., 1994). Hence, cell autonomous events seemed sufficient to regulate conventional T-cell activation and attenuation, and there seemed little need to revisit the controversial claims of the 1970s and early 1980s that there were suppressor T cells (Gershon et al., 1972). Nonetheless, set against this backdrop were several other studies destined to show that conventional T-cell responses are critically integrated with other T cells. Among them, the body of work leading to the identification of CD25hi CD4þ ‘‘T-Reg’’ cells is well known (Read and Powrie, 2001). Less often considered is equally compelling evidence that T cells bearing the gd TCR

30

d a ni el j . p e n ni ng t o n

E T AL .

can regulate conventional ab T-cell responses, often in ways that are critical to maintaining the health of the host (Hayday and Tigelaar, 2003). The TCRg chain gene and gd T cells were each discovered in the mid-1980s, prior to which neither had been anticipated (Brenner et al., 1986; Saito et al., 1984). Since that time, much focus has been on the aspects of gd cells that distinguish them from conventional, CD4þ and CD8þ ab T cells. Thus, gd cells are not MHC-restricted and are not confined to secondary lymphoid tissues (Bucy et al., 1988). Rather, many gd cells emerge early in thymic ontogeny, from where they populate tissues such as the gut epithelium and, in some species, the skin. Following activation, they are potent producers of IFNg (Yin et al., 2002), and may provide effective primary protection against tumors (Girardi et al., 2001) and against infected cells, particularly in young animals (Ramsburg et al., 2003). Moreover, certain bacterial, viral, or protozoan infections of humans provoke immense transient expansions in the peripheral blood gd cell compartment (Parker et al., 1990). While supported by a plethora of data, this assessment of gd cells depicts them as separate to and independent from conventional T cells. And yet, throughout most of an animal’s life span, the thymus will continue to generate both gd cells and ab T cells in a fairly constant, species-specific ratio. This in turn poses the question as to whether the two T-cell lineages interact. Indeed, considering T-B interactions as a precedent, it is clear that B cells display both T-cell–independent and T-cell–dependent activities. The most compelling evidence that conventional T-cell responses to infection may be integrated with the activities of gd cells has come from the study of knockout mice that selectively lack either TCRab (Philpott et al., 1992) or TCR gd (Itohara et al., 1993). Whereas ab T-cell–deficient mice were strikingly immunodeficient in response to infection by Listeria monocytogenes, this was not the case for gd T-cell–deficient mice. However, the TCRd / mice were not asymptomatic; rather the course of infection in those mice was altered (Mombaerts et al., 1993). At the time, one could not know whether such results were peculiar to a particular regimen of Listeria infection or were more generally relevant and of what they might be more generally reflective. Several groups successfully addressed these issues in the years following. Thus, Born, O’Brien, and colleagues showed that gd T-cell–deficient mice infected at various sites with Listeria were characterized by exaggerated inflammatory responses in the tissues (Fu et al., 1994). That the immunoregulatory effects of gd T cells reflect a more generalized T-cell integration became apparent from several demonstrations that ab Tcell–mediated responses are a functional target of gd T cells. Thus, Shiohara and colleagues showed that murine skin-resident gd cells can protect the epidermis from infiltration by a graft of ab T cells, reactive to self-MHC

i n t e gr ati on o f t c e l ls

31

Class II expressed by Langerhans cells (LC) (Shiohara et al., 1996). In this system, the autoreactive ab T cell inoculum traffics through the skin and into the epidermis, destroys LC, and thereafter dies off (since the inoculum lacks stem cell potential). When treated mice are subsequently re-inoculated, the second bolus of autoreactive ab T cells traffics to the skin but fails to infiltrate the epidermis. Of note, this ‘‘acquired resistance’’ does not develop in TCRd / mice, but can be elicited by protocols that will reconstitute skin-resident gd cells, known as dendritic epidermal T cells (DETC) (Shiohara et al., 1996). This set of studies was extended by experiments showing that 100% of TCRd / .FVB or TCRd / .NOD mice spontaneously develop a cutaneous, atopic dermatitis–like pathology when housed in conventional conditions (Girardi et al., 2002). This reflects an exaggerated ab T-cell–mediated response to chronic environmental cutaneous stimulation and is abolished either by rearing the mice in dry, pathogen-free conditions, or by crossing the mice with the TCRb / strain. Normal regulation can also be restored by reconstituting the TCRd / mice, specifically with the TCRgdþ DETC compartment, with which the gd cells do not limit the priming of conventional T cells in the lymph nodes, but rather regulate the capacity of those cells to drive an inflammatory response in the tissue (Girardi et al., 2002). The cutaneous inflammation that was 100% penetrant in conventionally housed mice, indicates that immunoregulation by gd cells is a physiological feature, rather than a theoretical possibility (Girardi et al., 2002). It is further exaggerated during cutaneous delayed type hypersensitivity (DTH) responses that provoke ear-swelling, which is greatly enhanced in TCRd / mice over a background level that is already higher than normal (Girardi et al., 2002). Such exaggerated ab T-cell responses are not limited to the skin. Thus, the TCRabþ Th1 response to intestinal protozoan parasites is exaggerated in TCRd / mice, causing villus breakage and hemorrhage not seen in RAG / mice that also lack conventional T cells (Roberts et al., 1996). The capacity of gd cells to regulate ab T cells was directly evident in MRL/lpr mice. One quarter of such mice die of ab T-cell–dependent glomerular nephritis within 6 months. By contrast, the gd-cell–deficient MRL/lpr mouse shows an almost threefold increase in mortality and is characterized by substantially increased numbers of activated CD4þ ab T cells (Peng et al., 1996). Similarly exaggerated ab T-cell responses have been described in various tumor immunology models. For example, H. Schreiber and colleagues, Coussens and Hanahan, and Girardi et al., have each reported that conventional T-cell responses to solid tumors can promote rather than inhibit tumor growth (Daniel et al., 2003; Girardi et al., 2003a; Siegel et al., 2000). Girardi and colleagues studied the two-stage development of skin papillomas and carcinomas that is provoked by sequential application of DMBA and TPA.

32

d a ni el j . p e n ni ng t o n

E T AL .

They reported that at very high doses of carcinogens, the rate of conversion of papillomas to carcinomas is lower in TCRb / mice than it is in WT mice, reflecting the deleterious effects of ab T cells. By contrast, the highest rate of tumor growth occurred in TCRd / mice, probably reflecting the normal capacity of gd cells to both inhibit tumor growth and ameliorate the tumorpromoting effects of aggressive ab T-cell responses (Girardi et al., 2003a). In sum, it is inaccurate to depict T-cell responses to infected or otherwise compromised tissues simply as the product of T-cell priming by draining

Figure 1 (A) A conventional model of tissue immunosurveillance depicts antigen presenting cells, such as dendritic cells (DC) that are activated in the tissues, for example by pathogen associated molecular patterns, or by stress-associated cellular antigens. The DC then present antigen to naı¨ve conventional ab T cells in the draining lymph nodes. Once activated, the cognate responding T cells transit to the tissue where they engage infected or otherwise dysregulated epithelial cells in an antigen-specific fashion. (B) A revised model of tissue immunosurveillance modifies the conventional model by depicting unconventional T cells, such as intraepithelial gd cells interceding between the responding conventional antigen-specific ab T cells and the infected or otherwise dysregulated tissue. Each of the cellular components depicted—the DC, the conventional T cell, and the unconventional T cell—responds to different molecular stimuli, but their coordinated efforts compose an integrated cellular response.

i n t e gr ati on o f t c e l ls

33

dendritic cells (DC), and the resultant migration of the cognate T cells to the relevant site (Fig. 1A). Instead, the functions of the responding T cells are commonly integrated within the tissue with the activities of unconventional gd cells, in the absence of which autoimmunity can be more severe and responses to pathogens and tumors so aggressive as to be deleterious to the host (Fig. 1B). Thus, we need to better understand how conventional and unconventional T-cell activities are integrated. It was indicated by a set of elegant studies carried out by Huber and colleagues that the integration of gd-cell and ab T-cell activities can be complex, with different outcomes depending on the context. Mice of particular strains develop myocarditis following infection with Coxsackie virus B. A primary determinant of the disease is a conventional, MHC Class II restricted CD4 T-cell response, but the pathology that ensues is strongly regulated by gd cells (Huber et al., 2001). Moreover, Huber and O’Brien reported that the suppressive effects of gd cells are attributable to a Vg1þ subset, while a Vg4þ subset enhances the ab T-cell response and promotes disease (Huber et al., 2000). The prospect that the immunoregulatory effects of gd cells can operate in either direction receives further support from observations of Openshaw and colleagues, that gd cells promote immunopathology associated with the response of subunit-vaccinated mice to subsequent infection with respiratory syncytial virus (Dodd et al., 2005). 4. Mechanisms of Action of Unconventional T Cells The mechanism by which functional studies of gd cells have been complemented by T cells regulate the functional potential of ab T cells in vivo remains largely elusive, as is the case for most mechanisms of immunoregulation. Nonetheless, a number of functional studies have been undertaken that collectively demonstrate that gd cells are pleiotropic in their mechanisms of action, with capacity both to kill cells and to promote cell growth and wound healing. Likewise, gd cells are pleiotropic in their targeting, with capability to regulate conventional ab T-cell–dependent responses both directly and indirectly (see later discussion). Of late, functional studies of gd cells have been complemented by gene profiling that can indicate the functional potential of gd cells, and hence the one or several means by which they may integrate with conventional T cells. Several methods of gene profiling including microarrays, serial analysis of gene expression (SAGE), and representational difference analysis (RDA) have been applied to systemic and intraepithelial gd cells derived from different tissues and from the murine, human, and bovine systems (Fahrer et al., 2001; Hedges et al., 2003; Meissner et al., 2003; Pennington et al., 2003; Shires et al., 2001). As is often the case, these approaches have provided very substantial amounts

34

d a ni el j . p e n ni ng t o n

E T AL .

of data. In this review, the data are examined according to classes of molecules that are strong candidates for mediating gd cell-mediated T-cell integration. 5. Immune Integration via Cytolysis by gd Cells Two sets of mutations have clearly indicated the importance of cytotoxic mechanisms in regulating conventional T-cell homeostasis (Kagi et al., 1994; Russell and Ley, 2002). In one case, mice defective in Fas show uncontrolled lymphoproliferation and are known as lpr mice (Watanabe-Fukunaga et al., 1992). Human Fas deficiency is likewise associated with lymphoproliferation and predisposition to autoimmune disease (Straus et al., 1999). The defect primarily relates to the failure of activated T cells to express the TNF-receptorrelated molecule Fas, which is ordinarily engaged by Fas-ligand (FasL)expressing cells, in an interaction that promotes receptor-mediated T-cell apoptosis. Thus, gld/gld mice, in which there is a defect in FasL, likewise show lymphoproliferative disease (Roths et al., 1984). The second set of mutations leads to defects in the perforin-granzyme pathway of apoptosis (Kagi et al., 1994). Functional perforin deficiency is particularly penetrant in humans, leading to a fatal lymphoproliferative disease known as hemophagocytic syndrome, that can be attributed to any of several mutations in the apparatus that regulates perforin-containing secretory cytotoxic granules (Russell and Ley, 2002). There are clear data that gd cells can induce death in target cells by either a receptor-mediated or perforin-granzyme mediated mechanism. Budd, Huber, and colleagues have shown that the Coxsackie B3 virusinduced myocarditis that is induced in susceptible strains of mice by CD4þ ab T cells is dependent on gd cells that use FasL to preferentially kill Th2 cells. Thus, myocarditis does not develop in either gd cell deficient or gld/gld mice, which instead retain a Th2 response to the virus (Huber et al., 2002). The gd cells infiltrate the myocardium, where they express very high levels of FasL, and, as was the case in cutaneous immunopathology (Hayday and Tigelaar, 2003), appear to affect regulation directly in the tissue. Adoptive transfer of myocardial gd cells to gld/gld mice induces disease, but this does not happen in lpr/lpr mice that cannot respond to FasL-mediated killing, and in which a predominantly Th2 response is maintained. The preferential targeting of Th2 cells by gd cells remains to be clarified in more detail, but in the meantime the studies have been extended to humans suffering from Lyme arthritis, induced by Borrelia burgdorferi (Roessner et al., 2003). Human gd cells will respond directly to Borrelia (as they do to many bacteria) and accumulate in the synovial fluid. Again, the cells express very high levels of FasL that they sustain for over 3 weeks post-Borrelia stimulation and will target a broad range of Fasþ cells, including activated T cells (Roessner et al., 2003).

i n t e gr ati on o f t c e l ls

35

A similar situation has been demonstrated by Carding and colleagues who have shown that murine lymphoid Vg1-Vd6.3þ gd cells can kill activated macrophages following Listeria monocytogenes infection (Dalton et al., 2003), thereby inducing downstream suppressive effects on the T-cell response. This effect is dependent on the expression of FasL by the gd cells, and of Fas by the responding macrophages (Dalton et al., 2004). Hence, a Fasdependent capacity of gd cells to critically regulate conventional T-cell– dependent inflammatory responses occurs in response to phylogenetically diverse microbes and appears to be conserved across humans and mice. Thus, the pathologic, lymphoproliferative effects of Fas-deficiency may in part be attributable to the breakdown of gd cell-mediated immune regulation. A related member of the TNF/FasL family is TRAIL (TNF-related, apoptosis inducing ligand) that induces apoptosis in TRAIL-Rþ cells. There are several reports that activated T cells may express TRAIL-R (Jeremias et al., 1998; Wendling et al., 2000), and although there is evidence that such cells remain resistant to TRAIL-induced apoptosis (Mirandola et al., 2004; Soderstrom et al., 2002), there are reports that TRAIL engagement limits T-cell activation (Lunemann et al., 2002). Indeed, TRAIL has been reported to inhibit autoimmune inflammation and to induce cell cycle arrest in both the murine experimental allergic encephalomyelitis model of multiple sclerosis (MS) (Hilliard et al., 2001), and the collagen-induced arthritis model of rheumatoid arthritis (RA) (Song et al., 2000). This mechanism of action may be exerted directly on effector T cells. At the same time, human neutrophils express TRAIL-R and are susceptible to TRAIL-induced apoptosis (Kamohara et al., 2004; Renshaw et al., 2003). Moreover, a recent description of TRAIL-R-deficient mice reported exaggerated innate immune responses (Diehl et al., 2004). Thus, TRAIL may act on multiple targets and in multiple ways to limit inflammatory responses. In this light, it is interesting that activated human Vg9þ T cells, derived from peripheral blood, express high levels of TRAIL, as do murine, skin-associated TCRgdþ DETC, and murine intestinal TCRgdþ intraepithelial lymphocytes (IELs) that also express FasL and TNFa (that may itself induce apoptosis [Zheng et al., 1995]) (Table 1A). Indeed, gd cells have been reported to target activated B cells in a TNFa-dependent, MHC/CD1-independent fashion (Fujii et al., 2002). Susceptibility to killing provoked by TRAIL, FasL, or TNFa requires that cells express the appropriate receptors, providing a means by which tissue-associated gd cells might selectively target activated T cells and antigen-presenting cells. A further mechanism of receptor-mediated apoptosis is provoked by galectins that can engage galactose-containing saccharide ligands (e.g., N-glycans that modify CD45, CD43, and CD7) and particular peptides (Fouillit et al., 2000; Pace et al., 2000; Rabinovich et al., 2002). Galectins are secreted (although a splice variant of

d a ni el j . p e n ni ng t o n

36

E T AL .

Table 1A Cytotoxicity

Gene alias Gp1 GzmB GzmC Gp2 GzmA GzmK GzmM Gp3 GzmD/F GzmE/N GzmG Serglycin Cathepsin C Perforin SerPIN-b9 SerPIN-b6b SerPIN-e2 SerPIN-a3g FasL TRAIL TNFa Cryptidins b-Defensins

Fahrer et al. murine gut gd IEL þþþ þþþ

þþ þ

Shires et al. murine gut gd IEL

Wise et al., unpub. murine skin gd DETC

Vermijlen et al., unpub. human blood (Vg9þ) gd cells

þþþ þ

þþþþ þþþ

þþþþ (GzmH)þþ

þþþþ 

þþþ

þþþþ þþþþ þ

 

þ þ

þþ þ þ þ  

 þþ þ 

N/A N/A N/A þþþ

þþþ þþ þþ þ þþ

þþþ

þ 

þþþ þþþþ þ

Data for tables are derived from the following gene profiling studies: Microarray data of murine intestinal IEL ex vivo (Fahrer et al., 2001); SAGE of murine intestinal IEL ex vivo (Shires et al., 2001); SAGE of murine cutaneous IEL (DETC) activated with anti-CD3 e antibody and IL-2 for 24 hours (Wise et al., in preparation); Microarray data of human Vg9þ T cells derived from human peripheral blood and activated with nominal antigen [HMB-PP] and IL2 for 6 days (Vermijlen et al., in preparation); SAGE and micro-array analysis of CD8 and CD8þ gd T cells from bovine peripheral blood, cultured overnight and stimulated with PMA/ionomycin for 3.5 h (Hedges et al., 2003; Meissner et al., 2003). Expression levels are coded: , þ, , þþ, þþþ, þþþþ, to denote increasing amounts starting at undetectable ( ). Blank denotes no available data; N/A denotes not applicable (e.g., gene does not exist in this species).

galectin-3 encodes a molecule with a transmembrane domain) and fall into three groups: prototypic—galectin-1 and -2; chimera-type—galectin-3; and tandemrepeat-type—galectin-4, -6, -8, -9 (Rabinovich et al., 2002). TCRgdþ IELs express moderate amounts of galectin-1, -3, -4 and -9, while activated DETC express abundant galectin-1 and -3 with trace amounts of galectin-4 (Table 1B). Galectin 1-induced clustering of CD7 and CD45 has been shown to induce apoptosis in subsets of T cells, and there is an abundance of proapoptotic

Table 1B Immunomodulation

Gene alias TGFb1 TGFb2 TGFb3 GALECTIN-1

37

GALECTIN-2 GALECTIN-3 GALECTIN-4 GALECTIN-6 GALECTIN-8 GALECTIN-9 LAG-3 THYMOSIN-b4 THYMOSIN-b10

Fahrer et al. murine gut gd IEL



þ

Shires et al. murine gut gd IEL

Wise et al., unpub. murine skin gd DETC

þ

þþ

þ þ

 þþþ

 þ þ   þ þþ þþþ

þ þþþ

þþ

þþ

Vermijlen et al., unpub. human blood (Vg9þ) gd cells

Hedges et al. bovine blood CD8

gd cells

CD8þ gd cells

þþþ 

þþþþ

Data for tables are derived from the following gene profiling studies: Microarray data of murine intestinal IEL ex vivo (Fahrer et al., 2001); SAGE of murine intestinal IEL ex vivo (Shires et al., 2001); SAGE of murine cutaneous IEL (DETC) activated with anti-CD3 e antibody and IL-2 for 24 hours (Wise et al., in preparation); Microarray data of human Vg9þ T cells derived from human peripheral blood and activated with nominal antigen [HMB-PP] and IL2 for 6 days (Vermijlen et al., in preparation); SAGE and micro-array analysis of CD8 and CD8þ gd T cells from bovine peripheral blood, cultured overnight and stimulated with PMA/ionomycin for 3.5 h (Hedges et al., 2003; Meissner et al., 2003). Expression levels are coded: , þ, , þþ, þþþ, þþþþ, to denote increasing amounts starting at undetectable ( ). Blank denotes no available data; N/A denotes not applicable (e.g., gene does not exist in this species).

38

d a ni el j . p e n ni ng t o n

E T AL .

galectin-1 in immune-privileged sites, suggesting that it might trigger the death of infiltrating T cells responding in a proinflammatory manner (Fouillit et al., 2000; Pace et al., 1999, 2000; Vespa et al., 1999). Indeed, galectin-1 has potent anti-inflammatory effects in a number of animal models of disease, including experimental allergic encephalomyelitis in rats (Offner et al., 1990); concanavalin A-induced hepatitis (a T-cell–dependent model of liver injury) (Santucci et al., 2000); experimental myasthenia gravis in rabbits (Levi et al., 1983); and collageninduced arthritis (Rabinovich et al., 1999). Galectin-1 also has been demonstrated to have a role in the prevention of acute inflammation. For example, it ameliorates edema induced by bee venom phospholipase A2, inhibits the release of arachidonic acid from LPS stimulated macrophages, and inhibits neutrophil extravasation and mast cell degranulation (Rabinovich et al., 2000b). In contrast, galectin-3 has anti-apoptotic activity for the cells that express it, possibly by interaction with members of the Bcl2 family (Yang et al., 1996). By contrast to galectin-1, it can score as a powerful proinflammatory mediator, enhancing neutrophil activity and promoting monocyte chemotaxis (Sano et al., 2000; Yamaoka et al., 1995). Hence, the capacity of gd cells to express and utilize galectins seems very real, but the biological outcome may vary according to the specific circumstances and the relative expression levels of different family members. This level of complexity may underpin the observation that neither galectin-1 nor galectin-3 knockout mice show any clear signs of autoimmunity (Poirier and Robertson, 1993). Perforin-granzyme-mediated killing may not depend on specific receptor engagement and may target cells essentially indiscriminately. Several types of gd cells show conspicuously high levels of granzyme expression (Table 1A), and there is little doubt that this constitutes a major mechanism of gd cell function. Murine IELs, activated DETC, and human Vg9þ cells primarily express the group-1 granzyme, gzmB, and the group-2 granzyme, gzmA, but at levels substantially higher than conventional CD8þ effector T cells. Interestingly, activated DETC also express high levels of another group-1 granzyme, gzmC, together with lower levels of other orphan granzymes, including the group-3 granzymes, gzmD, E, F, G, and N (Wise et al., in preparation). These granzymes were heretofore considered to be NK cell effectors, and their expression by unconventional T cells might be interpreted in any of several ways. For example, their expression may reflect that gd cells possess a broad spectrum of ‘‘killing capability’’ since different granzymes kill in different ways, rendering some better at killing particular targets than others (Russell and Ley, 2002). Alternatively, the expression of gzmC may be induced by an NK-like afferent signalling pathway, which may in turn reflect a capacity of gd cells to be activated in a TCR-independent fashion (see later discussion). Finally, the

i n t e gr ati on o f t c e l ls

39

expression may reflect a close evolutionary relationship of unconventional T cells and NK cells (see later discussion). There may be several rate-determining steps for granzyme-mediated killing, including the expression of perforin (Stepp et al., 2000), which is expressed at very low levels by resting gd cells, but at high levels by activated gd cells; serglycin (which forms a complex with granzyme B [Galvin et al., 1999]), which is expressed at high levels by murine IELs (Fahrer et al., 2001) and activated human Vg9þ cells; and cathepsin C (which is needed for the processing and activation of granzyme A and B [Caputo et al., 1993]), which is expressed by TCRgdþ IELs (Fahrer et al., 2001) (Table 1A). Activated DETC likewise express high levels of serine protease inhibitors (serpins [Sun et al., 1997]) that are known to protect cytolytic cells from their own effectors (Wise et al., in preparation). In sum, gd cell compartments that integrate their activities with conventional T-cell responses display several signatures of potently cytolytic cells. 6. Targeting by Cytolytic gd Cells Although perforin-granzyme-mediated killing might require no specific cell surface receptor, it is contingent on target recognition and consequent activation of the effector cell. Understanding this aspect of the regulation of gd cell cytolyis is compromised by our very rudimentary knowledge of ligands for TCRgd, but probably the most substantial data set exists for the recognition of the MHC Class IB antigens, T10/T22 by 0.5% of murine splenic and intestinal gd cells (Crowley et al., 2000). Provocatively, T10/T22 is upregulated on activated lymphoid cells (Crowley et al., 2000), possibly facilitating the recognition of activated T cells. Another ‘‘stress-inducible’’ MHC Class IB antigen, human MICA, is also recognized by gd cells. Some human Vg1þ cells may recognize this via their TCR (Wu et al., 2002), but there is more substantial evidence that MICA engages the activating receptor NKG2D (Bauer et al., 1999), that is likewise engaged by the products of the distantly related mouse genes, Rae1a-e, MULT-1, and H60 (Carayannopoulos et al., 2002; Cerwenka et al., 2000; Diefenbach et al., 2000; Girardi et al., 2001). NKG2D is expressed by activated CD8þ T cells and constitutively by NK cells and TCRgdþ DETC (Raulet, 2003) (Wise et al., in preparation). Although NKG2D ligands have most often been studied in the context of their stress-induced expression on epithelial cells, particularly tumors, there are several reports that they are up-regulated on activated T cells (Molinero et al., 2003, 2004; Rabinovich et al., 2000a, 2003), again creating the potential for them to be recognized by cytolytic gd cells. Interestingly, whereas activated CD8þ T cells transduce signals from NKG2D via a costimulatory adaptor, DAP10, that complements TCR-

40

d a ni el j . p e n ni ng t o n

E T AL .

mediated activation, murine gd cells, like NK cells, also express the adaptor, DAP12 that may permit the recognition of target cells by NKG2D alone (Diefenbach et al., 2000; Gilfillan et al., 2002; Raulet, 2003). Indeed, the perforin-mediated, NKG2D-dependent killing of activated but not resting T cells by syngeneic NK cells has been reported not to require any additional down-regulation of MHC Class I that might reduce NK cell inhibition (Rabinovich et al., 2000a, 2003). Since TLR-signalling has been reported to up-regulate NKG2D ligands on macrophages (Hamerman et al., 2004), one may speculate that this mechanism, in addition to Fas-mediated lysis, may permit gd cells to target APCs. Indeed, gd cells in BCG-inoculated mice will kill infected macrophages via granule exocytosis, an event that promotes the subsequent development of a CD8þ response to the infection, in preference to a CD4 response (Dieli et al., 2003). 7. Immunointegration by Cytokines Given the many contexts in which gd cells exert an anti-inflammatory activity, it is perhaps paradoxical that the cytokine with which they are most often associated, both in mice and humans, is IFNg. Indeed, a molecular explanation has been provided for this in that GATA-3 in murine gd cells fails to suppress the activity of T-bet that promotes IFNg production (Yin et al., 2002). Nonetheless, it is increasingly clear that IFNg alone is not a reliable predictor of response. Indeed, its primary role in some contexts may be as a direct inhibitor of microbial replication in infected cells, whereas its capacity to orchestrate a prototypic Th1 response may be influenced by the broader spectrum of cytokines and chemokines expressed in any particular context. In this regard, the expression profiling of murine DETC and IELs reflects conspicuously little expression of conventional Th1 or Th2 cytokines, such as IL-1, IL-2, IL-4, IL-5, IL-10, IL-18, and IL-21 (Table 1C). This would suggest that IELs and DETCs do not exert a direct, conventional cytokine-mediated regulation of the immune system, akin to the IL10-dependent mechanisms widely reported for T-Reg cells (Read and Powrie, 2001). One exception to this may be the TGFb genes (Gorelik and Flavell, 2002), which, while not abundantly expressed, are transcribed by IELs and DETCs (Table 1C). Moreover, it was reported that TGFb produced by gd cells promotes B cell production of IgA in IgA nephropathy (Toyabe et al., 2001; Wu et al., 2004). The capacity of TGFb to mediate the integration of gd cell and conventional T cell activities requires further study. A further exception may be the utilization by TCRgdþ IELs and DETC of certain less well-known cytokines (e.g., IL-16, IL-17B, and IL-25) (Table 1C). Of note, conventional T cells express high levels of the IL-17R, potentially

i n t e gr ati on o f t c e l ls

41

Table 1C Cytokines

Gene alias IL-1a IL-1b IL-2 IL-4 IL-6 IL-7 IL-10 IL-12 IL-15 IL-18 IL-21 IL-16 IL-17B IL-25 IFN-g TNFa FLT3-L TGFb1 TGFb2 TGFb3

Fahrer et al. murine gut gd IEL

Shires et al. murine gut gd IEL

Wise et al., unpub. murine skin gd DETC

Vermijlen et al., unpub. human blood (Vg9þ) gd cells





þ þ    þþ  

þþ 

þþ    þþ

þþþ þþþþ 



Data for tables are derived from the following gene profiling studies: Microarray data of murine intestinal IEL ex vivo (Fahrer et al., 2001); SAGE of murine intestinal IEL ex vivo (Shires et al., 2001); SAGE of murine cutaneous IEL (DETC) activated with anti-CD3 e antibody and IL-2 for 24 hours (Wise et al., in preparation); Microarray data of human Vg9þ T cells derived from human peripheral blood and activated with nominal antigen [HMB-PP] and IL2 for 6 days (Vermijlen et al., in preparation); SAGE and micro-array analysis of CD8 and CD8þ gd T cells from bovine peripheral blood, cultured overnight and stimulated with PMA/ionomycin for 3.5 h (Hedges et al., 2003; Meissner et al., 2003). Expression levels are coded: , þ, , þþ, þþþ, þþþþ, to denote increasing amounts starting at undetectable ( ). Blank denotes no available data; N/A denotes not applicable (e.g., gene does not exist in this species).

facilitating responsiveness to IL-17 that mediates up regulation of IL-6 and IL-8 (in human) (Kolls and Linden, 2004). IL-16 may regulate Th2 responses (Little et al., 2003), and the maturation of dendritic cells (DCs), in concert with FLT3-L that is expressed by TCRgdþ IELs (Table 1C) (Della Bella et al., 2004). Thus, unconventional T cells resident in the tissues may condition the maturation and mobilization of immature tissue-resident DCs (Fig. 1B), thereby influencing the conventional T-cell response. DETCs express significant

Table 1D Chemokines

Gene alias CCL1 CCL2 CCL3 CCL4 CCL5

Fahrer et al. murine gut gd IEL

Wise et al., unpub. murine skin gd DETC

Vermijlen et al., unpub. human blood (Vg9þ) gd cells

þþþ þ þ þþþþ

42

CCL6 CCL7 CCL8 CCL9 CXCL1 CXCL5 CXCL8

þþ þþ þþþþ

þþþ þþþ þþþ

  N/A

þþ

  þ þ

þ þ  þ

 þþþþ þþþ þþþþ

þ þþþ

N/A



CXCL10 CXCL11 CXCL12 CXCL14 CXCL16 Lymphotactin MMIF Osteopontin Furin

Shires et al. murine gut gd IEL

þ   þþ þþþþ þþþ þ þ

þþ þþþ þþþ þþþþ þþ

Hedges et al. bovine blood CD8

gd cells

CD8þ gd cells

i n t e gr ati on o f t c e l ls

43

levels of mRNA for IL-25 (IL-17E) (Table 1C), which may promote Th2 responses, at the expense of Th1 responses (Fort et al., 2001). Conspicuously murine IELs and DETCs and human gd cells all express IL21R (Wise et al., in preparation; Vermijlen et al., in preparation). Since activated CD4þ T cells are the only known source of IL-21 (Collins et al., 2003; Nutt et al., 2004), this axis could permit gd cells to ‘‘sense’’ and respond to conventional T-cell activation. For T cells, IL-21 promotes proliferation, whereas it enhances the perforin-dependent cytotoxic function of NK cells (Brady et al., 2004; Ma et al., 2003). Given the many similarities of unconventional T cells to both conventional T cells and NK cells, it may be that IL-21 is a profound regulator of unconventional T-cell activity, as is IL-15 (Fehniger and Caligiuri, 2001; Waldmann, et al., 2001). In this regard, both unconventional T cells and NK cells constitutively express high levels of IL-2Rb that would facilitate responsiveness to IL-15 and IL-21, but that requires activation for its high level expression on conventional T cells (Wise et al., in preparation). 8. Immunointegration by Chemokines Chemokines are an attractive candidate underlying the capacity to regulate the size and form of immune responses within target tissues (Mackay, 2001; Moser et al., 2004). Although the net biological effects can be a complex aggregate of multiple chemokine combinations, an examination of chemokine expression by murine gd cells reveals certain clear points (Table 1D). First, there is no evidence for chemokines that attract neutrophils. Thus, CXCL1-7 are not expressed, although the same may not hold for human gd cells. The macrophage chemo-attractant proteins (MCP) 1-4 (CCL2, 7, 8, and 13) are also not expressed, and neither is IP-10 (CXCL10). However, monocytes may be attracted by lymphocyte-attracting MIP-1a (CCL3), MIP-1b (CCL4), and RANTES (CCL5) (Dorner et al., 2002; Mackay, 2001; Moser et al., 2004), which are expressed very strongly by human and

Data for tables are derived from the following gene profiling studies: Microarray data of murine intestinal IEL ex vivo (Fahrer et al., 2001); SAGE of murine intestinal IEL ex vivo (Shires et al., 2001); SAGE of murine cutaneous IEL (DETC) activated with anti-CD3 e antibody and IL-2 for 24 hours (Wise et al., in preparation); Microarray data of human Vg9þ T cells derived from human peripheral blood and activated with nominal antigen [HMB-PP] and IL2 for 6 days (Vermijlen et al., in preparation); SAGE and micro-array analysis of CD8 and CD8þ gd T cells from bovine peripheral blood, cultured overnight and stimulated with PMA/ionomycin for 3.5 h (Hedges et al., 2003; Meissner et al., 2003). Expression levels are coded: , þ, , þþ, þþþ, þþþþ, to denote increasing amounts starting at undetectable ( ). Blank denotes no available data; N/A denotes not applicable (e.g., gene does not exist in this species).

44

d a ni el j . p e n ni ng t o n

E T AL .

murine gd cells. Of note, gd cells may also express macrophage migration inhibitory factor (MMIF), which might dampen the monocyte-specific migratory responses to CCL3, CCL4, and CCL5, while sustaining the capacity to attract lymphocytes. Paradoxically, these chemokines, along with lymphotactin (XCL1), that is also highly expressed by gd cells, are most clearly associated with the recruitment of proinflammatory, effector T cells (Dorner et al., 2002; Moser et al., 2004), whereas murine gd cells express negligible levels of Th2-associated inflammatory chemokines (eotaxin 1-3; CCL11, CCL24, CCL26, and CCL28); chemokines (CXCL12, CCL18, CCL19, and CCL21) that influence the homeostasis of naı¨ve T cells; chemokines (CXCL13, CCL12, CCL17, CCL20, CCL22, CCL27, and CCL28) that have variously documented roles in the homeostatic migration of effector or memory T cells; and (predictably for tissue-associated immune regulators) chemokines (e.g., CXCL12, CXCL13, CCL18, CCL19, and CCL21) that attract cells to secondary lymphoid organs (Mackay, 2001; Moser et al., 2004). These data indicate that gd cells do not primarily regulate conventional inflammatory T-cell responses by an immunodiversionary mechanism that would utilize cytokines and chemokines to promote the selective priming, recruitment, and proliferation of Th2 cells. Instead, an attractive explanation for the overtly high expression of Th1recruiting chemokines is that this promotes the integration of gd cell activities with those of activated proinflammatory T cells. Indeed, the cytolytic and IFNg-associated effector mechanisms of gd cells (see previous discussion) may reflect a primary contribution of gd cells to proinflammatory, antimicrobial, antiself, and antitumor responses, but the additional capacity of gd cells to respond to and to target responding conventional T cells results in an overall outcome that scores as anti-inflammatory. This we term ‘‘The Inner Circle Model’’ of regulation, building on the concept that a participant needs access to and involvement in a process if they are to regulate that process. There is additional evidence that activated DETC may express CCL1 that may recruit to the skin T-Reg cells, a mechanism that may enhance the regulation of an active inflammatory response (Annunziato et al., 2002; Schaerli et al., 2004). 9. Immunointegration by Adhesion and Costimulatory Molecules Clues to immunointegration can be gleaned from an improved understanding of molecules that may mediate cell–cell interactions. Thus, the expression of NKG2D ligands by activated T cells and of NKG2D by gd cells provides clear evidence for the potential of two cell types to interact directly. Further studies have not provided other such clear candidates. Indeed, the expression by IELs of the integrin aEb7 (Table 1E), highlights that the natural interaction of these

Table 1E Adhesion

Gene alias

Fahrer et al. murine gut gd IEL

Shires et al. murine gut gd IEL

Wise et al., unpub. murine skin gd DETC



INTa4 INTa5

45

E-Cadherin CD44 ICAM-1

Hedges et al. bovine blood CD8

gd cells

CD8þ gd cells



þþþ

þþþ

þþþ

þþþ



þ

 þ þ

þþ þþþþ

 þþ

þ  þ

þþ þþ

þ  

þ 

þ

INTa6 INTa9 INTaE INTaL INTaX INTb1 INTb2 INTb7

Vermijlen et al., unpub. human blood (Vg9þ) gd cells

þ

Data for tables are derived from the following gene profiling studies: Microarray data of murine intestinal IEL ex vivo (Fahrer et al., 2001); SAGE of murine intestinal IEL ex vivo (Shires et al., 2001); SAGE of murine cutaneous IEL (DETC) activated with anti-CD3 e antibody and IL-2 for 24 hours (Wise et al., in preparation); Microarray data of human Vg9þ T cells derived from human peripheral blood and activated with nominal antigen [HMB-PP] and IL2 for 6 days (Vermijlen et al., in preparation); SAGE and micro-array analysis of CD8 and CD8þ gd T cells from bovine peripheral blood, cultured overnight and stimulated with PMA/ionomycin for 3.5 h (Hedges et al., 2003; Meissner et al., 2003). Expression levels are coded: , þ, , þþ, þþþ, þþþþ, to denote increasing amounts starting at undetectable ( ). Blank denotes no available data; N/A denotes not applicable (e.g., gene does not exist in this species).

46

d a ni el j . p e n ni ng t o n

E T AL .

cells is with epithelial cells that express the ligand, E-cadherin (Pribila et al., 2004). Likewise, IELs express high levels of the alpha 5 integrin that binds fibronectin (Table 1E). It may therefore be the case that immune integration in the tissues that is promoted by unconventional T cells is an indirect process in which the unconventional T cells regulate the expression of immunological regulators by the epithelial and mesenchymal cells that they directly engage. Indeed, unconventional T cells do not obviously express many ligands or receptors that would complement the surface expression profile of activated conventional T cells. Nevertheless, two independent assays of murine intestinal IELs (Fahrer et al., 2001; Shires et al., 2001) found that they also expressed E-cadherin, which might mediate the interaction of tissue-associated unconventional T cells with other aEb7þ T cells. Such cells include T-Reg cells on which the expression of integrin aEb7 correlates with CTLA-4 expression, their suppression of T-cell proliferation in vitro, and protection against colitis in a severe combined immunodeficient model (Lehmann et al., 2002). Indeed, mice deficient in aE are predisposed to the development of inflammatory skin lesions (Schon et al., 2000). Another possible link to T-Reg cells is the expression of GITR (glucocorticoid-induced TNF receptor) that is expressed constitutively by CD4þCD25hi T-Reg cells (McHugh et al., 2002) and seemingly by TCRgdþ IEL and DETC (Wise et al., in preparation). This receptor is also expressed on conventional T-cell cells, but only after activation (Nocentini et al., 1997). It appears to down-regulate T-cell proliferation, as T cells from GITR-deficient mice showed exaggerated proliferative responses and increased sensitivity to activation-induced cell death (AICD) (Ronchetti et al., 2002). Interestingly, signalling via GITR breaks self-tolerance and suppresses the regulatory function of T-Reg cells (McHugh et al., 2002; Shimizu et al., 2002). Various subsets of gd cells collectively express other TNF-R-like molecules, including CD27 and 4-1BB (Wise et al., in preparation). Each of these may contribute to immunoregulation by engaging activated B cells, monocytes, and DCs via CD70 and 4-1BBL, respectively (Croft, 2003; Mackay and Kalled, 2002). 10. Additional Clues to Immunoregulation TCRgdþ IELs and DETC express high levels of thymosin-b4 (Tb4) and thymosin-b10. The thymosin family is involved in G-actin sequestration, but in an oxidized state, Tb4 acts as a potent anti-inflammatory mediator (Abiko and Ogawa, 2001; Young et al., 1999). Tb4 is expressed by essentially all cells, and in some is expressed at very high levels. Nonetheless, activated TCRgdþ cells also express high levels of an alternatively spliced RNA that encodes a variant, lymphoid-specific form of Tb4 (L-Tb4) (Girardi et al., 2003b). Consis-

i n t e gr ati on o f t c e l ls

47

tent with the observation that L-Tb4 has an extra methionine substrate for oxidation, it has been shown to have substantially greater anti-inflammatory activity than the ubiquitous form of Tb4, particularly in assays that measure neutrophil responses (Girardi et al., 2003b). A further molecule of interest that is conspicuously highly expressed by many sets of gd cells is LAG-3 (CD223), which is related to CD4, binds to Class-II MHC with 100-fold greater affinity than CD4, and is an important T-cell homeostasis regulator in mice and humans, inhibiting antigen-driven T-cell expansion (Triebel, 2003). With regard to the integration of immune responses within tissues, LAG-3 is normally expressed in activated CD4þ or CD8þ T cells and NK cells in inflamed tissue, but not in the secondary lymphoid organs. Whereas T-cell defects were not originally noted in LAG-3 / mice, the issue was recently reevaluated, with the clear demonstrations that LAG-3 / Tcells are less effective at cell cycle arrest, and show greater homeostatic expansion and commitment to the memory T-cell pool (Workman et al., 2004). Hence, LAG-3 is a negative regulator of T cells. Interestingly, T cells that are negatively regulated via LAG-3 can acquire T-reg activity, indicating that LAG-3 somehow contributes to T-regulatory function (Huang et al., 2004), in which case its constitutive expression by TCRgdþ IELs is most provocative. The mechanism by which LAG-3 works is as yet unclear, but its biochemistry is complex, with a soluble form (sLAG-3) being released from the cell surface (Li et al., 2004). By contrast to cell-associated LAG-3, sLAG-3 is a potent immunostimulant for inducing antigen- or tumor-specific CTL and CD4þ Th1 responses when administered subcutaneously as a vaccine adjuvant together with the antigen or the tumor cells (El Mir and Triebel, 2000). Hence, the expression by gd cells of LAG-3 may confer on them a pleiotropic capacity to integrate cell-mediated immune responses. It has also been reported that activated TCRgdþ IELs and DETC express high levels of keratinocyte growth factors, FGF-VII and FGF-X (Boismenu and Havran, 1994; Jameson et al., 2002). In fact, both conventional and unconventional T cells can express several types of growth factors that act on epithelial cells. Nonetheless, FGF expression by gd cells appears functionally significant, since TCRd / mice show defects in cutaneous and intestinal wound healing (Chen et al., 2002; Jameson et al., 2002). Any capacity of T cells to regulate the repair and growth of epithelia layers might be predicted to suppress inflammatory infiltrates via enhanced resistance of the tissue. 11. A Spectrum of Unconventional T Cells The discovery of gene expression patterns that are characteristic of subsets of gd cells unexpectedly revealed that the same patterns were, to a first approximation, shared by other T-cell subsets, such as CD8aaþ ab T cells that

48

d a ni el j . p e n ni ng t o n

E T AL .

populate the murine intestine (Pennington et al., 2003). This naturally suggested that these enigmatic T cells might have similar functional potential as gd cells, contributing to an integrated cellular immune response to diverse challenges. In support of this, Poussier and colleagues showed that intestinal CD8aaþ ab T cells will down-regulate the proinflammatory effects of conventional T cells responding to gut antigens (Poussier et al., 2002). These findings extend the concept of tissue-based immune integration beyond the interactions that involve gd cells, and suggest that in humans and in mice, the signatory molecular features of immunoregulatory gd cells may be used to identify further unconventional Tcell subsets with similar functional activities. In humans, these may include CD8aaþ ab T cells and CD4 CD8 ab T cells that are enriched in the epidermis (Spetz et al., 1996), and peripheral blood CD5 CD8þ and CD6 TCRab T cells that are reported not to express conventional T-cell markers (Indraccolo et al., 1995; Rasmussen et al., 1994). Nonetheless, ongoing studies suggest that any signatory gene expression profile of unconventional T cells in humans will differ significantly from that described in the mouse. Although this makes it more difficult to identify unconventional T-cell subsets in humans, it encourages the belief that when the commonalities between unconventional T-cell gene expression across species are identified, they will highlight the genes of greatest functional significance. 12. Unconventional T Cells and NK Cells We have previously considered that the signatory gene expression patterns of murine unconventional T cells share some features with NK cells, which, like unconventional T cells, have both intrinsic effector function and the capacity to regulate conventional T-cell responses (Pennington et al., 2003; Shires et al., 2001). Included among these similarities are the constitutive expression of LAG-3, CD122 (IL2Rb), and DAP12 (that have been referred to previously); CD44, CD69, c-kit, and CD7 (that are usually expressed by conventional T cells only following activation); and a signaling machinery that includes FceRIg and lyn kinase. Many of these similarities may reflect the general status of unconventional T cells (e.g., rapid responsiveness, capacity to survive outside of secondary lymphoid tissue), rather than their functional potential. At the same time, there may be clues to the cells’ mechanism of action. For example, both human and murine gd cells express the NK-associated receptor 2B4, that binds to CD48 (Lee et al., 2003). Since CD48 is expressed by T cells, 2B4 expression offers the potential to mediate direct interaction of conventional and unconventional T cells, just as has been proposed for NK:T-cell cross-talk (Assarsson et al., 2004).

i n t e gr ati on o f t c e l ls

49

13. A Developmental Program of T-Cell Integration By combining the emerging gene profiles with functional data, it should gradually be possible to define the several mechanisms by which unconventional T cells establish integration in the cellular immune response. Meanwhile, the fundamental importance of such T-T cross-talk in the immune system is suggested by the recent and unexpected finding that unconventional and conventional T-cell lineages influence each other during T-cell development in the thymus (Pennington et al., 2003). More specifically, the expression of a subset of the unconventional T-cell gene profile by gd cells developing in the thymus is dependent on the cells’ interaction with CD4þCD8þ (Double Positive [DP]) thymocytes that are late-stage progenitors of conventional, MHC-restricted ab T cells. Although the mechanism of this developmental cross-talk is not fully resolved, it involves lymphotoxin (LT) that can be expressed by DP cells, and that can directly influence TCRgdþ thymocytes and their early progenitors (Silva-Santos et al., 2004). In this regard, the mechanism is reminiscent of lymphoid tissue induction, in which LT facilitates the development of organized peripheral lymphoid structures including lymph nodes and Peyer’s Patches (Eberl and Littman, 2003). In the absence of ‘‘conditioning’’ by CD4þCD8þ thymocytes, the gd cells that emerge from the thymus show a selectively altered functional potential, with impaired IFNg production and reduced proliferative responses to stimulation (Pennington et al., 2003; Silva-Santos et al., 2004). By contrast, other functions of the cells, such as the expression of lymphotactin, are seemingly unaltered. Although the powerful influence of CD4þCD8þ thymocytes has so far only been established in the context of its effects on gd cells, it is reasonable to hypothesize that a similar cross-talk may influence the development of other unconventional T cells, such as CD8aa TCRabþ cells. As yet, we can only speculate on the benefits that underpin the selective advantage of developmental T-T cross-talk. But the phenomenon emphasizes at least two points. First, conventional and unconventional T cells are ‘‘aware of each other’’ from a very early time point. Indeed, the data reconcile well with the observation that the most immature cortical thymocytes, that will give rise to gd cells, develop in intimate contact with CD4þCD8þ thymocytes (Prockop and Petrie, 2000). Second, the thymus in which the majority of unconventional T cells develop is a very different organ to that first encountered in the fetus by the progenitors of the earliest T cells. This concept is illustrated in Fig. 2, which depicts the influence of the stroma on the differentiation of the earliest gd cell progenitors entering a small fetal thymus. This differentiation program may produce unconventional T cells that can then populate and afford primary protection to various tissues. By contrast, progenitors entering the thymus

50

d a ni el j . p e n ni ng t o n

E T AL .

Figure 2 The ontogenetic progression of T-cell development. The top panel depicts a fetal-liver derived cell (left) entering the early fetal thymus where it differentiates under the intimate influence of the thymic stroma, maturing into a gd cell with a signatory gene expression pattern (diagonal). The gd cell then leaves the thymus to effect independent, immunoprotective functions in the periphery of the newborn animal (right). The lower panel depicts a bone-marrow derived cell (left) entering the much larger perinatal or adult thymus (rectangle), where it differentiates under the intimate influence not only of the thymic stroma, but also of CD4þCD8þ (Double Positive [DP]) thymocytes that in terms of cell number, dominate the thymus, and a subset of which express lymphotoxin (LT). The DP cells may exert their effects on the developing thymocyte progenitor via the stroma and/or via direct interactions, as shown. As a result, the thymocyte matures into a gd cell that expresses a different subset of genes compared with those that develop in the fetal thymus (black). This different gene expression profile may permit the cell to better integrate its peripheral function with mature ab T cells (pale gray) that continue to develop from the DP thymocyte pool.

later in ontogeny will encounter an organ dominated by CD4þCD8þ thymocytes that may account for 85% of all thymocytes. The influence of these cells on gd cell differentiation may imprint an optimized capacity of mature gd cells to functionally integrate with conventional T-cell responses in the periphery. Moreover, the cells’ developmental cross-talk may go further than this, possibly influencing the continued production by the thymus of a relatively constant ratio of conventional and unconventional T cells. This would seem a prerequisite for sustaining effective integration of the T-cell response. 14. T-Cell Integration: Genetics and Disease In the early parts of this chapter, we considered numerous examples of immune responses that reflected unconventional and conventional T-cell integration. Given the range of these examples, and the fact that the examples were not exhaustive, one readily infers that T-cell integration is an important and invariable component of cellular immune responses. In fact this is not necessarily the

i n t e gr ati on o f t c e l ls

51

case. For example, whereas FvB.TCRd / mice and NOD.TCRd / mice spontaneously develop cutaneous inflammation, this is not the case for C57. BL/6.TCRd / mice, which seem largely refractory to the loss of skin-associated gd cells. Presumably such mice have other, not yet elucidated means of regulating local inflammatory responses. One way to approach this is to intercross NOD.TCRd / mice and C57.BL/6.TCRd / mice, and thereby to map loci that reduce the dependence on unconventional T cells. At this point in time, it is very difficult to predict even the type of loci that will account for the differences in phenotype between NOD.TCRd / mice and C57.BL/6.TCRd / mice. Will they regulate the integrity of the skin, affecting its susceptibility to inflammatory infiltrates? Will they regulate the conventional T-cell response, affecting its capacity to promote immunopathology? Or will they confer on other cells (e.g., keratinocytes) the capacity to express regulators of the conventional T-cell response that are in other strains restricted to gd cells? Although this may seem a priori unlikely, it is increasingly clear that activated epithelial cells have potent immunoregulatory capacity, for example via their production of chemokines and cytokines, and via their expression of FasL that might limit the infiltration of activated lymphoid cells (Yoshikai, 1999). Such issues may be germane to human inflammatory disease. For example, it was recently shown by Nestle and colleagues that non-diseased skin harvested from psoriatic patients will spontaneously convert to a psoriatic lesion following grafting onto a mouse deficient in NK cells and all lymphocytes (Boyman et al., 2004). The development of such lesions is driven by immune dysregulation and can be inhibited by blocking TNFa. And yet, in this new animal model, all lymphoid activity must be local, as the recipient mouse can provide none. This implies that the balance between normal epidermal function and gross inflammatory pathology hinges on the precise regulation of lymphoid cells in the skin. While this may not involve gd cells per se, an understanding of how those cells regulate conventional T-cell responses in the tissues may reveal molecular mechanisms that make critical contributions to psoriasis and other human inflammatory diseases. Moreover, given the striking genetic basis for psoriasis and other such diseases (Capon et al., 2004), an understanding of the murine genes that control an animal’s dependence on conventional and unconventional T-cell integration may directly complement attempts to understand the genetic regulators of disease susceptibility. Acknowledgments We thank Wellcome Trust for support, the NIH (R. H.), and the Marie Curie Intra-European Fellowship Programme (D. V.). This paper is dedicated to F. L. Hayday (1922–2005).

52

d a ni el j . p e n ni ng t o n

E T AL .

References Abiko, T., and Ogawa, R. (2001). Syntheses of two deacetyl-thymosin beta4 analogs and their antiinflammatory effect on carrageenin-induced edema in the mouse paw. Mediators Inflamm. 10, 89–92. Annunziato, F., Cosmi, L., Liotta, F., Lazzeri, E., Manetti, R., Vanini, V., Romagnani, P., Maggi, E., and Romagnani, S. (2002). Phenotype, localization, and mechanism of suppression of CD4(þ) CD25(þ) human thymocytes. J. Exp. Med. 196, 379–387. Assarsson, E., Kambayashi, T., Schatzle, J. D., Cramer, S. O., von Bonin, A., Jensen, P. E., Ljunggren, H. G., and Chambers, B. J. (2004). NK cells stimulate proliferation of T and NK cells through 2B4/CD48 interactions. J. Immunol. 173, 174–180. Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J. H., Lanier, L. L., and Spies, T. (1999). Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285, 727–729. Boismenu, R., and Havran, W. L. (1994). Modulation of epithelial cell growth by intraepithelial gamma delta T cells. Science 266, 1253–1255. Boyman, O., Hefti, H. P., Conrad, C., Nickoloff, B. J., Suter, M., and Nestle, F. O. (2004). Spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-alpha. J. Exp. Med. 199, 731–736. Brady, J., Hayakawa, Y., Smyth, M. J., and Nutt, S. L. (2004). IL-21 induces the functional maturation of murine NK cells. J. Immunol. 172, 2048–2058. Brenner, M. B., McLean, J., Dialynas, D. P., Strominger, J. L., Smith, J. A., Owen, F. L., Seidman, J. G., Ip, S., Rosen, F., and Krangel, M. S. (1986). Identification of a putative second T-cell receptor. Nature 322, 145–149. Bucy, R. P., Chen, C. L., Cihak, J., Losch, U., and Cooper, M. D. (1988). Avian T cells expressing gamma delta receptors localize in the splenic sinusoids and the intestinal epithelium. J. Immunol. 141, 2200–2205. Capon, F., Trembath, R. C., and Barker, J. N. (2004). An update on the genetics of psoriasis. Dermatol. Clin. 22, 339–347. Caputo, A., Garner, R. S., Winkler, U., Hudig, D., and Bleackley, R. C. (1993). Activation of recombinant murine cytotoxic cell proteinase-1 requires deletion of an amino-terminal dipeptide. J. Biol. Chem. 268, 17672–17675. Carayannopoulos, L. N., Naidenko, O. V., Fremont, D. H., and Yokoyama, W. M. (2002). Cutting edge: Murine UL16-binding protein-like transcript 1: A newly described transcript encoding a high-affinity ligand for murine NKG2D. J. Immunol. 169, 4079–4083. Cerwenka, A., Bakker, A. B., McClanahan, T., Wagner, J., Wu, J., Phillips, J. H., and Lanier, L. L. (2000). Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice. Immunity 12, 721–727. Chen, Y., Chou, K., Fuchs, E., Havran, W. L., and Boismenu, R. (2002). Protection of the intestinal mucosa by intraepithelial gamma delta T cells. Proc. Natl. Acad. Sci. USA 99, 14338–14343. Collins, M., Whitters, M. J., and Young, D. A. (2003). IL-21 and IL-21 receptor: A new cytokine pathway modulates innate and adaptive immunity. Immunol. Res. 28, 131–140. Croft, M. (2003). Costimulatory members of the TNFR family: Keys to effective T-cell immunity? Nat. Rev. Immunol. 3, 609–620. Crowley, M. P., Fahrer, A. M., Baumgarth, N., Hampl, J., Gutgemann, I., Teyton, L., and Chien, Y. (2000). A population of murine gammadelta T cells that recognize an inducible MHC class Ib molecule. Science 287, 314–316. Dalton, J. E., Howell, G., Pearson, J., Scott, P., and Carding, S. R. (2004). Fas-Fas ligand interactions are essential for the binding to and killing of activated macrophages by gamma delta T cells. J. Immunol. 173, 3660–3667.

i n t e gr ati on o f t c e l ls

53

Dalton, J. E., Pearson, J., Scott, P., and Carding, S. R. (2003). The interaction of gamma delta T cells with activated macrophages is a property of the V gamma 1 subset. J. Immunol. 171, 6488–6494. Daniel, D., Meyer-Morse, N., Bergsland, E. K., Dehne, K., Coussens, L. M., and Hanahan, D. (2003). Immune enhancement of skin carcinogenesis by CD4þ T cells. J. Exp. Med. 197, 1017–1028. Della Bella, S., Nicola, S., Timofeeva, I., Villa, M. L., Santoro, A., and Berardi, A. C. (2004). Are interleukin-16 and thrombopoietin new tools for the in vitro generation of dendritic cells? Blood 104, 4020–4028. Diefenbach, A., Jamieson, A. M., Liu, S. D., Shastri, N., and Raulet, D. H. (2000). Ligands for the murine NKG2D receptor: Expression by tumor cells and activation of NK cells and macrophages. Nat. Immunol. 1, 119–127. Diehl, G. E., Yue, H. H., Hsieh, K., Kuang, A. A., Ho, M., Morici, L. A., Lenz, L. L., Cado, D., Riley, L. W., and Winoto, A. (2004). TRAIL-R as a negative regulator of innate immune cell responses. Immunity 21, 877–889. Dieli, F., Ivanyi, J., Marsh, P., Williams, A., Naylor, I., Sireci, G., Caccamo, N., Di Sano, C., and Salerno, A. (2003). Characterization of lung gamma delta T cells following intranasal infection with Mycobacterium bovis bacillus Calmette-Guerin. J. Immunol. 170, 463–469. Dodd, J., Riffault, S., Kodituwakku, J., Hayday, A., and Openshaw, P. (2005). Regulation of viral disease by gamma delta T cells. Dorner, B. G., Scheffold, A., Rolph, M. S., Huser, M. B., Kaufmann, S. H., Radbruch, A., Flesch, I. E., and Kroczek, R. A. (2002). MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines. Proc. Natl. Acad. Sci. USA 99, 6181–6186. Eberl, G., and Littman, D. R. (2003). The role of the nuclear hormone receptor RORgammat in the development of lymph nodes and Peyer’s patches. Immunol. Rev. 195, 81–90. El Mir, S., and Triebel, F. (2000). A soluble lymphocyte activation gene-3 molecule used as a vaccine adjuvant elicits greater humoral and cellular immune responses to both particulate and soluble antigens. J. Immunol. 164, 5583–5589. Fahrer, A. M., Konigshofer, Y., Kerr, E. M., Ghandour, G., Mack, D. H., Davis, M. M., and Chien, Y. H. (2001). Attributes of gammadelta intraepithelial lymphocytes as suggested by their transcriptional profile. Proc. Natl. Acad. Sci. USA 98, 10261–10266. Fehniger, T. A., and Caligiuri, M. A. (2001). Interleukin 15: Biology and relevance to human disease. Blood 97, 14–32. Feuerer, M., Beckhove, P., Bai, L., Solomayer, E. F., Bastert, G., Diel, I. J., Pedain, C., Oberniedermayr, M., Schirrmacher, V., and Umansky, V. (2001). Therapy of human tumors in NOD/ SCID mice with patient-derived reactivated memory T cells from bone marrow. Nat. Med. 7, 452–458. Fort, M. M., Cheung, J., Yen, D., Li, J., Zurawski, S. M., Lo, S., Menon, S., Clifford, T., Hunte, B., Lesley, R., Muchamuel, T., Hurst, S. D., Zurawski, G., Leach, M. W., Gorman, D. M., and Rennick, D. M. (2001). IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo. Immunity 15, 985–995. Fouillit, M., Joubert-Caron, R., Poirier, F., Bourin, P., Monostori, E., Levi-Strauss, M., Raphael, M., Bladier, D., and Caron, M. (2000). Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells. Glycobiology 10, 413–419. Fu, Y. X., Roark, C. E., Kelly, K., Drevets, D., Campbell, P., O’Brien, R., and Born, W. (1994). Immune protection and control of inflammatory tissue necrosis by gamma delta T cells. J. Immunol. 153, 3101–3115. Fujii, T., Okada, M., and Craft, J. (2002). Regulation of T-cell–dependent autoantibody production by a gammadelta T-cell line derived from lupus-prone mice. Cell Immunol. 217, 23–35.

54

d a ni el j . p e n ni ng t o n

E T AL .

Galvin, J. P., Spaeny-Dekking, L. H., Wang, B., Seth, P., Hack, C. E., and Froelich, C. J. (1999). Apoptosis induced by granzyme B-glycosaminoglycan complexes: Implications for granulemediated apoptosis in vivo. J. Immunol. 162, 5345–5350. Gershon, R. K., Cohen, P., Hencin, R., and Liebhaber, S. A. (1972). Suppressor T cells. J. Immunol. 108, 586–590. Gilfillan, S., Ho, E. L., Cella, M., Yokoyama, W. M., and Colonna, M. (2002). NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation. Nat. Immunol. 3, 1150–1155. Girardi, M., Glusac, E., Filler, R. B., Roberts, S. J., Propperova, I., Lewis, J., Tigelaar, R. E., and Hayday, A. C. (2003a). The distinct contributions of murine T-cell receptor (TCR)gammadeltaþ and TCRalphabetaþ T cells to different stages of chemically induced skin cancer. J. Exp. Med. 198, 747–755. Girardi, M., Lewis, J., Glusac, E., Filler, R. B., Geng, L., Hayday, A. C., and Tigelaar, R. E. (2002). Resident skin-specific gammadelta T cells provide local, nonredundant regulation of cutaneous inflammation. J. Exp. Med. 195, 855–867. Girardi, M., Oppenheim, D. E., Steele, C. R., Lewis, J. M., Glusac, E., Filler, R., Hobby, P., Sutton, B., Tigelaar, R. E., and Hayday, A. C. (2001). Regulation of cutaneous malignancy by gammadelta T cells. Science 294, 605–609. Girardi, M., Sherling, M. A., Filler, R. B., Shires, J., Theodoridis, E., Hayday, A. C., and Tigelaar, R. E. (2003b). Anti-inflammatory effects in the skin of thymosin-beta4 splice-variants. Immunology 109, 1–7. Gorelik, L., and Flavell, R. A. (2002). Transforming growth factor-beta in T-cell biology. Nat. Rev. Immunol. 2, 46–53. Hamerman, J. A., Ogasawara, K., and Lanier, L. L. (2004). Cutting edge: Toll-like receptor signaling in macrophages induces ligands for the NKG2D receptor. J. Immunol. 172, 2001–2005. Hayday, A., and Tigelaar, R. (2003). Immunoregulation in the tissues by gammadelta T cells. Nat. Rev. Immunol. 3, 233–242. Hedges, J. F., Cockrell, D., Jackiw, L., Meissner, N., and Jutila, M. A. (2003). Differential mRNA expression in circulating gammadelta T lymphocyte subsets defines unique tissue-specific functions. J. Leukoc. Biol. 73, 306–314. Herold, K. C., and Taylor, L. (2003). Treatment of Type 1 diabetes with anti-CD3 monoclonal antibody: Induction of immune regulation? Immunol. Res. 28, 141–150. Hilliard, B., Wilmen, A., Seidel, C., Liu, T. S., Goke, R., and Chen, Y. (2001). Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis. J. Immunol. 166, 1314–1319. Huang, C. T., Workman, C. J., Flies, D., Pan, X., Marson, A. L., Zhou, G., Hipkiss, E. L., Ravi, S., Kowalski, J., Levitsky, H. I., Powell, J. D., Pardoll, D. M., Drake, C. G., and Vignali, D. A. (2004). Role of LAG-3 in regulatory T cells. Immunity 21, 503–513. Huber, S., Shi, C., and Budd, R. C. (2002). Gammadelta T cells promote a Th1 response during coxsackievirus B3 infection in vivo: Role of Fas and Fas ligand. J. Virol. 76, 6487–6494. Huber, S. A., Graveline, D., Born, W. K., and O’Brien, R. L. (2001). Cytokine production by V gamma(þ)-T-cell subsets is an important factor determining CD4(þ)-Th-cell phenotype and susceptibility of BALB/c mice to coxsackievirus B3-induced myocarditis. J. Virol. 75, 5860–5869. Huber, S. A., Graveline, D., Newell, M. K., Born, W. K., and O’Brien, R. L. (2000). V gamma 1þ T cells suppress and V gamma 4þ T cells promote susceptibility to coxsackievirus B3-induced myocarditis in mice. J. Immunol. 165, 4174–4181. Indraccolo, S., Mion, M., Zamarchi, R., Coppola, V., Calderazzo, F., Amadori, A., and ChiecoBianchi, L. (1995). A CD3þCD8þ T cell population lacking CD5 antigen expression is expand-

i n t e gr ati on o f t c e l ls

55

ed in peripheral blood of human immunodeficiency virus-infected patients. Clin. Immunol. Immunopathol. 77, 253–261. Itohara, S., Mombaerts, P., Lafaille, J., Iacomini, J., Nelson, A., Clarke, A. R., Hooper, M. L., Farr, A., and Tonegawa, S. (1993). T-cell receptor delta gene mutant mice: Independent generation of alpha beta T cells and programmed rearrangements of gamma delta TCR genes. Cell 72, 337–348. Jameson, J., Ugarte, K., Chen, N., Yachi, P., Fuchs, E., Boismenu, R., and Havran, W. L. (2002). A role for skin gammadelta T cells in wound repair. Science 296, 747–749. Jeremias, I., Herr, I., Boehler, T., and Debatin, K. M. (1998). TRAIL/Apo-2-ligand-induced apoptosis in human T cells. Eur. J. Immunol. 28, 143–152. Kagi, D., Vignaux, F., Ledermann, B., Burki, K., Depraetere, V., Nagata, S., Hengartner, H., and Golstein, P. (1994). Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265, 528–530. Kamohara, H., Matsuyama, W., Shimozato, O., Abe, K., Galligan, C., Hashimoto, S., Matsushima, K., and Yoshimura, T. (2004). Regulation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor expression in human neutrophils. Immunology 111, 186–194. Klavinskis, L. S., Barnfield, C., Gao, L., and Parker, S. (1999). Intranasal immunization with plasmid DNA-lipid complexes elicits mucosal immunity in the female genital and rectal tracts. J. Immunol. 162, 254–262. Kolls, J. K., and Linden, A. (2004). Interleukin-17 family members and inflammation. Immunity 21, 467–476. Lee, K. M., Bhawan, S., Majima, T., Wei, H., Nishimura, M. I., Yagita, H., and Kumar, V. (2003). Cutting edge: The NK cell receptor 2B4 augments antigen-specific T-cell cytotoxicity through CD48 ligation on neighboring T cells. J. Immunol. 170, 4881–4885. Lehmann, J., Huehn, J., de la Rosa, M., Maszyna, F., Kretschmer, U., Krenn, V., Brunner, M., Scheffold, A., and Hamann, A. (2002). Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25þ as well as CD25-regulatory T cells. Proc. Natl. Acad. Sci. USA 99, 13031–13036. Leung, H. T., and Linsley, P. S. (1994). The CD28 costimulatory pathway. Ther. Immunol. 1, 217–228. Levi, G., Tarrab-Hazdai, R., and Teichberg, V. I. (1983). Prevention and therapy with electrolectin of experimental autoimmune myasthenia gravis in rabbits. Eur. J. Immunol. 13, 500–507. Li, N., Workman, C. J., Martin, S. M., and Vignali, D. A. (2004). Biochemical analysis of the regulatory T cell protein lymphocyte activation gene-3 (LAG-3; CD223). J. Immunol. 173, 6806–6012. Little, F. F., de Bie, J., van Oosterhout, A., Kornfeld, H., Center, D. M., and Cruikshank, W. W. (2003). Immunomodulatory effect of interleukin-16 on allergic airway inflammation. Chest. 123, 431S–432S. Lunemann, J. D., Waiczies, S., Ehrlich, S., Wendling, U., Seeger, B., Kamradt, T., and Zipp, F. (2002). Death ligand TRAIL induces no apoptosis but inhibits activation of human (auto) antigen-specific T cells. J. Immunol. 168, 4881–4888. Ma, H. L., Whitters, M. J., Konz, R. F., Senices, M., Young, D. A., Grusby, M. J., Collins, M., and Dunussi-Joannopoulos, K. (2003). IL-21 activates both innate and adaptive immunity to generate potent antitumor responses that require perforin but are independent of IFN-gamma. J. Immunol. 171, 608–615. Mackay, C. R. (2001). Chemokines: Immunology’s high impact factors. Nat. Immunol. 2, 95–101. Mackay, F., and Kalled, S. L. (2002). TNF ligands and receptors in autoimmunity: An update. Curr. Opin. Immunol. 14, 783–790.

56

d a ni el j . p e n ni ng t o n

E T AL .

McHugh, R. S., Whitters, M. J., Piccirillo, C. A., Young, D. A., Shevach, E. M., Collins, M., and Byrne, M. C. (2002). CD4(þ)CD25(þ) immunoregulatory T cells: Gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16, 311–323. Meissner, N., Radke, J., Hedges, J. F., White, M., Behnke, M., Bertolino, S., Abrahamsen, M., and Jutila, M. A. (2003). Serial analysis of gene expression in circulating gamma delta T cell subsets defines distinct immunoregulatory phenotypes and unexpected gene expression profiles. J. Immunol. 170, 356–364. Mirandola, P., Ponti, C., Gobbi, G., Sponzilli, I., Vaccarezza, M., Cocco, L., Zauli, G., Secchiero, P., Manzoli, F. A., and Vitale, M. (2004). Activated human NK and CD8þ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity. Blood 104, 2418–2424. Molinero, L. L., Fuertes, M. B., Fainboim, L., Rabinovich, G. A., and Zwirner, N. W. (2003). Upregulated expression of MICA on activated T lymphocytes involves Lck and Fyn kinases and signaling through MEK1/ERK, p38 MAP kinase, and calcineurin. J. Leukoc. Biol. 73, 815–822. Molinero, L. L., Fuertes, M. B., Girart, M. V., Fainboim, L., Rabinovich, G. A., Costas, M. A., and Zwirner, N. W. (2004). NF-kappa B regulates expression of the MHC class I-related chain A gene in activated T lymphocytes. J. Immunol. 173, 5583–5590. Mombaerts, P., Arnoldi, J., Russ, F., Tonegawa, S., and Kaufmann, S. H. (1993). Different roles of alpha beta and gamma delta T cells in immunity against an intracellular bacterial pathogen. Nature 365, 53–56. Moser, B., Wolf, M., Walz, A., and Loetscher, P. (2004). Chemokines: Multiple levels of leukocyte migration control. Trends Immunol. 25, 75–84. Nocentini, G., Giunchi, L., Ronchetti, S., Krausz, L. T., Bartoli, A., Moraca, R., Migliorati, G., and Riccardi, C. (1997). A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T-cell receptor-induced apoptosis. Proc. Natl. Acad. Sci. USA 94, 6216–6221. Nutt, S. L., Brady, J., Hayakawa, Y., and Smyth, M. J. (2004). Interleukin 21: A key player in lymphocyte maturation. Crit. Rev. Immunol. 24, 239–250. Offner, H., Celnik, B., Bringman, T. S., Casentini-Borocz, D., Nedwin, G. E., and Vandenbark, A. A. (1990). Recombinant human beta-galactoside binding lectin suppresses clinical and histological signs of experimental autoimmune encephalomyelitis. J. Neuroimmunol. 28, 177–184. Pace, K. E., Hahn, H. P., Pang, M., Nguyen, J. T., and Baum, L. G. (2000). CD7 delivers a proapoptotic signal during galectin-1-induced T cell death. J. Immunol. 165, 2331–2334. Pace, K. E., Lee, C., Stewart, P. L., and Baum, L. G. (1999). Restricted receptor segregation into membrane microdomains occurs on human T cells during apoptosis induced by galectin-1. J. Immunol. 163, 3801–3811. Parker, C. M., Groh, V., Band, H., Porcelli, S. A., Morita, C., Fabbi, M., Glass, D., Strominger, J. L., and Brenner, M. B. (1990). Evidence for extrathymic changes in the T cell receptor gamma/delta repertoire. J. Exp. Med. 171, 1597–1612. Peng, S. L., Madaio, M. P., Hayday, A. C., and Craft, J. (1996). Propagation and regulation of systemic autoimmunity by gammadelta T cells. J. Immunol. 157, 5689–5698. Pennington, D. J., Silva-Santos, B., Shires, J., Theodoridis, E., Pollitt, C., Wise, E. L., Tigelaar, R. E., Owen, M. J., and Hayday, A. C. (2003). The inter-relatedness and interdependence of mouse T-cell receptor gammadeltaþ and alphabetaþ cells. Nat. Immunol. 4, 991–998. Philpott, K. L., Viney, J. L., Kay, G., Rastan, S., Gardiner, E. M., Chae, S., Hayday, A. C., and Owen, M. J. (1992). Lymphoid development in mice congenitally lacking T-cell receptor alpha beta-expressing cells. Science 256, 1448–1452.

i n t e gr ati on o f t c e l ls

57

Poirier, F., and Robertson, E. J. (1993). Normal development of mice carrying a null mutation in the gene encoding the L14 S-type lectin. Development 119, 1229–1236. Poussier, P., Ning, T., Banerjee, D., and Julius, M. (2002). A unique subset of self-specific intraintestinal T cells maintains gut integrity. J. Exp. Med. 195, 1491–1497. Pribila, J. T., Quale, A. C., Mueller, K. L., and Shimizu, Y. (2004). Integrins and T-cell–mediated immunity. Annu. Rev. Immunol. 22, 157–180. Prockop, S., and Petrie, H. T. (2000). Cell migration and the anatomic control of thymocyte precursor differentiation. Semin. Immunol. 12, 435–444. Rabinovich, B. A., Li, J., Shannon, J., Hurren, R., Chalupny, J., Cosman, D., and Miller, R. G. (2003). Activated, but not resting, T cells can be recognized and killed by syngeneic NK cells. J. Immunol. 170, 3572–3576. Rabinovich, B. A., Shannon, J., Su, R. C., and Miller, R. G. (2000a). Stress renders T cell blasts sensitive to killing by activated syngeneic NK cells. J. Immunol. 165, 2390–2397. Rabinovich, G. A., Baum, L. G., Tinari, N., Paganelli, R., Natoli, C., Liu, F. T., and Iacobelli, S. (2002). Galectins and their ligands: Amplifiers, silencers or tuners of the inflammatory response? Trends Immunol. 23, 313–320. Rabinovich, G. A., Daly, G., Dreja, H., Tailor, H., Riera, C. M., Hirabayashi, J., and Chernajovsky, Y. (1999). Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis. J. Exp. Med. 190, 385–398. Rabinovich, G. A., Sotomayor, C. E., Riera, C. M., Bianco, I., and Correa, S. G. (2000b). Evidence of a role for galectin-1 in acute inflammation. Eur. J. Immunol. 30, 1331–1339. Ramsburg, E., Tigelaar, R., Craft, J., and Hayday, A. (2003). Age-dependent requirement for gammadelta T cells in the primary but not secondary protective immune response against an intestinal parasite. J. Exp. Med. 198, 1403–1414. Rasmussen, R. A., Counts, S. L., Daley, J. F., and Schlossman, S. F. (1994). Isolation and characterization of CD6-T cells from peripheral blood. J. Immunol. 152, 527–536. Raulet, D. H. (2003). Roles of the NKG2D immunoreceptor and its ligands. Nat. Rev. Immunol. 3, 781–790. Read, S., and Powrie, F. (2001). CD4(þ) regulatory T cells. Curr. Opin. Immunol. 13, 644–649. Renshaw, S. A., Parmar, J. S., Singleton, V., Rowe, S. J., Dockrell, D. H., Dower, S. K., Bingle, C. D., Chilvers, E. R., and Whyte, M. K. (2003). Acceleration of human neutrophil apoptosis by TRAIL. J. Immunol. 170, 1027–1033. Roberts, S. J., Smith, A. L., West, A. B., Wen, L., Findly, R. C., Owen, M. J., and Hayday, A. C. (1996). T-cell alpha beta þ and gamma delta þ deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium. Proc. Natl. Acad. Sci. USA 93, 11774–11779. Roessner, K., Wolfe, J., Shi, C., Sigal, L. H., Huber, S., and Budd, R. C. (2003). High expression of Fas ligand by synovial fluid-derived gamma delta Tcells in Lyme arthritis. J. Immunol. 170, 2702–2710. Ronchetti, S., Nocentini, G., Riccardi, C., and Pandolfi, P. P. (2002). Role of GITR in activation response of T lymphocytes. Blood 100, 350–352. Roths, J. B., Murphy, E. D., and Eicher, E. M. (1984). A new mutation, gld, that produces lymphoproliferation and autoimmunity in C3H/HeJ mice. J. Exp. Med. 159, 1–20. Russell, J. H., and Ley, T. J. (2002). Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 20, 323–370. Saito, H., Kranz, D. M., Takagaki, Y., Hayday, A. C., Eisen, H. N., and Tonegawa, S. (1984). Complete primary structure of a heterodimeric T-cell receptor deduced from cDNA sequences. Nature 309, 757–762.

58

d a ni el j . p e n ni ng t o n

E T AL .

Sano, H., Hsu, D. K., Yu, L., Apgar, J. R., Kuwabara, I., Yamanaka, T., Hirashima, M., and Liu, F. T. (2000). Human galectin-3 is a novel chemoattractant for monocytes and macrophages. J. Immunol. 165, 2156–2164. Santucci, L., Fiorucci, S., Cammilleri, F., Servillo, G., Federici, B., and Morelli, A. (2000). Galectin-1 exerts immunomodulatory and protective effects on concanavalin A-induced hepatitis in mice. Hepatology 31, 399–406. Schaerli, P., Ebert, L., Willimann, K., Blaser, A., Roos, R. S., Loetscher, P., and Moser, B. (2004). A skin-selective homing mechanism for human immune surveillance T cells. J. Exp. Med. 199, 1265–1275. Schon, M. P., Schon, M., Warren, H. B., Donohue, J. P., and Parker, C. M. (2000). Cutaneous inflammatory disorder in integrin alphaE (CD103)-deficient mice. J. Immunol. 165, 6583–6589. Shimizu, J., Yamazaki, S., Takahashi, T., Ishida, Y., and Sakaguchi, S. (2002). Stimulation of CD25 (þ)CD4(þ) regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3, 135–142. Shiohara, T., Moriya, N., Hayakawa, J., Itohara, S., and Ishikawa, H. (1996). Resistance to cutaneous graft-vs.-host disease is not induced in T-cell receptor delta gene-mutant mice. J. Exp. Med. 183, 1483–1489. Shires, J., Theodoridis, E., and Hayday, A. C. (2001). Biological insights into TCRgammadeltaþ and TCRalphabetaþ intraepithelial lymphocytes provided by serial analysis of gene expression (SAGE). Immunity 15, 419–434. Siegel, C. T., Schreiber, K., Meredith, S. C., Beck-Engeser, G. B., Lancki, D. W., Lazarski, C. A., Fu, Y. X., Rowley, D. A., and Schreiber, H. (2000). Enhanced growth of primary tumors in cancer-prone mice after immunization against the mutant region of an inherited oncoprotein. J. Exp. Med. 191, 1945–1956. Silva-Santos, B., Pennington, D. J., and Hayday, A. C. (2004). Lymphotoxin-mediated regulation of gd cell differentiation by ab T cell progenitors. Science 307, 925–928. Soderstrom, T. S., Poukkula, M., Holmstrom, T. H., Heiskanen, K. M., and Eriksson, J. E. (2002). Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in activated T cells abrogates TRAIL-induced apoptosis upstream of the mitochondrial amplification loop and caspase-8. J. Immunol. 169, 2851–2860. Song, K., Chen, Y., Goke, R., Wilmen, A., Seidel, C., Goke, A., and Hilliard, B. (2000). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J. Exp. Med. 191, 1095–1104. Spetz, A. L., Strominger, J., and Groh-Spies, V. (1996). T-cell subsets in normal human epidermis. Am. J. Pathol. 149, 665–674. Stepp, S. E., Mathew, P. A., Bennett, M., de Saint Basile, G., and Kumar, V. (2000). Perforin: More than just an effector molecule. Immunol. Today 21, 254–256. Straus, S. E., Sneller, M., Lenardo, M. J., Puck, J. M., and Strober, W. (1999). An inherited disorder of lymphocyte apoptosis: The autoimmune lymphoproliferative syndrome. Ann. Intern. Med. 130, 591–601. Sun, J., Ooms, L., Bird, C. H., Sutton, V. R., Trapani, J. A., and Bird, P. I. (1997). A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9). J. Biol. Chem. 272, 15434–15441. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302, 575–581. Toyabe, S., Harada, W., and Uchiyama, M. (2001). Oligoclonally expanding gammadelta T lymphocytes induce IgA switching in IgA nephropathy. Clin. Exp. Immunol. 124, 110–117. Triebel, F. (2003). LAG-3: A regulator of T-cell and DC responses and its use in therapeutic vaccination. Trends Immunol. 24, 619–622.

i n t e gr ati on o f t c e l ls

59

Vespa, G. N., Lewis, L. A., Kozak, K. R., Moran, M., Nguyen, J. T., Baum, L. G., and Miceli, M. C. (1999). Galectin-1 specifically modulates TCR signals to enhance TCR apoptosis but inhibit IL2 production and proliferation. J. Immunol. 162, 799–806. Waldmann, T. A., Dubois, S., and Tagaya, Y. (2001). Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: Implications for immunotherapy. Immunity 14, 105–110. Walunas, T. L., Lenschow, D. J., Bakker, C. Y., Linsley, P. S., Freeman, G. J., Green, J. M., Thompson, C. B., and Bluestone, J. A. (1994). CTLA-4 can function as a negative regulator of T-cell activation. Immunity 1, 405–413. Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A., and Nagata, S. (1992). Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356, 314–317. Wendling, U., Walczak, H., Dorr, J., Jaboci, C., Weller, M., Krammer, P. H., and Zipp, F. (2000). Expression of TRAIL receptors in human autoreactive and foreign antigen-specific T cells. Cell Death Differ. 7, 637–644. Workman, C. J., Cauley, L. S., Kim, I. J., Blackman, M. A., Woodland, D. L., and Vignali, D. A. (2004). Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J. Immunol. 172, 5450–5455. Wu, H., Knight, J. F., and Alexander, S. I. (2004). Regulatory gamma delta T cells in Heymann nephritis express an invariant Vgamma6/Vdelta1 with a canonical CDR3 sequence. Eur. J. Immunol. 34, 2322–2330. Wu, J., Groh, V., and Spies, T. (2002). T-cell antigen receptor engagement and specificity in the recognition of stress-inducible MHC class I-related chains by human epithelial gamma delta T cells. J. Immunol. 169, 1236–1240. Yamaoka, A., Kuwabara, I., Frigeri, L. G., and Liu, F. T. (1995). A human lectin, galectin-3 (epsilon bp/Mac-2), stimulates superoxide production by neutrophils. J. Immunol. 154, 3479–3487. Yang, R. Y., Hsu, D. K., and Liu, F. T. (1996). Expression of galectin-3 modulates T-cell growth and apoptosis. Proc. Natl. Acad. Sci. USA 93, 6737–6742. Yin, Z., Chen, C., Szabo, S. J., Glimcher, L. H., Ray, A., and Craft, J. (2002). T-Bet expression and failure of GATA-3 cross-regulation lead to default production of IFN-gamma by gammadelta T cells. J. Immunol. 168, 1566–1571. Yoshikai, Y. (1999). The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense. Immunol. Res. 20, 219–235. Young, J. D., Lawrence, A. J., MacLean, A. G., Leung, B. P., McInnes, I. B., Canas, B., Pappin, D. J., and Stevenson, R. D. (1999). Thymosin beta 4 sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids. Nat. Med. 5, 1424–1427. Zheng, L., Fisher, G., Miller, R. E., Peschon, J., Lynch, D. H., and Lenardo, M. J. (1995). Induction of apoptosis in mature T cells by tumour necrosis factor. Nature 377, 348–351.

Negative Regulation of Cytokine and TLR Signalings by SOCS and Others Tetsuji Naka,* Minoru Fujimoto,* Hiroko Tsutsui,{,z and Akihiko Yoshimura§ *Department of Molecular Medicine, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan { Department of Immunology and Medical Zoology, Hyogo College of Medicine, Hyogo 663-8501, Japan z CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan § Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Introduction ..................................................................................................... Cytokine Signaling ............................................................................................. Negative Regulation of Cytokine Signaling ............................................................. Regulation of Cytokine Signaling by SOCS Proteins (Tables 1 and 2) .......................... TLR-Mediated Pathways..................................................................................... Signal Transduction Pathways Through TLRs.......................................................... Major Biological Events by the TLR-Mediated Cell Activation ................................... Pathophysiological Roles for TLR-Mediated Signal Pathways ..................................... Negative Regulation of the TLR Signalings............................................................. Regulation of TLR Signaling by SOCS................................................................... Concluding Remarks .......................................................................................... References .......................................................................................................

61 62 64 68 83 88 92 93 96 101 103 103

1. Introduction Immunity is largely categorized into two types, adoptive immunity and innate immunity. As compared with innate immunity, acquired immunity has the diversity and the accuracy in its recognition of corresponding antigens based on the DNA rearrangement machineries and hypermutation properties that T cells and B cells selectively possess. Therefore, acquired immunity had been believed to be extremely sophisticated and an ideal system for host defense. Innate immunity had been regarded to play a role only as the front line that would drop out after activation of the corresponding acquired immunity. In fact, innate immune constituents, such as dendritic cells (DCs) and macrophages, can promptly respond to microbes and their products without help from additional acquired immune responses, whereas acquired immunity takes a long time to be able to exert its full immunological actions (Janeway

61 advances in immunology, vol. 87 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)87003-8

62

TETSUJI N AKA ET A L .

and Medzhitov, 2002). However, recent intensive studies on signaling receptors in the innate immune system, in particular on Toll-like receptors (TLRs), led us to notify its importance comparable to adaptive immunity. After microbial infection, antigen-presenting cells (APCs) composing innate immunity capture the microbial antigens. Simultaneously, the microbial products stimulate the APCs through their TLRs to undergo the appropriate maturation, which is represented by expression of chemokine receptors critical for translocation into the regional lymph nodes and of various costimulatory molecules essential for the appropriate activation of T helper cells. These APCs produce a variety of cytokines in response to the TLR ligands of the microbes as well. Unless they experience these biological events through their TLRs, these APCs cannot drive the activation or differentiation of antigenspecific T cells. In particular, certain cytokines, such as IL-12 and IL-18, produced by the APCs are required for the differentiation of naive helper T cells toward the effector cells to eradicate the microbes. Thus, the absence of innate immune responses render mammalian hosts highly susceptible to pathological organisms (Takeda et al., 2003). Like in the case of autoimmunity associated with the dysregulated adaptive immunity, excessive activation of innate immunity causes diseases. It is well documented that the activation of innate immunity by pathogens occasionally causes fatal pathological alterations via aberrant induction of cytokines. Both innate and acquired immune systems complete their proper actions via cognate cellular interactions and cytokine catch bowl. Therefore, it is quite important for homeostatic immune responses to regulate the innate immune responses and adaptive immunity by controlling TLR and cytokine signaling. In this review, we will describe two major immune signal pathways, cytokine signaling and TLR-mediated signaling, and also regulatory mechanisms for these pathways, particularly focusing on the family of suppressor of cytokine signaling (SOCS) proteins that is implicated in negative regulation of both cytokine signaling and TLR signaling. 2. Cytokine Signaling Cytokines are central to the immune cell biology, including their differentiation, proliferation, activation, and apoptosis, and therefore are critical for the modulation of a wide range of immune responses. Cytokines signal through their own receptors on the cell surface. Most of these receptors lack intrinsic kinase activity but are associated with janus kinases (JAKs), a family of protein tyrosine kinases. Upon ligand binding to cytokine receptors and subsequent receptor dimerization, JAKs are activated to phosphorylate tyrosine residues in the intracellular domain of cytokine receptors. These phosphorylated tyrosines then become docking sites for a number of intracellular proteins—most represent a family of signal transdu-

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

63

Figure 1 Cytokine signaling pathway. Cytokines bind to their specific receptors on the cell surface and activate intracellular signaling cascades. The most representative and important cascade for cytokines is the JAK‐STAT pathway. JAKs are protein tyrosine kinases that associate with cytokine receptors. The binding of cytokines to their receptors induces receptor oligomerization and activation of JAKs. JAKs phosphorylate tyrosine residues in cytoplasmic portion of cytokine receptors and create docking platforms for intracellular signaling proteins. STATs are recruited to their specific docking sites of receptors, bind to phosphorylated tyrosine residues with their SH2 domains, and are tyrosine‐phosphorylated by JAKs. Activated STATs dissociate from receptors, then become dimers and translocate to the nucleus. STATs directly associate with GAS motifs, their consensus binding motifs, of the promoter region, and regulate the transcription of their target genes. Other signaling cascades such as MAPK and PI3K pathways are also activated by cytokines and contribute to the action of cytokines. JAK, Janus kinase; STAT, Signal transducer and activator of transcription; GAS, Gamma-activated sequences; MAPK, Mitogen activated; PI3K.

cers and activators of transcription (STATs). STATs, after tyrosine-phosphorylation by JAKs, dimerize and translocate to the nucleus, where they induce the expression of their target genes by binding to GAS (g activated sequence) or other specific motifs in the promoter region (Fig. 1). Various combinations of JAKs

64

TETSUJI N AKA ET A L .

(JAK1, JAK2, JAK3, and TYK2) and STATs (STAT1, STAT2, STAT3, STAT5a, STAT5b, and STAT6) are activated to transduce cytokine signals, but recent genetargeting studies have clarified non-redundant and specific roles of each JAK and STAT member in different cytokine signaling (Leonard and O’Shea, 1998; O’Shea et al., 2002). Among them, STAT1 is relatively specific to IFNs, STAT3 is activated by IL-6 and other gp130-related cytokines, STAT4 is activated by IL-12, and STAT6 is specifically activated by IL-4 and IL-13. STAT5 is activated by various cytokines including IL-2, IL-3, EPO, and GH. 3. Negative Regulation of Cytokine Signaling It has been suggested that sustained and/or excessive action of cytokines can be harmful to organisms. Accordingly, a number of mechanisms have been reported to modulate cytokine signaling to prevent this overaction of cytokines (Naka et al., 1999). For example, soluble forms of cytokine receptors that lack intracellular domains can inhibit the action of cytokines by simple competition for cytokine binding. The endocytosis of receptors and proteasomal degradation of signaling molecules after initial ligand stimulation is thought to play a role in preventing continuous cytokine signaling (Naka et al., 1999). In addition, there are some molecules that actively function as negative regulators of cytokine signaling. SH2-containing phosphatase SHP-1 can terminate cytokine signaling by dephosphorylation of JAKs (Shultz et al., 1997; Zhang et al., 2000). Other phosphatases such as protein tyrosine phosphatase 1B (PTP1B) (Myers et al., 2001), CD45 (Irie-Sasaki et al., 2001), and T-cell protein tyrosine phosphatase (TCPTP) (Simoncic et al., 2002) have also been reported to inhibit cytokine signaling as JAK phosphatases. Protein inhibitors of activated STATs (PIAS) family of proteins can inhibit the function of STATs by binding directly to STATs (Chung et al., 1997; Liu et al., 2004). Moreover, recent accumulating evidence suggests that another family of proteins, SOCS proteins, is an important negative regulator for cytokine signaling (Fig. 2) (Alexander and Hilton, 2004; Fujimoto and Naka, 2003; Ilangumaran et al., 2004; Kubo et al., 2003). 3.1. SHP-1 (SH2-Containing Phosphatase-1) SHP-1 is a protein tyrosine phosphatase that contains two SH2 domains and a phosphatase domain. SHP-1 is constitutively expressed in immune cells and has been implicated in the dephosphorylation of signaling proteins such as IL-4 receptor, c-kit, EPO receptor, and JAK2. In addition, lines of evidence

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

65

Figure 2 Negative regulators of cytokine signaling. Accumulating evidence suggests that cytokine signaling is regulated by multiple mechanisms. Soluble forms of receptors capture secreted cytokines and inhibit the activation of intracellular signaling cascades. Upon activation by cytokines, the expression of cell surface receptors is downregulated by receptor internalization and/or proteasomal degradation. Several kinds of phosphatases such as CD45, SHP‐1, PTP1B, and TCPTP are constitutively expressed and regulate kinase activity of JAKs. PIAS proteins associate with activated STATs and inhibit DNA‐STAT interaction. SOCS proteins are target genes of cytokines and upon induction, terminate signaling in a negative feedback manner by binding to activated receptors and/or JAKs. SHP‐1, SH2 containing phosphatase‐1; PTP1B, Protein tyrosine phosphatase 1B; TCPTP, T cell protein tyrosine phosphatase; PIAS, Protein inhibitors of activated STATs; SOCS, Suppressor of cytokine signaling.

66

TETSUJI N AKA ET A L .

suggest that signaling from B-cell receptor (BCR) and T-cell receptor (TCR) are also regulated by SHP-1. Critical roles of SHP-1 were shown by the analysis of mice known as motheaten mice. Motheaten mice possess spontaneous mutation in hematopoietic cell phosphatase (Hcph) locus encoding SHP-1 and show severe dysregulation in macrophages and neutrophils, resulting in patchy dermatitis and fatal hemorrhagic pneumonitis (Wormald and Hilton, 2004; Zhang et al., 2000). Since the immune dysregulation of motheaten mice is severe and complex, it may require further investigations to determine whether the wide range of SHP-1 actions reported previously are its direct and primary functions in vivo. 3.2. Protein Inhibitors of Activated STATs (PIAS) PIAS proteins have a N-terminal nuclear receptor interaction motif and a central zinc-binding domain (Wormald and Hilton, 2004). PIAS3, a member of PIAS family, was identified as STAT3-binding protein that inhibits functions of STAT3 (Chung et al., 1997). Later, other members of this family, PIAS1, PIASx, and PIASy were identified and shown to be inhibitors of STAT1, STAT4, and STAT1, respectively (Wormald and Hilton, 2004). The expression of PIAS proteins is not dependent on cytokines, but their interaction with STATs requires cytokine stimulation. Previous reports have shown that PIAS1 and PIAS3 function by interfering with the DNA binding of STAT1 and STAT3, respectively, but several other functions are also reported for PIAS proteins. In particular, recent analyses have shown that PIAS proteins can function as small ubiquitin-like modifier (SUMO) E3 ligases that attach SUMO protein to a number of proteins including STAT1 (Wormald and Hilton, 2004). Studies in physiological functions of PIAS proteins are currently underway through the generation of knockout mice. PIASy KO mice are phenotypically normal and show normal STAT1 activation and normal sumoylation (Wong et al., 2004). This result suggests that the function of PIASy in vivo is dispensable or is compensated possibly by other PIAS proteins. PIAS1 KO mice are recently generated, and cells from these mutant mice also show unaltered STAT1 phosphorylation in response to IFNs and normal SUMO3 conjugation in response to stress signals (Liu et al., 2004). However, PIAS1 KO mice are not born at expected Mendelian frequency and appear runted at birth. In addition, in PIAS1 KO cells, a small subset of IFN-inducible genes (9% of the genes examined) shows enhanced induction after IFN stimulation. The altered IFN responsiveness in the absence of PIAS1 is biologically significant and possibly one of the causes for the runted phenotype of PIAS1 KO mice, since

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

67

PIAS1 KO mice are resistant to microbe infection and are susceptible to LPSinduced endotoxin shock (Liu et al., 2004). This study suggests that PIAS1 suppresses a subset of STAT1 functions, possibly by inhibiting the binding of STAT1 to low- but not high-affinity binding sites of IFN-responsive promoters. Further investigation appears to be required to clarify whether PIAS proteins in vivo have previously reported functions including SUMO E3 ligase activity. 3.3. SOCS Family of Proteins SOCS proteins are structurally defined by two common motifs. One is a central Src homology 2 (SH2) domain that can bind to phosphorylated tyrosine residues, and the other is a carboxy-terminal 40-amino-acid module named SOCS box that can bind to elongin BC complex. To date, eight mammalian SOCS proteins (CIS, SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, SOCS-6, and SOCS-7) have been discovered (Fig. 3). In general, SOCS proteins are

Figure 3 SOCS family. Two conserved motifs, a central SH2 domain and a C‐terminal SOCS box, structurally define SOCS proteins. The SH2 domain of SOCS proteins is required for the association with the phosophorylated tyrosine residues of their target proteins. The SOCS box can recruit Elongin BC and contribute to the proteasomal degradation of the target proteins. SOCS box may also be involved in the stabilization of SOCS proteins. SOCS‐1 and SOCS‐3 have a common motif called pre‐SH2 domain that contributes to the interaction with JAKs mediated by its kinase inhibitory region (KIR).

68

TETSUJI N AKA ET A L .

expressed at low levels in unstimulated cells. Upon stimulation of cells with cytokines, hormones, and bacterial components such as LPS, SOCS proteins are rapidly induced and then regulate intracellular signaling. In 1995 the member of SOCS proteins was first cloned. This protein was named cytokine-inducible SH2 containing protein (CIS), and was shown to be a negative feedback regulator of IL-3 signaling (Yoshimura et al., 1995). Two years later, another member of SOCS proteins was cloned independently in three laboratories and called variously as suppressor of cytokine signaling-1 (SOCS-1), STAT-induced STAT inhibitor-1 (SSI-1), or JAK-binding protein (JAB) (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997). Interestingly, this protein also functions as a negative feedback regulator of cytokine signaling, since it is induced by cytokines such as IL-6 and inhibits cytokine signaling by suppressing JAK kinases. Simultaneously, two more SOCS proteins were cloned and named as SOCS-2 and SOCS-3 (Starr et al., 1997). Subsequent searching of genetic databases revealed that there are at least four other SOCS proteins—SOCS-4, SOCS-5, SOCS-6, and SOCS-7 (Hilton et al., 1998). Similar to CIS, SOCS-1, SOCS-2, and SOCS-3 also act in a classical negativefeedback loop to inhibit cytokine signal transduction. However, the functions of other SOCS proteins were less elucidated. In this part of the review, we would like to refer mainly to CIS, SOCS-1 to -3 and then briefly to other SOCS proteins. 4. Regulation of Cytokine Signaling by SOCS Proteins (Tables 1 and 2) 4.1. CIS (CIS1) The expression of CIS can be induced by cytokines such as IL-2, IL-3, GMCSF, erythropoietin (EPO), growth hormone (GH), and prolactin (PRL), which activates STAT5. In line with this finding, the promoter region of CIS contains several STAT5 responsive elements. Upon induction, CIS can associate with a number of cytokine receptors, such as IL-2 receptor (IL-2Rb), IL3Rb, PRLR, GHR, and EPOR, at the same docking site with STAT5, and inhibit the tyrosine-phosphorylation of STAT5 (Aman et al., 1999; Matsumoto et al., 1997; Ram and Waxman, 2000; Yoshimura et al., 1995). Thus, CIS appears to inhibit STAT5 activation by competing for the binding site with STAT5. In addition, as suggested by the finding that CIS can be ubiquitinated, CIS may also inhibit cytokine signaling by targeting CIS-receptor complexes for ubiquitin-mediated proteasomal degradation (Verdier et al., 1998). All these results suggest that CIS is a negative feedback regulator of STAT5. Indeed, CIS transgenic mice have a strikingly similar phenotype with STAT5 knockout mice including growth retardation and impaired mammary gland development (Matsumoto et al., 1999), suggesting that CIS can function as an

Table 1 Physiological Functions of SOCS Family Proteins SOCS family CIS SOCS-1

SOCS-2

69

SOCS-3

SOCS-4 SOCS-5 SOCS-6 SOCS-7

Immunoregulatory cytokines IL-2, IL-3, IL-6, IL-9, IFN-a, TNF-a IL-2, IL-4, IL-6, IL-7, IL-9, IL-13, IFN- a/b, IFN-a, LIF, TNF-a IL-6, IFN-a, IFN-a, LIF IL-1, IL-2, IL-6, IL-9, IL-10, IL-13, IFN-a, IFN-a, LIF unknown IL-6 unknown unknown

Colony-stimulating factors

Hormones and growth factors

EPO, TSLP

GH, Prolactin

EPO, TPO, TSLP, G-CSF, GM-CSF, M-CSF

GH, Prolactin, Insulin, CNTF, Cadiotropin, TSH

EPO, GM-CSF

GH, Prolactin, Insulin, CNTF, Cadiotropin GH, Prolactin, Insulin, Leptin, CNTF

PAMPs

LPS, CpG DNA

LPS, CpG DNA

Table 2 Phenotypes of Mice Deficient in SOCS Family Members Factors regulated by SOCS proteins in vivo SOCS proteins CIS SOCS-1

70

SOCS-2 SOCS-3 SOCS-4 SOCS-5 SOCS-6 SOCS-7

Phenotype of KO mice Viable without obvious phenotype Viable but lethal within 3 wks of age, multiorgan inflammation mediated by IFN-a and other cytokines Viable, fertile, gigantism Embryonic lethality due to placental insufficiency Not reported yet Viable, fertile, normal lymphocyte function Viable, fertile, mild growth retardation Viable, fertile, early death due to hydrocephalus

Cytokines IL-2, IL-4, IL-7, IL-12, IL-15, IFN-a, TNF-a IL-6, LIF, G-CSF

Hormones and growth factors Prolactin, Insulin

GH Insulin, Leptin

Insulin?

PAMPs LPS, CpG DNA

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

71

inhibitor of STAT5 in vivo. However, most of the functions of CIS may be redundant in vivo, possibly due to the compensation by other SOCS proteins, because it was reported that CIS knockout mice did not show an obvious phenotype (Marine et al., 1999a). However, bone-marrow-derived mast cells from CIS-KO mice are apparently hyperresponsive to IL-3 (Kimura et al., unpublished data). Further careful examination of CIS-KO mice is necessary to define specific physiological functions of CIS. While an analysis of other CIS transgenic mice suggests that CIS may be involved in the regulation of T-cell receptor (TCR) signaling by binding to protein kinase C (PKCy) (Li et al., 2000), its physiological significance remains to be elucidated. 4.2. SOCS-1 (SSI-1, JAB) 4.2.1. Negative Regulation of Cytokine Signaling by SOCS-1 Originally, we and others reported SOCS-1 as a negative-feedback suppressor of IL-6 and LIF (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997). However, subsequent analyses in vitro have revealed that a number of cytokine stimulations can induce the expression of SOCS-1, and the action of many cytokines, including interleukins, interferons, and growth factors, can be suppressed by the overexpression of SOCS-1 (Naka et al., 1999). These findings suggest that SOCS-1 is not only a negative-feedback regulator of cytokines, but also a cross-talk inhibitor among cytokines. A great majority of cytokines activates JAK-STAT signaling pathways and, like other target genes of these pathways, SOCS-1 is induced by cytokines via the activation of STATs and their downstream transcription factors such as IRF-1 (Saito et al., 2000). In addition, it is likely that SOCS-1 is also induced independently of JAK-STAT pathways, because SOCS-1 can be induced by factors that do not primarily utilize JAKs and/or STATs, such as stem cell factor, TGF-b, insulin, and LPS. These results indicate that a wide range of cytokines and growth factors can induce SOCS-1. However, because of its wide range of influences on various signaling pathways, SOCS-1 expression appears to be strictly controlled by several mechanisms. For example, the transcription of SOCS-1 is negatively regulated by growth factor independence (Gfi)-1B, a proto-oncogenic transcriptional repressor (Jegalian and Wu, 2002). In addition, it has been shown that SOCS-1 expression is regulated through translational repression (Schluter et al., 2000). Moreover, in line with the rapid turnover rate of SOCS-1 protein, SOCS-1 protein levels are also targets for the regulation. Previous findings suggest the possibility that the SOCS box plays a role in the stabilization of SOCS-1 protein (Kamura et al., 1998; Narazaki et al., 1998). In addition, recent findings suggest that other

72

TETSUJI N AKA ET A L .

proteins such as Pim-1 or TRIM8 also affect the stability of SOCS-1 protein (Chen et al., 2002; Toniato et al., 2002). This broad inhibitory function of SOCS-1 can be explained partly by the fact that SOCS-1 directly binds to the activation loop of JAKs and suppresses their kinase activity. The association of SOCS-1 to JAKs is dependent on pre-SH2 and SH2 domains of SOCS-1 and also requires the tyrosine-phosphorylation of JAKs (Narazaki et al., 1998; Nicholson et al., 1999; Yasukawa et al., 1999). The pre-SH2 domain of SOCS-1 also contains kinase inhibitory region (KIR) that appears to suppress the kinase activity of JAKs by functioning as a pseudosubstrate (Yasukawa et al., 1999). C-terminal SOCS box is also required for the full action of SOCS-1, since this motif can serve as a docking site for Elongin BC complex, which is likely to form an E3 ubiquitin ligase together with cullin and Rbx-1 (Kamura et al., 1998, 2004; Kile et al., 2002; Zhang et al., 1999). This E3 ubiqitin ligase contributes to the poly-ubiqitination of SOCS-1associated proteins such as phosphorylated JAKs, and leads to proteasomal degradation of these proteins (Frantsve et al., 2001; Kamizono et al., 2001; Kile et al., 2002; Monni et al., 2001). Thus, three functional domains of SOCS-1 accomplish inhibition of JAKs by SOCS-1; namely, pre-SH2 domain, SH2 domain, and SOCS box. The suppressive action of SOCS-1 may be exerted through JAK-independent mechanism, since SOCS-1 associates not only with JAKs but also with a number of other proteins such as tec (Ohya et al., 1997), c-kit (De Sepulveda et al., 1999), vav (De Sepulveda et al., 2000), IRS-1 (Kawazoe et al., 2001), IRS-3 (Rui et al., 2002), insulin receptor (IR) (Mooney et al., 2001), EGFR (Xia et al., 2002), and IRAK (IL receptor-associated kinase) (Nakagawa et al., 2002). SOCS-1 is also shown to interact with nuclear proteins such as human papilomavirus E7 (Kamio et al., 2004) and p65 subunit of NF-kB (Ryo et al., 2003). Interestingly, a large amount of SOCS-1 protein is localized in the nucleus (Kamio et al., 2004). At least in some cases, the association of SOCS-1 with these molecules can accelerate their proteasomal degradation and possibly confers to the suppression of their functions. However, it remains to be elucidated that these associations of SOCS-1 reflect physiological functions of SOCS-1. 4.2.2. Physiological Function of SOCS-1 After the generation of SOCS-1 knockout (KO) mice, physiological functions of SOCS-1 have been extensively studied. SOCS-1 KO mice are born normally in a Mendelian fashion, but they become runted and die within 3 weeks after birth (Naka et al., 1998; Starr et al., 1998). The pathological features of SOCS-1 KO mice are (1) progressive lymphocytopenia accompanied with their

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

73

enhanced activation and accelerated apoptosis and (2) mononuclear infiltrations in multiple organs including liver, lung, heart, and pancreas. In particular, pathological changes in the liver are most striking, which include fatty degeneration and necrosis of hepatocytes, and presumably a major cause for the early death of SOCS-1 KO mice (Naka et al., 1998; Starr et al., 1998). The complex multi-organ disease of SOCS-1 KO mice can be attributed to the harmful action of cytokines. This notion was proven at first by the generation of SOCS-1/IFN-g double knockout (DKO) mice (Alexander et al., 1999; Marine et al., 1999b). Unlike SOCS-1 KO mice, SOCS-1/IFN-g DKO mice exhibit no signs of wasting disease, grow up normally, and become fertile. The pathological role of IFN-g is also illustrated by the findings on SOCS-1 KO mice, such as spontaneous activation of STAT1 in liver, upregulation of IFN-responsive genes, and hyperresponse of SOCS-1-deficient cells to exogenous IFN-g (Alexander et al., 1999). Subsequent analysis of mice lacking SOCS-1 and STAT1, a major transcription factor for IFNs, also supports the pathological role for IFN-g (Naka et al., 2001). However, IFN-g is not a sole cytokine that causes pathological alterations in SOCS-1 KO mice, because a detailed and long-term analysis of SOCS-1/IFN-g DKO mice has revealed that these DKO mice cannot escape from inflammatory organ damages and have shorter lifespans than controls due to the diseases such as leukemia and polycystic kidneys (Metcalf et al., 2002). Indeed, mice lacking both SOCS-1 and STAT6, a major transcription factor for IL-4 signaling, are also rescued from early death (Naka et al., 2001), suggesting that IL-4 also contributes to the disease of SOCS-1 KO mice. It is likely that IL-4, in conjunction with IFNg, plays an important role for hepatic disease of SOCS-1 KO mice. In particular, a lack of SOCS-1 leads to the disruption of cross-inhibitory action of IFN-g on IL-4, and causes aberrant activation of hepatic NKT cells by simultaneous action of these cytokines (Naka et al., 2001). Recently, a suppressive role for SOCS-1 in IL-12 signaling is also suggested by the analysis of mice lacking SOCS-1 and STAT4, a major signaling molecule for IL-12 (Eyles et al., 2002). SOCS-1/STAT4 DKO mice have improved survival compared to SOCS-1 KO mice, which is in line with the hyperresponsiveness of SOCS-1deficient lymphocytes to exogenous IL-12 (Eyles et al., 2002; Fujimoto et al., 2002). Thus, these findings indicate that SOCS-1 negatively regulates cytokines such as IL-4, IL-12, and IFN-g, implying an important role for SOCS-1 in the regulation of acquired immunity. SOCS-1 is also involved in the negative regulation of endocrine systems. For example, SOCS-1 KO mice exhibit hypoglycemia, and embryonic fibroblasts from SOCS-1 KO mice showed hyperresponsiveness to insulin, suggesting an inhibitory role for SOCS-1 in insulin signaling (Kawazoe et al., 2001). This may be ascribed to the interaction of SOCS-1 with IRS-1 followed by the

74

TETSUJI N AKA ET A L .

proteasomal degradation of this complex (Rui et al., 2002) and may account for the development of insulin resistance on stressed conditions such as infection. In addition, SOCS-1 appears to inhibit PRL signaling by inhibiting JAK2, since pregnant SOCS-1/IFN-g DKO mice exhibit enhanced mammary gland development (Lindeman et al., 2001). All these results indicate that all of the SOCS-1 functions suggested by the in vitro studies are not necessarily essential in vivo. However, the phenotype of SOCS-1 KO mice is complex, and the detailed analyses are still underway. For instance, SOCS-1 has the capacity to modulate TNF-a signaling, the mechanism of which remains to be clarified (Chong et al., 2002; Morita et al., 2000). Furthermore, recent generations of TCR-transgenic SOCS-1/ IFN-g DKO mice suggest that SOCS-1 has actions in the maintenance of T-cell homeostasis, which are not exerted by the inhibition of TCR and IFN-g (Cornish et al., 2003b), but possibly by the inhibition of other cytokines like those that signal through common g chains (Fujimoto et al., 2000). Indeed, T-cell–specific deletion of SOCS-1 in mice (T-cell–specific conditional SOCS-1 KO mice) revealed that SOCS-1 is a physiological regulator of IL-7 and other gc-cytokines, including IL-2 and IL-15 (Chong et al., 2003). Similarly, inhibitory action of SOCS-1 on gc-cytokines has also been illustrated by detailed analysis of T cells in SOCS-1/IFN-g DKO mice (Cornish et al., 2003a; Ilangumaran et al., 2003a,b). 4.2.3. SOCS-1 and Acquired Immunity Several lines of evidence indicate that SOCS-1 is critical for the homeostasis of T cells that play major roles in acquired immunity. For example, T cells in SOCS-1 KO mice spontaneously exhibit activated phenotype (Marine et al., 1999b) and have the capacity to produce large amounts of cytokines such as IFN-g and IL-4 (Fujimoto et al., 2002). SOCS-1/Rag-2 DKO mice, which lack mature lymphocytes, are rescued from early lethality (Marine et al., 1999b). Adoptive transfer of SOCS-1-deficient lymphocytes in Rag-2 KO mice or JAK3 KO mice leads to multi-organ inflammatory disease similar to SOCS-1 KO mice (Marine et al., 1999b; Naka et al., 2001). These results suggest that the disease of SOCS-1 KO mice is dependent on T cells. However, it should be noted here that immune cells other than T cells are also involved in the disease of SOCS-1 KO mice, because T-cell–specific elimination of SOCS-1 failed to recapitulate the multiorgan disease of SOCS-1 KO mice (Chong et al., 2003). Supportingly, recent reports have shown that the lack of SOCS-1 leads to the activation of dendritic cells and induces autoimmune disease similar to systemic lupus erythematosus (SLE) (Hanada et al., 2003). Nonetheless, these findings suggest that SOCS-1 may regulate the magnitude of acquired immune responses and inhibits their

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

75

harmful overactivation. Indeed, as described previously, SOCS-1 suppresses the signaling of IL-4, IL-12, and IFN-g, representative cytokines for Th responses. Moreover, in vitro culture of CD4 T cells from healthy SOCS-1 heterozygous mice leads to the enhanced production of IFN-g and IL-4 under Th1 and Th2 conditions, respectively (Fujimoto et al., 2002). SOCS-1 heterozygous mice exhibit enhanced Th1 or Th2 polarization in response to the infection with Listeria or Nippostrongylus, respectively (Fujimoto et al., 2002). Thus, SOCS-1 is a critical regulator of acquired immunity, and a small decrease in the amount of SOCS-1 may result in strong or excess immune responses. This nature of SOCS1-deficient DCs can be applied to antitumor immunity. Shen et al. reported that silencing the SOCS1 gene by siRNA technology in antigen-presenting DCs strongly enhances antigen-specific antitumor immunity (Shen et al., 2004). 4.2.4. SOCS-1 and Immune Diseases Given the wide range of immunoregulatory functions, SOCS-1 may be implicated in the pathology of immune diseases. In a murine model of autoimmune colitis, inhibition of endogenous SOCS-1 and SOCS-3 by transgenic expression of the dominant-negative form of SOCS-1 exaggerated the inflammation and disease (Suzuki et al., 2001). Similarly, in a murine model of autoimmune arthritis, joint inflammation and destruction were significantly enhanced in mice lacking SOCS-1 (Egan et al., 2003; Ivashkiv and Tassiulas, 2003). Moreover, recent generations of other SOCS-1 mutant mice in which SOCS-1 is restored in lymphocytes on a SOCS-1 KO background suggest that SOCS-1 functions as a suppressor of lupus-like systemic autoimmunity (Fujimoto et al., 2004; Hanada et al., 2003). These findings suggest that, in experimental models, SOCS-1 counteracts against the development of autoimmune diseases. Future studies are required to elucidate the roles of SOCS-1 in human autoimmune diseases. We found that SOCS-1 gene silencing by DNA methylation is frequently observed in hepatitis induced by HCV infection (Yoshida et al., 2004). SOCS-1 gene methylation was well correlated with the severity of liver fibrosis, suggesting that reduction of SOCS-1 gene expression by DNA methylation promotes liver inflammation. 4.2.5. SOCS-1 as a Tumor Suppressor Recently, antitumor activity of SOCS-1 has been reported by several groups. SOCS-1 may inhibit the development and/or progression of hepatocellular carninoma (HCC), since SOCS-1 expression is significantly reduced in HCC

76

TETSUJI N AKA ET A L .

cells, which can be explained by the inactivation of the SOCS-1 promoter due to hypermethylations of CpG islands (Nagai et al., 2002a; Yoshikawa et al., 2001). Yang et al. (2004) investigated the promoter methylation status of major tumor suppressor genes (including SOCS-1, GSTP, APC, E-cadherin, RAR-beta, p14, p15, p16, and p73) in 51 cases of HCC. Among these, SOCS-1 was most frequently methylated (65%). Methylation of SOCS-1, APC, and p15 was more frequently seen in hepatitis C virus-positive HCC than hepatitis C virus/hepatitis B virus-negative HCC. These data suggest that promoter hypermethylation of SOCS-1 is an important event in HCC development. Supportingly, a recent experiment has shown that SOCS-1 heterozygous mice are hypersensitive to dimethylnitrosamine-induced hepatocarcinogenesis (Yoshida et al., 2004). SOCS-1 could be a novel anti-oncogene that accelerates inflammation-induced carcinogeneisis. In addition, SOCS-1 may inhibit the progression of hematopoietic malignancies, since SOCS-1 in vivo is preferentially expressed in lymphoid organs. Indeed, SOCS-1 interacts with TEL-JAK2 oncoprotein, a leukemic fusion protein caused by a chromosomal translocation, and can target it for ubiquitin-mediated proteasomal degradation (Frantsve et al., 2001; Kamizono et al., 2001; Monni et al., 2001; Rottapel et al., 2002). Moreover, a recent report indicated that reduced expression of SOCS-1 is frequently found in myeloma cells (Galm et al., 2003) and leukemia cells (Liu et al., 2003). SOCS-1 gene silencing by DNA methylation is also frequently observed in acute myeloid leukemia and in human multiple myeloma (Galm et al., 2004). Interestingly, biallelic mutation in the SOCS-box of SOCS-1 gene was found in 9 out of 20 primary mediastinal B-cell lymphoma cells. These mutations probably result in the impaired JAK2 degradation and sustainsed JAK2 activation. In most cases, SOCS-1 overexpression in cell lines could inhibit tumor cell proliferation. Therefore, SOCS-1 could be an important target of antitumor therapy. 4.3. SOCS-2 SOCS-2 was first cloned as a SOCS-box containing protein by the survey of genetic database (Starr et al., 1997), and then independently as a protein that associates with IGF-I receptor (Dey et al., 1998). Structurally, SOCS-2 has the highest homology with CIS. Unlike SOCS-1, SOCS-2 expression can be induced only by a small number of cytokines such as GH and prolactin, and SOCS-2 has no suppressive activity upon IL-6 signaling. Rather, SOCS-2 may enhance the action of IL-6, since overexpression of SOCS-2 together with SOCS-1 in cell lines reverses the inhibitory action of SOCS-1 on IL-6 signaling. Other studies in vitro suggest that SOCS-2 may be involved in the

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

77

regulation of GH signaling. At low concentrations, SOCS-2 has a mild inhibitory effect on GH signaling, which is less remarkable than that of SOCS-1 or SOCS-3 (Adams et al., 1998; Favre et al., 1999). In contrast, surprisingly, a higher expression of SOCS-2 enhances the action of GH (Favre et al., 1999). Thus, the function of SOCS-2 on GH signaling is somewhat complex and needs to be re-evaluated. To elucidate the role of SOCS-2 in vivo, SOCS-2 knockout mice were generated. SOCS-2 KO mice are normally born in a Mendelian fashion, but thereafter exhibit gigantism similar to those observed in IGF-I and GH transgenic mice (Metcalf et al., 2000). The phenotype of SOCS-2 KO mice is also similar to mice with high growth (hg) phenotype, which has recently been probed to lack functional SOCS-2 protein (Horvat and Medrano, 2001). The phenotype of SOCS-2 KO mice may be due to the overaction of GH, since IGF-I expression is elevated in some of their tissues and GH-induced STAT5 activation is slightly prolonged in SOCS-2-deficient hepatocytes (Metcalf et al., 2000). Moreover, a gigantic phenotype is not seen in SOCS-2 KO mice also lacking STAT5b, a key molecule for GH signaling (Greenhalgh et al., 2002a). These results suggest that SOCS-2 in vivo is a physiological inhibitor of GH signaling. In accordance with this, SOCS-2 can associate with GHR receptor. In addition, it is likely that SOCS-2 has the capacity to promote neuronal differentiation in vivo by blocking GH-medicated down-regulation of neurogenin-1 (Ngn1) (Turnley et al., 2002). However, still confusingly, SOCS-2 transgenic mice that ubiquitously overexpress SOCS-2 also show a gigantic phenotype (Greenhalgh et al., 2002b). This phenomenon may be in line with the previous observations that overexpressed SOCS-2 can overcome the inhibitory function of other SOCS proteins such as SOCS-1 and SOCS-3. Nevertheless, further studies will be required to precisely understand this two-modal action of SOCS-2 on GH signaling. One report indicated that SOCS-2 might be associated with the development of chronic myeloid leukemia. Because the function of SOCS-2 on hematopoietic cells remains unknown, further studies are required to reveal the inhibitory mechanism of SOCS-2 on leukemia (Schultheis et al., 2002). 4.4. SOCS-3 4.4.1. Negative Regulation of Cytokine Signaling by SOCS-3 Structurally, SOCS-3 is most closely related to SOCS-1 and contains a pre-SH2 domain including kinase inhibitory region (KIR), a SH2 domain, and a SOCS box. In accordance with this notion, initial investigation of SOCS-3 suggests

78

TETSUJI N AKA ET A L .

that SOCS-3 is also a negative feedback regulator of cytokines through the inhibition of JAKs. However, subsequent analyses suggest that the consensus binding motif for SOCS-3 is pY-(S/A/V/Y/F)-hydrophobic-(V/I/L)-hydrophobic(H/V/I/Y) (De Souza et al., 2002) and SOCS-3 associates more strongly with the activated cytokine receptors including EPOR (Hortner et al., 2002a; Sasaki et al., 2000), gp130 (Nicholson et al., 2000; Schmitz et al., 2000), LIFR, leptin receptor (Bjorbak et al., 2000), GHR (Ram and Waxman, 1999), IL-2Rb (Cohney et al., 1999), and G-CSFR (Hortner et al., 2002b) than with JAKs. For instance, SOCS-3 binds to tyrosine 759 of human gp130, which is the same docking site with SHP2 and almost completely matches the SOCS-3 binding motif as well, and contributes to the attenuation of IL-6 signaling initiated from this tyrosine residue (Nicholson et al., 2000; Schmitz et al., 2000). These results suggest that the inhibitory action of SOCS-3 depends on the presence of cytokine receptors that may supply a platform for SOCS-3 to access JAKs more easily. It should be noted that SOCS-3, like SOCS-1, may also have JAK-independent action, as suggested by the fact that SOCS-3 associates with IRS proteins (Rui et al., 2002), EGFR (Xia et al., 2002), and CD28 (Matsumoto et al., 2003) and attenuates the signaling of insulin, EGF, and CD28. Several functional aspects have been reported for SOCS-3. At first, SOCS-3 may be involved in the negative feedback regulation of endocrine systems, as suggested by its ability to inhibit the signaling of GH, PRL, insulin, and leptin in vitro. In this context, there are several observations that may illustrate clinical importance for SOCS-3. For example, SOCS-3 may account for the GH and/or insulin resistance during infection and/or uremia, because remarkable induction of SOCS-3 has been observed in these conditions (Mao et al., 1999; Schaefer et al., 2001; Senn et al., 2003). In addition, SOCS-3 may account for leptin resistance in obese patients because SOCS-3 expression is up-regulated in the brains of ob/ob mice, which have disturbance of leptin signaling (Bjorbaek et al., 1998). Indeed, deletion of the SOCS-3 gene in the brain elevated leptin sensitivity and conferred resistance to diet-induced obesity (Mori et al., 2004). Recent investigation showed that SOCS-3 may confer insulin resistance also in obesity (Ueki et al., 2004). Second, SOCS-3 may be involved in the modulation of inflammation, because SOCS-3 is potently induced by IL-10 (Cassatella et al., 1999; Ito et al., 1999), a representative anti-inflammatory cytokine, and can suppress the action of proinflammatory cytokines, such as IL-6, IFN-g, and G-CSF. In addition, proinflammatory cytokines themselves can induce SOCS-3, suggesting SOCS-3 also functions in a negative feedback loop of these cytokines. Moreover, recent analysis suggests that SOCS-3 may directly inhibit LPS signaling (Berlato et al., 2002). In line with these findings, recent reports have illustrated a protective role for SOCS-3 against the development and/or progression of experimental

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

79

autoimmune diseases such as collagen-induced arthritis (a model for rheumatoid arthritis) (Shouda et al., 2001), ConA-induced hepatitis (a model for autoimmune hepatitis) (Hanada et al., 2001), and sodium dextran sulfate (DSS)-induced colitis (a model for inflammatory bowel disease) (Suzuki et al., 2001). In contrast, enhanced action of SOCS-3 may promote allergic responses, since recent analysis indicated that transgenic SOCS-3 expression in T cells inhibits Th1 development and promotes Th2 development (Seki et al., 2003). Indeed, this report also describes that increased SOCS-3 expression in T cells correlates with the severity of human allergic diseases such as asthma and atopic dermatitis. Lastly, SOCS-3 may be profoundly involved in the negative regulation of erythropoiesis, because the overexpression of SOCS3 in murine hematopoietic cells results in severe anemia and embryonic lethality (Marine et al., 1999a). This notion is supported by the finding that SOCS-3 associates with EPOR and can inhibit its signaling (Sasaki et al., 2000). 4.4.2. Physiological Roles of SOCS-3 Since SOCS-3 KO mice die in uteri around day 12 of gestation (Marine et al., 1999a; Roberts et al., 2001), the early death of SOCS-3 KO embryos made it difficult to determine the physiological functions of SOCS-3. Initially, one report concluded that this early death of SOCS-3 KO mice was due to marked erythrocytosis (Marine et al., 1999a). However, the other concluded that the death could be ascribed not to erythrocytosis but to placental insufficiency (Roberts et al., 2001). More recently, another report by the former group was published and supported the conclusion made by the latter group (Takahashi et al., 2003). This report describes a tetraploid rescue of SOCS-3 KO embryos and thereby indicates that SOCS-3 is dispensable for erythropoiesis and embryonic development. This report also describes the possibility that SOCS-3 negatively regulates the differentiation of trophoblast giant cells in placenta through the inhibition of LIFR signaling, which is strengthened by the fact that LIFR deficiency rescues SOCS-3 KO embryos from lethality. Unfortunately, SOCS-3/LIFR DKO mice also show lethality within hours after birth that is associated with LIFR deficiency. Moreover, tetraploid-rescued SOCS-3 KO mice exhibit growth retardation and die within 3 weeks after birth, possibly due to hypertrophic cardiomyopathy (Takahashi et al., 2003). The cardiac manifestation in SOCS-3 KO mice may suggest that SOCS-3 negatively regulates the signaling of IL-6 family of cytokines in vivo, since these cytokines are profoundly involved in cardiac hypertrophy and are inhibitory targets of SOCS-3 (Yasukawa et al., 2001). In fact, a recent generation of SOCS-3 conditional KO mice was of great help in characterizing the inhibitory action of SOCS-3 in signaling of IL-6 family and other cytokines.

80

TETSUJI N AKA ET A L .

By the usage of the Cre-loxP system, several kinds of tissue-specific knockout mice of SOCS-3 have been generated. At first, two groups reported myeloid cell-specific SOCS-3 KO mice (Croker et al., 2003; Yasukawa et al., 2003). As expected, these mice develop normally and show no lethality. Detailed analysis of these conditional KO mice revealed that SOCS-3 is a physiological inhibitor of IL-6 signaling. Similar findings were also observed by the generation of SOCS-3 KO bone marrow chimeras (transfer of SOCS-3 KO fetal liver cells into lethally irradiated wild-type mice) (Lang et al., 2003), excluding the possible contribution of the tissue-specific knockout approach in the reported phenotype. In SOCS-3 deficient macrophages and hepatocytes, IL-6-induced activation of STAT3 is significantly sustained, indicating that SOCS-3 functions as a negative feedback factor of IL-6 in vivo (Croker et al., 2003; Lang et al., 2003; Yasukawa et al., 2003). In contrast, STAT3 activation in response to IL-10 and IFN-g is normally downregulated in SOCS-3-deficient macrophages, indicating that SOCS-3 is not a regulator of STAT3 and is dispensable for the regulation of IL-10 and IFN-g signaling. In addition, since IL-10 normally inhibits LPS-induced proinflammatory cytokine production from SOCS-3-deficient macrophages, SOCS-3 is dispensable for the immunosuppressive action of IL-10 (Yasukawa et al., 2003). Interestingly, these reports also demonstrated that the absence of SOCS-3 in macrophages changes the original function of IL-6. In SOCS-3-deficient macrophages, IL-6 stimulation induces the enhanced activation of STAT1 and stimulates the expression of IFN-responsive genes, suggesting that IL-6 behaves like IFNs (Croker et al., 2003; Lang et al., 2003). Moreover, IL-6 stimulation of these cells elicits anti-inflammatory action equivalent to IL-10 stimulation (i.e., IL-6 inhibited LPS-induced secretion of TNF-a and other proinflammatory cytokines from SOCS-3-deficient macrophages) suggesting that in the absence of SOCS-3, IL-6 acts like IL-10 (Yasukawa et al., 2003). Thus, these results suggest that SOCS-3 is required not only for the negative regulation of IL-6 signaling but also for the proper function of IL-6. This finding may explain why IL-6 and IL-10, both of which mainly activate STAT3, function in a different manner. Subsequently, two groups generated hematopoietic cell-specific SOCS-3 KO mice and revealed a role for SOCS-3 in negative regulation of G-CSF signaling in vivo (Croker et al., 2004; Kimura et al., 2004). Stimulation of SOCS-3 KO cells with G-CSF resulted in enhanced and sustained activation of STAT3, indicating SOCS-3 regulates G-CSF signaling in a negative-feedback manner. The absence of SOCS-3 in bone marrow cells significantly perturbs the hematopoiesis, since hematopoietic cell-specific SOCS-3 KO mice with aging exhibit increased myelopoiesis in bone marrow and marked peripheral

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

81

neutrophilia, and develop inflammatory infiltrates of multiple hematopoietic lineage cells in liver, lung, and pleural/peritoneal cavities (Croker et al., 2004). Recently, essential roles of SOCS-3 in endocrine systems have also been clarified. Administration of leptin to neural cell-specific SOCS-3 KO mice greatly reduces their food intake and causes enhanced body weight loss compared to wild-type mice, indicating that SOCS-3 in the brain negatively regulates leptin signaling (Mori et al., 2004). Similar findings were observed in SOCS-3 heterozygous mice (Howard et al., 2004), confirming that the physiological action of SOCS-3 in leptin signaling is not specific to events in conditional KO mice. These results provide the rationale that SOCS-3 potentiates leptin resistance and ameliorates obesity. In addition, SOCS-3-deficient adipocytes generated from SOCS-3 KO fibroblasts are significantly protected from TNF-a-induced insulin resistance, mainly due to reduced proteasomal degradation of IRS proteins by TNF-a, suggesting that SOCS-3 is an important mediator of insulin resistance in vivo (Shi et al., 2004). Collectively, these results indicate that SOCS-3 can be a potential therapeutic target for prevalent human metabolic disorders such as obesity and diabetes. Like other SOCS proteins, SOCS-3 may also be involved in the development and the progression of malignancies. In chronic myelogenous leukemia cells, especially in cells on blast crisis, SOCS-3 is constitutively expressed and may confer resistance to IFN therapy (Sakai et al., 2002). In contrast, silenced expression of SOCS-3 due to hypermethylation has been observed in human lung cancers and may be associated with the progression of cancer cells (He et al., 2003). 4.5. SOCS-5 Previous analysis has indicated that SOCS-5 can be induced in mouse livers by the injection of IL-6 (Hilton et al., 1998). However, enforced expression of SOCS5 in cell lines resulted in only marginal inhibition on IL-6 signaling (Nicholson et al., 1999). Recent analysis provided evidence that SOCS-5 may promote Th1 polarization. This report showed that SOCS-5 is preferentially expressed in Th1 cells, and SOCS-5 can interact with IL-4R in the absence of tyrosinephosphorylation of IL-4R (Seki et al., 2002b). This interaction of SOCS-5 with IL-4R is likely to cause the reduction in IL-4-induced activation of STAT6 and thus regulate Th2 polarization. In line with this finding, Tcells from SOCS-5 transgenic mice also exhibit reduced Th2 polarization (Seki et al., 2002b). However, recent analysis of SOCS-5 KO mice failed to confirm the roles of SOCS-5 in lymphocyte function (Brender et al., 2004). CD4þ Tcells in SOCS-5 KO mice showed normal Th1/Th2 response in vitro as well as in vivo (Brender et al., 2004). The conflicting findings in SOCS-5 KO mice may be explained by the compensation of SOCS-5 by

82

TETSUJI N AKA ET A L .

other SOCS proteins such as SOCS-4, since SOCS-4 shares significant homology with SOCS-5. Further analyses including those of SOCS-4/SOCS-5 DKO mice may be required to address the function of SOCS-5 in vivo. In Drosophila, SOCS protein highly homologous to mammalian SOCS-5 was cloned and named SOCS36E. Interestingly, ectopic expression of SOCS36E in transgenic flies results in phenotypes resembling those of flies defective in JAK/STAT or EGF signaling (Callus and Mathey-Prevot, 2002). This result may imply that SOCS-5 is also involved in the regulation of JAK/STAT or EGF signaling in mammals, but future studies are required to address this issue. 4.6. SOCS-6 The function of SOCS-6 has been largely unknown. Recent analysis indicated that SOCS-6 can interact with insulin receptor (IR) and can inhibit insulin signaling (Mooney et al., 2001). Another analysis suggested that SOCS-6 modulates insulin signaling by binding to IRS-4 (Krebs et al., 2002). Interestingly, targeted disruption of the SOCS-6 gene in mice resulted in mild growth retardation (Krebs et al., 2002). However, the precise mechanism of this KO phenotype remains to be studied. SOCS-6 transgenic mice overexpressing SOCS-6 under the control of elongation factor 1 promoter were also generated (Li et al., 2004). This report describes that SOCS-6 is induced by insulin stimulation and associates with p85 subunit of PI3 kinase. Interestingly, SOCS-6 transgenic mice show a phenotype strikingly similar to p85 KO mice. SOCS-6 transgenic mice show enhanced activation of Akt after insulin stimulation and significant improvement in glucose metabolism. Collectively, these results are in line with the notion that SOCS-6 regulates the Insulin/PI3K/ Akt pathway, but elucidating its mechanism of action and the physiological significance may require further investigation. 4.7. SOCS-7 SOCS-7 was initially reported as Nck, Ash, and phospholipase Cg binding protein (NAP4) but its biological roles were not elucidated (Matuoka et al., 1997). Since SOCS-7 is most homologous to SOCS-6, these two proteins may be involved in the regulation of similar signaling pathways, including those of insulin. Recently, the phenotype of SOCS-7 KO mice was reported. Although SOCS-7 KO mice are born normally and show normal glucose homeostasis, approximately half of these mice develop hydrocephalus and die within 15 weeks of age (Krebs et al., 2004). This appears to be in line with the finding that SOCS-7 is preferentially expressed in the brain, but its mechanism of action is still a mystery.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

83

5. TLR-Mediated Pathways Innate immunity is equipped with various signaling receptors including a Toll-like receptor (TLR) family (Janeway and Medzhitov, 2002). TLR family consists of more than 10 members. Each member precisely recognizes corresponding molecular patterns associated with pathogens and exerts its host defensive actions based on their ignorance of host-derived intact components (Takeda et al., 2003). Indeed, the TLR-mediated signal pathways are essential for microbe expulsion at the early infectious phase (Edelson and Unanue, 2002; Reiling et al., 2002; Scanga et al., 2002; Seki et al., 2002a). Unexpectedly and importantly, the signalings through TLRs are definitely required for the following activation of the acquired immunity (Kaisho and Akira, 2002). Lack of the TLR-mediated pathway sometimes causes immature T-cell responses and failure in the development of memory T cells, presumably due to the absence of cytokine production and DC maturation and activation, which is normally induced by the activation of the TLR pathway and is required for those immunological events (Akira et al., 2001; Kaisho et al., 2001). TLR and cytokines have strong connections. For example, IFNb is rapidly induced by TLR stimulation, through TRIF/IRF3, resulting in the activation of STAT1. Inflammatory cytokines including TNFa, IL-1, IL-6, and IL-12 were rapidly induced after TLR stimulation. IL-12 secreted from DCs plays an especially important role in TH1 responses. IL-4 and IL-10 are antiinflammatory cytokines and somehow downregulate macrophage and DC activation, presumably suppressing TLR signals. Some of the unidentified immunodeficiency to microbial infection is recently shown to be attributable to a defect in the TLR-mediated pathways. Very recently, it was shown that the TLR-mediated signalings are involved in the tissue homeostasis in mice (Rakoff-Nahoum et al., 2004). Thus, the signal transduction activation through TLRs is indispensable for normal healthy life in mammals. We have demonstrated that SOCS is a negative regulator of TLRmediated signalings and cytokine signalings (Kinjo et al., 2002, Nakegawa et al., 2002). Here, we first overview the expanding knowledge on the TLRmediated signal pathways and then on the regulatory mechanisms of these pathways. 5.1. Ligands for TLRs Individual TLRs principally recognize distinct pathogen-associated molecular patterns (PAMPs), which are not expressed on normal mammalian cells. Here we show some examples (Fig. 4).

84

TETSUJI N AKA ET A L .

Figure 4 Distinct signaling pathways among TLR family members. The TLR family, characterized by the presence of common Toll/IL‐1 receptor (TIR) domain in their cytoplasmic portions, consists of more than 10 members. Each member utilizes a different signal transduction pathway mediated by the different signal adaptor molecules containing TIR via homophilic TIR/TIR interaction. TLR5, TLR7, and TLR9 use the MyD88‐mediated NF‐kB activation pathway after stimulation with flagellin, ssRAN, and hypomethylated CpG motifs, respectively. TLR2, together with TLR6 or TLR1, recognizes diacyl and triacyl lipopeptides, respectively, and transduces signals in a MyD88 and TIRAP‐dependent manner, leading to the nuclear translocation of NF‐kB. TLR4/ CD14/MD‐2 complex recognizes LPS and relays a signal via MyD88 and TIRAP to activate NF‐k B. Moreover, TLR4 signaling is transduced via TRIF and TRAM to activate both IRF3 and NF‐kB as well. The TLR3‐mediated dsRNA signalings use only TRIF as a signal adaptor and induce activation of IRF3 and NF‐kB. The TLR/MyD88‐mediated pathways lead to the activation and maturation of DCs as well as the activation of various proinflammatory cytokine/chemokine expressions. The activation of TLR/TRIF‐dependent pathways induces the same events supplemented with the expression of IFN‐b and various IFN‐related genes, such as IP‐10. The TLR signalings activate both innate immune responses, which connect to induction of the appropriate

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

85

5.1.1. Ligands for TLR4 LPS, a major constituent of the cell wall of Gram-negative bacteria (Raetz and Whitfield, 2002), is recognized by the functional receptor complex composed of TLR4, MD-2, which is an indispensable protein for recognition of LPS and normal trafficking of TLR4 onto the cell surface, and CD14 (Gruber et al., 2004; Hirschfeld et al., 2001; Hornef et al., 2002; Hoshino et al., 1999b; Nagai et al., 2002b; Ogawa et al., 2002; Poltorak et al., 1998; Qureshi et al., 1999). As the lipid A moiety possesses the most of the biological activities of LPS, it is plausible that saturated fatty acids composing lipid A might show LPS-activity. Interestingly, saturated free fatty acids but not nonsaturated fatty acids induce TLR4-dependent biological effects in macrophages. This may imply involvement of some types of dietary fat in development of chronic and acute inflammatory diseases via activation of TLR4-mediated signal pathways (Lee et al., 2001). Pneumolysin, although a toxin derived from Gram-positive Streptococcus pneumoniae, an important relevant bacterium to meningitis, can induce production of proinflammatory cytokine/nitric oxide (NO) in polymorphonuclear cells and apoptosis in central nervous system via TLR4 complexmediated signal pathways (Malley et al., 2003). Taxol, an antitumor agent derived from plant, possesses many LPS-like activities and requires the TLR4/MD-2 complex for exerting its biological actions, although the structure of Taxol is quite distinct from that of LPS (Kawasaki et al., 2000; Perera et al., 2001). Recent studies revealed the intrinsic host-derived ligands for TLR4. Necrotic cells can stimulate innate immunity via activation of the TLR4 signalings (Barsness et al., 2004; Taylor et al., 2004). Particularly, intacellular components of necrotic cells and stress signals produced in them are potent activators. Hyaluronan fragments, a major glycosaminoglycan of the extracellular matrix during inflammation, can activate TLR-4-mediated signalings to induce wound repair responses via activation of endothelial cells (Taylor et al., 2004) and to induce DC maturation, perhaps modulating inflammatory responses (Termeer et al., 2002). Heat shock proteins, which are conserved proteins acting as molecular chaperones intracellularly packaged and induced after various

Th1 immune responses via action of IL‐12. Abbreviations; dsRNA, double‐stranded RNA; IFN, interferon; IL, interleukin; IRF‐3, IFN regulatory factor 3; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; PGN, peptidoglycan; NF‐kB, nuclear factor kB; ssRNA, single‐stranded RNA; TIR, Toll‐like receptor IL‐1 receptor; TIRAP, TIR domain‐containing adaptor protein, also known as Mal, MyD88‐like adaptor protein (Mal); Th1, T helper type 1; TLR, Toll‐like receptor; TRAM, TRIF‐related adaptor molecule; TRIF, TLR domain‐containing adaptor inducing IFN‐b.

86

TETSUJI N AKA ET A L .

types of stress, including cellular necrosis, are demonstrated to also activate TLR4 to provoke immune responses as adjuvant likewise microbial products (Ohashi et al., 2000; Vabulas et al., 2001, 2002). This may implicate that accidental release of heat shock proteins upon necrotic stress promotes the tissue repair by prompt amputation of the necrotic part via induction of TLR-mediated inflammatory responses. Moreover, small antimicrobial peptides released upon stimulation with pathogens themselves can activate the TLR4-mediated pathways to synergistically complete host defense. bDefensins, released from mucosal tissues and skin, have been demonstrated to induce maturation of and to activate DCs, resulting in the successful local expulsion of the pathogens (Biragyn et al., 2002; Yang et al., 1999). The identification of self-derived natural ligands for TLR4 might raise concerns whether innate immunity has a pitfall, as does adaptive immunity, like autoimmune diseases. 5.1.2. Ligands for TLR3, TLR7, and TLR9 Nucleic acids are also recognized by TLRs. Double-stranded (ds) RNA primarily derived from viruses or synthetic ds-poly I:C can stimulate immune cells through TLR3 (Alexopoulou et al., 2001), while hypomethylated CpG oligoDNA in bacteria or synthetic unmethylated CpG-DNA can activate them through TLR9 (Hemmi et al., 2000; Krieg, 2002). Murine TLR7 was originally identified as a signaling receptor for small antiviral compounds such as imiquimod and R-848 (Hemmi et al., 2002). Recently, natural ligands were determined. Viral single-stranded (ss) RNA was identified as natural ligands for TLR7 in mouse and TLR8 in human (Heil et al., 2004). Indeed, HIV (human immunodeficiency virus)-derived ssRNA activates TLR8-expressing DCs to secrete proinflammatory cytokines and to express costimulatory molecules (Heil et al., 2004). 5.1.3. Ligands for TLR5 TLR5 recognizes flagellin (Gewirtz et al., 2001; Gomez-Gomez and Boller, 2002), a major protein component of bacterial flagella (Hayashi et al., 2001). 5.1.4. Ligands for TLR2 In contrast to the previous TLR members, TLR2 recognize various types of PAMPs by forming multiple kinds of the heterodimer associated with other

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

87

TLR members. TLR2, together with TLR1, identifies bacterial lipoprotein (BLP), such as triacyl lipopeptide, synthetic lipopeptide N-palmitoyl-Sdipalmitoylglyceril (Pam3) Cys-Cer-(Lys)4 (CSK4), and 19 kDa lipoprotein purified from Mycobacterium tuberculosis (Aliprantis et al., 1999; Ozinsky et al., 2000; Takeuchi et al., 2002), while TLR2 in association with TLR6 recognizes diacyl lipoprotein such as macrophage-activating lipoprotein 2 kDa (MALP-2) derived from Mycoplasma fermentans (Ozinsky et al., 2000; Takeuchi et al., 2000, 2001). TLR2, presumably forming heterodimer with TLR6 or other TLR members, might also be required for recognition of peptidoglycan (PGN) derived from Gram-positive bacteria including Staphylococcus aureus but not from mammalian cells (Lien et al., 1999; Ozinsky et al., 2000; Schwandner et al., 1999). TLR2 is also activated by protozoan parasites. Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipid (GIPLs) from Trypanosoma cruzi, the causative protozoa for lethal Chagas’ disease, activate TLR-2mediated IL-12, TNF-a, and nitric oxide production, resulting in host innate defense and inflammatory responses (Campos et al., 2001). 5.1.5. Miscellaneous TLR11 is reported to recognize uropathogenic bacteria, such as a uropathogenic strain of Escherichia coli (Zhang et al., 2004). To date we have not yet identified ligands of TLR10. 5.2. TLR Expression on Various Cell Types TLRs are expressed on various immune competent cells. Recent studies revealed that the individual types of cells express various combinations of TLRs (Applequist et al., 2001). DCs as well as macrophages strongly express TLR1 to TLR9 (Applequist et al., 2001; Hornung et al., 2002; Muzio et al., 2000). Intriguingly, functionally different types of human DCs express distinct combinations of TLRs on their surface. Plasmocytoid cell-derived DC2 cells, which selectively induce Th2 cell differentiation, preferentially express TLR7 and TLR9 but not TLR2 or TLR4, while myeloid DC1 cells capable of inducing Th1 cell defferentiation express the inverse pattern of the TLRs (Boonstra et al., 2003; Kadowaki et al., 2001; Rissoan et al., 1999). Moreover, stimulation through TLR7, which is highly expressed on plasmocytoid DCs but poorly on myeloid DCs, provides these two types of DCs with quite distinct results. DC1 cells produce IL-12 but not IFN-a, while DC2 cells show IFN-a but not IL-12 production (Ito et al., 2002). However, both of these two types of DCs have equivalent activity increase in their expression of costimulatory molecules (Ito et al., 2002). Interestingly, the TLR7 ligand-stimulated DC1

88

TETSUJI N AKA ET A L .

and DC2 cells similarly cause differentiation toward Th1 cells, but not Th2 cells, after stimulation of naı¨ve Th cells with allogeneic cells (Ito et al., 2002). B cells express TLR2, TLR4, TLR 6, TLR7, and TLR9 (Applequist et al., 2001; Hornung et al., 2002). It is well established that B cells proliferate and produce IgM in response to LPS. B cells require additional expression of RP105, which is a member of TLR family, and MD-1 to be able to respond to LPS (described later). B cells also can respond to lipopeptide via their TLR2 (Leadbetter et al., 2002). TLR9 is reported to contribute to produce autoantibody by B cells after being stimulated with chromatin-IgG complex (Leadbetter et al., 2002), suggesting the importance of TLR-mediated signaling pathways in autoimmune diseases. TLRs are also expressed on hematopoietic cells in blood. Human peripheral neutrophils and basophils, but not eosinophils, constitutively express both TLR2 and TLR4 and produce cytokines/chemokines in response to PGN and LPS, respectively (Kurt-Jones et al., 2002; Sabroe et al., 2002). Mast cells also express TLRs and contribute to microbial expulsion (Masuda et al., 2002; Supajatura et al., 2001, 2002). Mouse bone marrow-derived mast cells produce a different set of cytokines upon stimulation with TLR2 and TLR4 ligands (Supajatura et al., 2002). They produce IL-4, IL-5, IL-6, and IL13 via activation of their TLR2, while producing TNF-a, IL-1b, IL-6, and IL-13 in response to LPS (Supajatura et al., 2002). Skin-derived mast cells, while not bone marrow-derived cells, have the capacity to respond to nucleotide-derived TLR3, TLR7, and TLR9 ligands (Matsushima et al., 2004). Human mast cells, however, express TLR1, TLR2, and TLR6, but not TLR4 (McCurdy et al., 2003). The distinct and limited TLR expressions on the different cell types may reflect their specialized functions in innate and adaptive immune responses. TLRs are also expressed by various epithelial cells, such as respiratory epithelial cells and keratinocytes, and produce chemokines/cytokines that might induce recruitment/activation of inflammatory cells to successfully complete the expulsion of pathogens (Monick et al., 2003; Smith et al., 2003). 6. Signal Transduction Pathways Through TLRs All members of a TLR family consist of cytoplasmic, transmembrane, and extracellular portions. Their extracellular portion expresses different leucine rich repeats (LRRs) for recognition of their corresponding ligands (Medzhitov et al., 1998; O’Neill, 2000; O’Neill and Dinarello, 2000; O’Neill and Greene, 1998). In contrast, the cytoplasmic portion of all the TLRs is characterized by a common motif, termed Toll IL-1 receptor (TIR) domain. After stimulation with corresponding ligands, their cytoplasmic portion recruits signal adaptor

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

89

molecules via the TIR/TIR interaction, to relay signals to activate nuclear factor-kB (NF-kB) and/or interferon regulatory factor 3 (IRF3). Five intracellular proteins expressing TIR have been identified (O’Neill et al., 2003). Four of the five are demonstrated to be signal adaptor molecules required for the TLR-mediated pathways. The four TIR adaptors are named as myeloid differentiation factor 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), or MyD88 adaptor-like (Mal) (Fitzgerald et al., 2001; Horng et al., 2001, 2002; Yamamoto et al., 2002a), TLR domain-containing adaptor inducing IFN-b (TRIF) (Hoebe et al., 2003; Oshiumi et al., 2003; Yamamoto et al., 2003a), and TRIF-related adaptor molecule (TRAM) (Fitzgerald et al., 2003b; Yamamoto et al., 2003b). There are two major signal pathways—MyD88- and TRIF-mediated pathways. Individual TLRs utilize one of these two or both. TLR3 utilizes only the TRIF-dependent pathway. TLR4 complex uses both MyD88- and TRIF-mediated pathways associated with additional adaptor molecules, TIRAP and TRAM, respectively. TLR2 heterodimerized with TLR1 or TLR6 uses the MyD88-mediated pathways with help from TIRAP. TLR5, 7, and 9 use only the MyD88-dependent pathways. These pathways can be established from the experimental observations using individual TIR-containing adaptor-deficient mice and mice deficient in various pairs of the four adaptors (Adachi et al., 1998; Yamamoto et al., 2002a, 2003a,b). After stimulation with TLR ligands, the MyD88-mediated pathway leads to activation of NF-kB and mitogen-activated protein kinase (MAPK) pathways, while the TRIF-mediated pathways result in the activation of NF-kB and IRF3. 6.1. MyD88-Mediated Pathway All the TLRs except for TLR3 employ the MyD88-mediated pathways. TLR2 and TLR4 use both MyD88 and TIRAP to transduce the signal pathway, while TLR5, 7, and 9 use MyD88 alone (Fig. 4). Upon stimulation with appropriate ligands, the TIR domain of the cytoplasmic portion of the TLRs provides a platform to recruit MyD88, in some cases together with TIRAP. As MyD88 is composed of death domain (DD) as well as TIR domain (Wesche et al., 1997), IL-1R-associated kinase 4 (IRAK4), which is a DD domaincontaining kinase, is recruited onto the platform via the DD/DD interaction, followed by the phosphorylation of IRAK1 (Cao et al., 1996; Li et al., 2002). Then, phosphorylated IRAK is dissociated from the receptor platform to associate with another signaling molecule, TNF-R-associated factor (TRAF) 6, to form a new signal adaptor molecule, eventually leading to the phosphorylation of TGF-b-activated kinase 1 (TAK1) (Wang et al., 2001). TAK1 then phosphorylates complex of IkB kinase (IKK) consisting of IKKa, IKKb, and IKKg, which induces ubiquitination-induced degradation of IkB, leading to

90

TETSUJI N AKA ET A L .

the nuclear translocation of NF-kB (Deng et al., 2000; Hoffmann et al., 2002; Karin and Ben-Neriah, 2000) (Fig. 1). TAK1 is also believed to be involved in the activation of mitogen-activated protein kinase (MAPK) pathways. Subsequently, corresponding gene expressions are accomplished (Fig. 1). The mutant mice deficient in the individual signaling molecules demonstrate the requirement of each molecule for the TLR signalings. For example, MyD88-, TIRAP-, IRAK1-, IRAK-4-, and TRAF6-deficient mice are impaired in signalings of the TLR/MyD88-mediated pathways (Adachi et al., 1998; Fitzgerald et al., 2001; Horng et al., 2002; Kanakaraj et al., 1998, 1999; Kawai et al., 1999; Suzuki et al., 2002a,b; Thomas et al., 1999; Yamamoto et al., 2002a). Very recently, it was demonstrated that some of the MyD88mediated responses are regulated in a gene expression process of at least two steps that requires IkB
, an IkB family member (Yamamoto et al., 2004). IkB
was reported as an inducible protein after stimulation with IL-1 and LPS. Indeed, IkB
is induced in response to IL-1 and ligands for TLR2, 4, 5, 7, and 9 in a MyD88-dependent manner. Intriguingly, IkB
is not induced after stimulation with TNF-a, while other IkB family members including IkBa are inducible after stimulation with TNF-a as well as IL-1 and the TLR ligands. IkB
/ cells show normal NF-kB activation but have defects in production of IL-6, IL-12, and G-CSF upon stimulation with these TLR ligands or IL-1, indicating requirement of inducible kB
for production of these cytokines. However, the activated mutant cells express normally TNF-a and normal TNF-a release, indicating that IkB
functions independently of the TNF-a pathways. Thus, the MyD88-mediated gene expressions are regulated by the two transcriptional factors, NF-kB and IkB
, which is induced after the activation of NF-kB. IkB
requires association with p50 in order to exert its transcriptional actions (Yamamoto et al., 2004). Indeed, cells lacking p50 show the impaired response similar to IkB
/ cells after stimulation with these ligands. 6.2. TRIF-Mediated Pathway In contrast to the complete lack of responsiveness to TLR5, TLR7, and TLR9 ligands in Myd88 / cells (Adachi et al., 1998), the mutant cells can respond to certain types of TLR ligands, TLR3 and TLR4 ligands (Kawai et al., 1999, 2001; Sato et al., 2002). Upon stimulation with LPS, WT cells show the activation of NF-kB and normal activation of IRF3, resulting in production of IFN-b, DC maturation, and expression of IFN-inducible genes such as IP10 (Kawai et al., 2001). On the same stimulation, Myd88 / cells do not produce the proinflammatory cytokines but do show the late activation of NF-kB. In addition, Myd88 / DCs still increase CD80 and CD86 expression, and

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

91

Myd88 / cells normally express IFN-inducible genes via the normal activation of IRF3 (Kawai et al., 2001). Upon stimulation with dsRNA, Myd88 / cells produce normal IFN-b, and Myd88 / DCs show normal maturation through the intact activation of both NF-kB and IRF3 when compared with WT cells (Alexopoulou et al., 2001; Hoshino et al., 2002; Kaisho et al., 2002; Sato et al., 2002). Tirap / cells show the responses similar to Myd88 / cells after stimulation with LPS or dsRNA (Yamamoto et al., 2002a). These observations indicate the signal pathways mediated by other adaptor molecule(s) might be involved in the TLR3- and TLR4-mediated signalings. Two groups identified TRIF, also named as TICAP-1 (Oshiumi et al., 2003; Yamamoto et al., 2002b). TRIF is predominantly involved in the activation of IRF-3 (IFN regulatory factor 3) and NF-kB upon stimulation with TLR3 and TLR4 ligands (Fig. 4). As Trif / cells completely lack the responses, the NF-kB activation and the dimerization of phosphorylated IRF3, after stimulation with TLR3 ligand, the TLR3-mediated pathways completely depend on TRIF (Oshiumi et al., 2003; Yamamoto et al., 2003a). Moreover, Trif / Myd88 / cells cannot respond to LPS. TRIF is necessary and sufficient for the MyD88-independent pathways evoked by activation of TLR4. Intriguingly, TRAM is demonstrated as a signal adaptor molecule selectively required for the TLR4/TRIF-mediated pathways (Yamamoto et al., 2003b). Tram / cells can normally respond to the stimulation with dsRNA but are selectively impaired in the TRIF-mediated responses after LPS challenge (Fitzgerald et al., 2003b; Yamamoto et al., 2003b), indicating that TRAM serves as a second adaptor molecule for the TLR4/TRIF-mediated pathways as does TIRAP for the TLR4/MyD88-mediated pathways. Recently, signaling molecules involved in the TRIF/IRF3-mediated signal pathways were identified. In unstimulated cells, IRF3 is located in the cytoplasm. Upon stimulation, IRF3 becomes activated by serine/threonine phosphorylation to form its dimerization, leading to the nuclear translocation. Two IKK-related kinases, IKK" and Tank-binding kinase 1 (TBK-1), were thought to participate in the activation of NF-kB, because they have homology with IKKa and IKKb, which are involved in the NF-kB activation through the TLR/MyD88-mediated pathways. IKK", also known as inducible IKK (IKK-i), is induced upon stimulation with LPS. TBK-1 was believed to be involved in the activation of NF-kB due to the reduced expression of certain genes regulated by NF-kB in Tbk-1 / cells. Recently, it was clearly demonstrated by analyzing Tbk-1 / , Ikk" / , and Tbk-1 / Ikk" / cells that both kinases are important for the activation of the IRF3 pathway evoked by TLR3 ligands and LPS (Fitzgerald et al., 2003a; Hemmi et al., 2004; McWhirter et al., 2004; Perry et al., 2004; Sharma et al., 2003). Ikk" / cells have moderate impairment in the dimerization of nuclear IRF3 and resulting IRF3-induced

92

TETSUJI N AKA ET A L .

gene expressions exemplified by IFN-b and IP-10 upon stimulation with dsRNA and LPS, while Tbk-1 / cells exhibit severe defects in both of the events (Hemmi et al., 2004). Ikk" / Tbk-1 / cells completely lack in these responses, clearly demonstrating that both kinases are essential and enough for the activation of IRF3 after stimulation with TLR3/TLR4 ligands. By contrast, both Ikk" / cells and Tbk-1 / cells show intact activation of NF-kB and MAP kinases, such as JNK (c-Jun terminal kinase) and ERK, on stimulation with LPS and dsRNA, indicating dispensable roles for these kinases in the TLR4-mediated NF-kB and MAP kinase activation (Hemmi et al., 2004). We now know the importance of IKK" and TBK-1 selectively for the activation of IRF3. However, it is still to be elucidated how TRIF is associated with these kinases after appropriate activation through TLR3 and TLR4. Two distinct pathways have been proposed for the TLRs/TRIF-dependent activation of NF-kB. One is the TRAF6-mediated pathway (Jiang et al., 2004), and the other is the pathway mediated by receptor interacting protein (RIP) 1 (Meylan et al., 2004), an essential adaptor molecule for the TNF-ainduced NF-kB activation signalings (Hsu et al., 1996). It was reported that, on stimulation with TLR3 ligands, the TRIF recruited onto the intracellular portion of TLR3 associates with TRAF6 through a TRAF-binding sequence in TRIF, which might activate TAK1, leading to the activation of NF-kB. TRIF recruited onto the TLR3 also activates RIP1 via RHIM (RIP homotypic interaction motif)/RHIM interaction (Meylan et al., 2004). In fact, enforced expression of mutant TRIF lacking RHIM or mutant RIP1 lacking RHIM cannot associate with each other or induce the NF-kB activation, although it can normally activate IRF3. Furthermore, Rip1 / cells exhibit no phosphorylation of IkB or induction of NF-kB-dependent Icam1 expression after stimulation with dsRNA. In contrast, dsRNA-incubated Rip1 / cells show normal IRF3 activation and normal JNK activation, indicating that RIP is not involved in the activation of IRF3 or JNK after stimulation with TLR3 ligands (Meylan et al., 2004). Thus, RIP1 is important for the TRIF-dependent NF-kB activation after stimulation of TLR3 ligands. 7. Major Biological Events by the TLR-Mediated Cell Activation Activation of distinct TLRs causes several common biological consequences, such as production of cytokines/chemokines, expression of costimulatory molecues, cell maturation, and sometimes cell growth (Takeda et al., 2003). It is well known that various kinds of TLR ligands activate DCs and macrophages to produce various kinds of proinflammatory cytokine and chemokine (Akira et al., 2001; Lien and Ingalls, 2002; O’Neill, 2000, 2002). In particular, TLR-mediated cytokines link innate immune response to adaptive

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

93

immune response in host. For example, following infection of normal mice with intracellular facultative pathogen, such as Listeria monocytogenes, DCs, as well as macrophages, promptly produce various types of proinflammatory cytokines, such as IL-12 and TNF-a, to eradicate these pathogens via the activation of TLR-mediated signal pathways. As DCs are professional antigen-presenting cells, this DC-derived IL-12 determines successful development of pathogen-specific Th1 cells, an essential tool for clearance of the microbes in the adaptive immune phase (Scanga et al., 2002; Seki et al., 2002a). LPS, TLR4 ligand, has the unique capacity to induce B cell activation. B cells express high levels of RP105, another member of TLRs, together with MD-1, an MD-2 homologue, in addition to low levels of TLR4/MD-2 complex (Miura et al., 1996, 1998; Miyake et al., 1994, 1995, 1998; Ogata et al., 2000). In contrast to TLR4/MD-2, RP105/MD-1 is selectively expressed on B cells and contributes to LPS-induced B cell activation including up-regulation of MHC class II and costimulatory molecules such as CD80 and CD86 expression. Although TLR4/MD-2 is shown to be essential for LPS-induced B cell responses by TLR4-deficient mice, MD-1-deficient mice also do not respond to LPS (Nagai et al., 2002c). It is still to be elucidated how these two receptor complexes are involved in LPS-induced B cell activation in detail. TLR ligands induce maturation and activation of DCs (Akira et al., 2001; O’Neill, 2002). After being stimulated with LPS—a ligand for TLR4 (Hoshino et al., 2002; Kaisho et al., 2001)—the cell wall skeleton (CWS) of Mycobacterium bovis (which requires both TLR2 and TLR4 for its signaling) (Tsuji et al., 2000), CpG-DNA (Hoshino et al., 2002), and TLR2 ligands (including PGN, lipoteichoic acid, and lipopeptides—immature DCs [Hertz et al., 2001; Michelson et al., 2001]), undergo maturation to express CD40, CD80, and CD86. TLR-mediated DC maturation, together with proinflammatory cytokine production, particularly IL-12, is required for initiation of appropriate adaptive immune responses. TLR signalings are reportedly involved in bone metabolism (Takami et al., 2002). Thus, TLR-mediated signalings are primarily involved in various biological events. 8. Pathophysiological Roles for TLR-Mediated Signal Pathways 8.1. Host Defenses TLR-mediated signal transmission pathways are involved in host defense against various types of microbes. Against Gram-positive bacteria, TLR2 plays a role as the predominant signaling receptor in the initiation of host defense. TLR-2-deficient mice, but not TLR4-deficient mice, are susceptible

94

TETSUJI N AKA ET A L .

to low-dose infection with Staphylococcus aureus (Takeuchi et al., 2000). L. monocytogenes, a Gram-positive and facultative intracellular bacterium, activates the host innate immune system primarily through TLR2, but not TLR4. Indeed, TLR-2-deficient macrophages produce a smaller amount of TNF-a in response to liver L. monocytogenes, while TLR4-deficient cells produce a comparable amount of it as in WT cells (Seki et al., 2002a). However, the TLR-2-mediated signal pathway is dispensable for eradication of L. monocytogenes, although MyD88-deficient mice are susceptible to L. monocytogenes, indicating importance of TLR/MyD88-mediated signal pathways for its eradication (Edelson and Unanue, 2002; Seki et al., 2002a). TLR4 is also important for eradication of Gram-positive bacterium, S. pneumoniae. In fact, mice lacking functional TLR4 are more susceptible to pneumococcal nasopharyngeal colonization (Malley et al., 2003). For expelling Mycobacterium tuberculosis, both TLR2 and TLR4 are required, because upon chronic infection with lethal dose of M. tuberculosis, TLR-2-deficient or TLR4-defective mice show significantly lower survival compared with WT mice (Abel et al., 2002; Reiling et al., 2002). Clearance of fungi also requires the TLR-mediated signals. Both TLR2 and TLR4 are involved in host defense against Candida albicans. Particularly, TLR4-mediated signal transmission participates in recruitment of fungicidal neutrophils and macrophages via induction of chemokines (Netea et al., 2002). The TLR/MyD88-mediated pathway is also essential for resistance to the pathological parasite, T. gondii (Scanga et al., 2002). However, for eradication of Plasmodium berghei, lethal strain of mouse malaria, the TLR/MyD88-meidated signal pathways play a minor role. TLR2-, TLR4-, TLR2, and TLR 4 doubly, or even MyD88-deficient mice show comparable susceptibility to P. berghei, as in WT mice (Adachi et al., 2001). Collectively, the host eradicates certain microbes by activation of multiple TLR-mediated signaling pathways upon stimulation with the many PAMPs the microbe expresses. Recently, the loss-of-function mutation of IRAK4, an essential molecule of the TLR/MyD88-mediated pathways, was identified in some patients with recurrent microbial infection of unknown etiology (Day et al., 2004; Picard et al., 2003). 8.2. Homeostasis Recently, the TLR/MyD88-mediated pathways were clearly demonstrated to be required for intestinal homeostaisis (Rakoff-Nahoum et al., 2004). Tlr2 / , Tlr4 / , or Myd88 / mice are highly susceptible to an experimental mouse colitis induced by oral administration of dextran sulfate sodium (DSS), a reagent directly toxic to intestinal epithelium. Almost all the Myd88 / mice and half numbers of the Tlr2 / , Tlr4 / mice die, while all WT mice survive

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

95

after challenge with DSS. Myd88 / mice show extremely severe clinical scores but with normal leukocyte infiltration, suggesting impaired protective responses of Myd88 / intestinal epithelial cells against exogenous stress. Indeed, Myd88 / mice exhibit profound impairment in DNA synthesis of their intestinal epithelial cells, which would be intensively initiated in WT cells, upon radiation-induced intestinal injury as well (Rakoff-Nahoum et al., 2004). Moreover, Myd88 / mice lack induction of various growth factors essential for recovery of parenchymal cells from tissue injuries, such as IL-6 and TNF-a (Fausto, 2000; Michalopoulos and DeFrances, 1997). As colonic microfloradepleted WT mice show the fatal colitis similar to Myd88 / mice upon DSS challenge and exogenous TLR ligands can restore their survivals, commensal intestinal microbes seem to activate the TLR/MyD88-mediated pathways. MyD88-mediated pathways are also required for liver homeostasis. Myd88 / mice entirely lack the production of TNF-a/IL-6 essential for liver replication as well and show impairment in liver regeneration upon partial hepatectomy (Seki et al., 2005). Thus, the TLR/MyD88-mediated pathways are profoundly involved in the maintenance of tissue homeostasis. 8.3. Involvement of the TLR/MyD88-Mediated Pathways in Diseases The TLR/MyD88-mediated pathways play an essential role in the development of atherosclerosis, an inflammatory disease of arterial intima, characterized by the presence of the activated macrophages expressing various proinflammatory cytokines/chemokines. Apoe / mice are murine models of this disease. Apoe / mice develop atherosclerosis after being fed with a high-cholesterol diet, whereas Myd88 / Apoe / mice evade this disease (Bjorkbacka et al., 2004; Michelsen et al., 2004). The artery wall of the former expresses higher levels of IL-12 and various potent chemokines involved in the recruitment of various inflammatory cells than the latter. Thus, the TLR/ MyD88 signalings might become a target for the treatment of atherosclerosis. The TLR9/MyD88 pathways are shown to be involved in the activation of autoreactive B cells. Patients with systemic lupus erythematosus (SLE), a systemic autoimmune disease, produce a large amount of autoantibodies, such as antichromatin, anti-DNA, and antiself IgG designated as rheumatoid factor (RF). Recently, it was clearly demonstrated that the dual signals via their antigen receptor and TLR9/MyD88 are essential for the appropriate activation of the autoreactive B cells producing RF by using Tlr / and Myd88 / mice. RFþ B cells are strongly activated by IgG prepared from the sera of autoimmune mice, but not WT normal mice, and proliferate and produce RF in vitro, implicating that the RFþ B cells might recognize immune complexes comprised of antinucleosome IgG and chromatin fragment derived from other

96

TETSUJI N AKA ET A L .

cells (Boule et al., 2004). The autoreactive B cells proliferate in response to immune complexes constructed from haptenated dsDNA with hypomethylated CpG motifs and antibodies against the hapten (Viglianti et al., 2003). As Myd88 / RFþ B cells do no proliferate in response to the chromatin immune complex, and inhibitor of the TLR9 signaling inhibit proliferative response of the RFþ B cells, the TLR9/MyD88-mediated pathway is required for this event (Boule et al., 2004). Thus, TLR9 on autoreactive B cells are a gate of systemic autoimmune diseases. 9. Negative Regulation of the TLR Signalings Although TLR-mediated signal pathways are indispensable for eradication of microbes (Edelson and Unanue, 2002; Reiling et al., 2002; Scanga et al., 2002; Seki et al., 2002a), their excessive and/or prolonged activation is harmful and sometimes becomes a new pathogenesis. In fact, LPS-activated cells induce multiple tissue injuries and/or systemic illness through their production of proinflammatory cytokines and bioactive factors involved in microcirculatory dysfunctions. LPS tolerance and cross-tolerance among various PAMPs might develop to prevent the host from this fatal responses to various TLR ligands (Mengozzi and Ghezzi, 1993). Prior exposure of cells to various TLR ligands induces tolerance to a second stimulation with the initial TLR ligand and the other TLR ligands, as well as in the cells. For example, LPS-treated cells do not respond to LPS or TLR2 ligand such as BLP, and vice versa (Sato et al., 2000; Wang et al., 2002). This is also the case in vivo (Lehner et al., 2001). The molecular mechanisms underlying these tolerances have been unknown for a long time. The intensive studies on the molecular basis for TLR-mediated signal pathways now allow us to investigate the molecular mechanisms of the self- and cross-tolerance, although we have not yet discovered the clear-cut theory that comprehensively explain these phenomena. Down-regulation of TLR expression might be involved in TLR ligand tolerances. LPS-pre-exposed murine macrophages are reported to express downregulated levels of TLR4/MD-2 complex as compared with non-treated cells (Nomura et al., 2000). BLP tolerance is also partly explained by the downregulated expression of TLR2 by using the human monocytic cell line, THP.1 (Wang et al., 2002). However, in freshly isolated human monocytes, LPS treatment does not cause down-regulatory expression of their TLR4 and MD-2 (Medvedev et al., 2002). Moreover, Chinese hamster ovary cells that are engineered to over-express TLR4/MD-2 or TLR2 still show LPS tolerance and tolerance to arabinose-capped lipoarabinomannan, a ligand of TLR2, respectively, although their TLR4/MD-2 or TLR2 expression levels are unchanged

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

97

after these treatments (Medvedev et al., 2001). Flagellin tolerance is not due to down-regulation of TLR5, but rather to association with down-regulation of TLR5 signaling (Mizel and Snipes, 2002). Therefore, the down-regulation of TLRs is not solely relevant to tolerance to TLR ligands. Many molecules that can negatively regulate the TLR-mediated pathways have been identified. IRAK-M, MyD88s, SOCS-1, RIP3, and NOD (nucleotide-binding oligomerization domain) 2 are the cytoplasmic regulators, while SIGIRR (single immunoglobulin IL-1R-related molecule) and ST2 are the receptor-type regulators (Fig. 5). 9.1. IRAK-M IRAK-M, a homologue of IRAK without kinase activity, is identified as a negative regulator of the TLR/MyD88-mediated signalings (Kobayashi et al., 2002a; Wesche et al., 1999). IRAK-M is induced in macrophages after stimulation with TLR ligands and inhibits the dissociation of phosphorylated IRAK from the intracellular signaling platform of TLRs, leading to the suppression of the TLR signalings (Kobayashi et al., 2002a; Mizel and Snipes, 2002). Irak-m / mice have impairment in LPS tolerance (Kobayashi et al., 2002a). 9.2. MyD88s MyD88s, a short form, splicing variant of MyD88 lacking the domain necessary for the interaction with IRAK4, was reported to be involved in the negative regulation of the TLR/MyD88-mediated signal transduction by inhibiting the recruitment of IRAK4 (Burns et al., 2003). However, the pathophysiological role of MyD88s is still unclear. 9.3. SOCSs SOCS-1, a potent negative regulator of cytokine signaling, is also involved in the negative regulation of the TLR signaling. We will describe it in detail in a later discussion. 9.4. RIP3 RIP3, a RIP kinase also having RHIM, can directly bind to TRIF but inhibit the TLR3/TRIF/RIP1-mediated pathway. RIP3 dose-dependently inhibit association of TRIF with RIP1 and NF-kB activation in the cells transfected with TRIF or RIP1 (Meylan et al., 2004), indicating competitive inhibitory

98

TETSUJI N AKA ET A L .

Figure 5 TLR-mediated signal pathways and intracellular negative regulators of them. (A) Ligand binding to the TLRs except for TLR3 initiates the recruitment of MyD88 or both TIRAP and MyD88 via homophilic TIR/TIR interaction between corresponding MyD88/TIRAP and the

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s

99

action of RIP3 on RIP1. However, we do not know the pathophysiological role of RIP3. 9.5. SIGIRR (TIR8) SIGIRR, also named TIR8, was identified as a TIR-containing receptor by database searching (Tomassen et al., 1999). SIGIRR/TIR8 is expressed in epithelial cells but not macrophages or fibroblasts. SIGIRR is recruited to TLR4 and prevents the TLR4 signaling by sequestrating IRAK and TRAF6 (Wald et al., 2003). Sigirr/Tir8 / mice are highly susceptible to LPS. Sigirr/Tir8 / cells show higher levels of activation of NF-kB and JNK than Sigirr/Tir8þ/þ cells after stimulation with LPS (Wald et al., 2003). Intriguingly, Sigirr/Tir8 / mice showed more severe colitis upon challenge with dextran sulfate sodium (DSS) than Sigirr/Tir8þ/þ cells (Wald et al., 2003). 9.6. ST2 ST2 or T1/ST2 is a TIR-containing orphan receptor and is regarded as an important Th2 cell marker (Hoshino et al., 1999a). Co-transfection of ST2 dose-dependently inhibits NF-kB activation induced by LPS and IL-1 but not dsRNA, suggesting that ST2 is a negative regulator of the TLR/MyD88mediated pathways. Indeed, it can interact with MyD88 but not IRAK. ST2 inhibits the TLR/MyD88-mediated pathways by sequestration of MyD88. cytoplasmic TLRs. This is followed by the recruitment of IRAK and IRAK4, eventually resulting in IRAK4-dependent IRAK phosphorylation. Activated IRAK dissociates from the TLRs and translocates into the cytoplasm. In the cytoplasm, activated IRAK1 interacts with TRAF6 and allows the formation of multiprotein complex of TRAF6, TAB2, TAB1, and TAK1, resulting in both degradation of TRAF6 via ubiquitination and its resulting phosphorylation of TAK1. Phosphorylated TAK1 activates both IKK complexes, composed of IKKa, IKKb and IKKg, and MKK kinase. The latter induces p38 and JNK MAPK family activation. The activated IKK complexes induce phophorylation of IkB, resulting in its degradation by ubiquitination. This is followed by the translocation of NF-kB to activate corresponding gene expressions including inflammatory cytokines (also see C). MyD88s might contribute to the sequestration of the MyD88/TRAF6 pathways, resulting in the negative regulation of this pathway. IRAK-M, an IRAK homologue without kinase activity, prohibits the dissociation of activated IRAK1 from the cytopasmic TLRs (also see B). SOCS-1 associates with IRAK-4 and negatively regulates the signaling between the TAK1 activation and the IKK phosphorylation in this pathway (also see B). (C) Upon stimulation with LPS, TLR4 recruit TRIF and TRAM. TRIF directly interacts with TRAF6, presumably leading to the activation of NF-kB and MAPK pathways. TRIF also associates with RIP1 via RHIM/RHIM interaction to activate NF-kB, leading to activation of Icam1. TRIF induces dimerization of IRF3, leading to the maturation of dendritic cells and the activation of Ifnb. IFN-b activates STAT1. SOCS-1 might negatively regulate this TRAM/TRIF-dependent pathway (also see B).

100

TETSUJI N AKA ET A L .

Moreover, St2 / mice lack LPS tolerance (Wald et al., 2003), demonstrating the importance of endogenous ST2 for LPS tolerance. 9.7. NOD2 NOD2 is a member of a family of intracellular LRR-containing proteins recently designated the CATERPILLER gene family (Inohara and Nunez, 2001; Tschopp et al., 2003). NOD2, like other members, has no TIR. NOD2 is preferentially expressed in macrophages and DCs. After recognition of muramyl dipeptide (MDP), bacterial peptidoglycan component via its LRRs, NOD2 interacts with RICK, a serine/threonine kinase, leading to the activation of NF-k B (Kobayashi et al., 2002b). NOD2 is a candidate gene relevant to Crohn’s disease, which is an intractable inflammatory bowel disease with Th1 cell deviation (Bouma and Strober, 2003; Strober et al., 2002). Indeed, substantial numbers of patients with Crohn’s disease express mutant Nod2 (Ogura et al., 2001). However, it is unclear how the mutant Nod2 causes chronic inflammatory bowel disease. Very recently, the mechanism underlying the inflammation-prone condition induced by the mutant Nod2, was reported (Watanabe et al., 2004). Nod2 / mice are reported not to develop spontaneous enterocolitis under standard maintenance conditions. However, Nod2 / macrophages produce much larger amounts of IL-12 (which is a major, causative cytokine of this disease) (Bouma and Strober, 2003), than do Nod2þ/þ cells selectively upon stimulation with PGN. Co-stimulation with MDP obviously reduces IL-12 production by PGN-activated Nod2þ/þ macrophages, while this treatment sustains production of high levels of IL-12 from Nod2 / cells in response to PGN, suggesting the MDP-induced NOD2 activation pathway as a negative regulator for the TLR2-mediated pathway. Furthermore, Nod2 / cells exhibit higher levels of activation of translocation of NF-kB, particularly c-Rel component, in response to PGN than do Nod2þ/þ cells. These observations suggest that the NOD2-mediated pathway that would be activated by MDP derived from the original stimulant, PGN, negatively regulates the PGN-induced activation of c-Rel. Thus, NOD2 negatively regulates the TLR2mediated signal pathways to ensure the safety circumstances, in particular in the intestine that is constitutively exposed to the stress of abundant commensal bacteria. 9.8. Negative Regulation of TLR Signaling by Cytokines It has been well known that anti-inflammatory cytokines, such as TGFb, IL-4, IL-13, and IL-10 suppress macrophage activation. However, the molecular mechanism how these cytokines suppress TLR signaling has not been clarified.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 101 Especially, IL-10 has been shown to potently inhibit TNFa production from macrophages in response to LPS. STAT3 has been shown to be essential for antiinflammatory function of IL-10 (Takeda et al., 1999). However, signals other than STAT3 are suggested to be involved in IL-10 effect. IL-10-inducible genes such as Bcl-10 and OH-1 may be involved in TLR signal suppression. Probably multiple mechanisms may participate in the antiinflammatory effect of IL-10. Molecular basis of TGFb and IL-4 is largely unclear. One possible mechanism of IL-4 is the induction of SOCS1, which inhibits IFNs-induced STAT1 activation. 10. Regulation of TLR Signaling by SOCS 10.1. SOCS-1 and TLR Regulation of TLR signaling is a key step for inflammation, septic shock, and innate/adaptive immunity. Previous observations indicate a possible link between TLR signaling and SOCS proteins. For example, in macrophages, SOCS-1 and SOCS-3 are induced by LPS or CpG-DNA (Crespo et al., 2002; Stoiber et al., 1999) and may cause hyporesponsiveness of these cells to cytokines such as IFNg after exposure to TLR ligands. More interestingly, absence of SOCS-1 results in hyper-sensitiveness to LPS shock (Kinjyo et al., 2002; Nakagawa et al., 2002). SOCS1 / mice (pre-disease onset) and SOCS1þ/ mice are hyperresponsive to LPS, and are very sensitive to LPSinduced lethality. In addition, IFN-g / SOCS1 / mice and STAT1 / SOCS1 / are also very sensitive to LPS shock, suggesting that hyperresponsiveness of these mice to LPS is not dependent on IFN-g/STAT1 signaling. Macrophages from these mutant mice produced increased levels of the proinflammatory cytokines, such as TNFa and IL-12 as well as nitric oxide (NO) in response to LPS. Importantly, LPS-tolerance was impaired in SOCS-1 / mice as well as SOCS-1 / macrophages. Since overexpression of SOCS-1 in a macrophage cell line resulted in the suppression of LPS signaling, SOCS1 negatively regulates not only the JAK/STAT pathway, but also the TLRNFkB pathway (Kinjyo et al., 2002; Nakagawa et al., 2002). Moreover, direct binding of NF-kB subunit p65 and SOCS-1, which resulted in accelerated p65 degradation, has been reported (Ryo et al., 2003). Recently, another study by a different group confirmed that SOCS-1 / mice are highly sensitive to LPS-induced shock. However, in this study, bone marrow-derived macrophages generated from SOCS1-deficient mice exhibited neither hyperresponsiveness to LPS nor impaired LPS-tolerance (Gingras et al., 2004). This could be due to the difference of preparation of macrophages. Freshly isolated macrophages from SOCS1 / mice may be in an altered

102

TETSUJI N AKA ET A L .

differentiation stage, or already activated. Alternatively, strong effects of SOCS-1 on the IFN/STAT1 pathway may affect TLR signaling indirectly. In any case, precise understanding of the molecular mechanism of hyperresponsiveness of SOCS1 / mice to LPS will uncover an unknown important mechanism of TLR signal down-regulation. 10.2. SOCS1 and DC Regulation In the absence of SOCS-1, dendritic cells (DCs) also become hyperresponsive to IFN-g and IL-4 (Hanada et al., 2003). To define the role of SOCS1-deficient DCs in vivo, other mutant mice were generated in which the SOCS1 expression was restored in T and B cells on a SOCS1 / background. In these mice, DCs were abnormally accumulated in the thymus and spleen and produced high levels of BAFF/BLyS and APRIL, resulting in the aberrant expansion of B cells and the production of autoreactive antibodies. SOCS1-deficient DCs efficiently stimulated B-cell proliferation in vitro and auto-antibody production in vivo. These results indicate that SOCS1 plays an essential role in the normal DC functions and suppression of systemic autoimmunity. Interestingly, recent investigation of conditional SOCS-1 / mice lacking the SOCS-1 gene specifically in NKT and T cells revealed that SOCS-1 / T cells alone do not cause any of the inflammatory pathologies and neonatal death found in SOCS1 / mice (Chong et al., 2003). This clearly indicates that SOCS-1 deficiency causes multiple effects in vivo and requires hematopoietic cell lineages other than T cells. One apparent candidate is APC, because APC plays critical roles in antigen recognition by T cells. These recent studies imply that SOCS1 / DCs are important players for the onset of SOCS1 / diseases, since SOCS1 / DCs can activate proliferation not only of B cells but also of T cells. In addition, SOCS1 / DCs can induce cytokine production from T cells more efficiently than wild type DCs (Hanada et al., unpublished data). This hypothesis will be probed by transferring SOCS1 / DCs with or without SOCS1 / T cells into wild-type mice. As mentioned, DCs with reduced SOCS-1 expression could be useful for antitumor vaccination. 10.3. SOCS-3 and TLR Signal Modulation IL-6 is a proinflammatory cytokine that plays a progressive role in many inflammatory diseases, including rheumatoid arthritis (RA) and Crohn’s disease (CD), while IL-10 is an immunoregulatory cytokine that has potent anti-inflammatory activity. Although the transcription factor STAT3 is essential for the function of both IL-6 and IL-10 (Takeda et al., 1999), it is not clear how these two cytokines exhibit such opposite functions. Recently, it was

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 103 demonstrated that at least in macrophages, SOCS-3 is a key regulator of the divergent action of these two cytokines (Yasukawa et al., 2003). In macrophages lacking the SOCS-3 gene, not only IL-10 but also IL-6 suppresses LPSinduced TNFa production. SOCS-3 protein is strongly induced by both IL-6 and IL-10 in the presence of LPS, but selectively inhibits IL-6 signaling, due to SOCS-3 binding to the IL-6 receptor, gp130, but not to the IL-10 receptor. Similarly, in macrophages from mice carrying a mutation in the SOCS-3 binding site (Y759F) of gp130, IL-6 elicits immunosuppressive function equivalent to IL-10 (Yasukawa et al., 2003). These data indicate that SOCS-3 selectively blocks IL-6 signaling, interfering with the ability of IL-6 to inhibit LPS signaling. Consistent with this, mice specifically lacking the SOCS-3 gene in macrophages and neutrophils are resistant to acute inflammation induced by LPS injection. This phenotype is completely opposite to that of mice lacking STAT3 in macrophages and neutrophils (Takeda et al., 1999). These STAT3 KO mice are very sensitive to LPS shock, and STAT3-deficient macrophages from these mice produce excessive TNFa in response to LPS. Recently, STAT3-deficient DCs were shown to be hyperactivated. Thus, in macrophages and probably in DCs, SOCS-3 modulates the activation status of these cells by suppressing STAT3. Suppression of SOCS-3 in macrophages may represent a novel therapeutic approach for the treatment of inflammatory diseases in which IL-6 plays progressive roles. 11. Concluding Remarks Cytokines are important not only for maintenance of homeostasis and defense against microbes, but also the onset and progression of disease. The negative regulation of signal transduction also plays a central role in balancing the positive and deleterious consequences of cytokine action. The SOCS proteins are indispensable for regulating many biochemical processes, including leukocyte homeostasis, glucose turnover, cell growth, and responses to pathogens, and are apparently a hallmark of such understanding of cytokine signal regulation. The next important step is apparently the modulation of SOCS protein expression levels or activity for beneficial clinical outcomes. This modulation of SOCS protein expression may lead to the development of beneficial strategies to prevent the host from inflammatory and autoimmune disease. References Abel, B., Thieblemont, N., Quesniaux, V. J. F., Brown, N., Mpagi, J., Miyake, K., Bihl, F., and Ryffel, B. (2002). Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice. J. Immunol. 169, 3155–3162.

104

TETSUJI N AKA ET A L .

Adachi, K., Tsutsui, H., Kashiwamura, S., Seki, E., Nakano, H., Takeuchi, O., Takeda, K., Okumura, K., Van Kaer, L., Okamura, H., Akira, S., and Nakanishi, K. (2001). Plasmodium berghei infection in mice induces liver injury by an IL-12- and toll-like receptor/myeloid differentiation factor 88-dependent mechanism. J. Immunol. 167, 5928–5934. Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K., and Akira, S. (1998). Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18mediated function. Immunity 9, 143–150. Adams, T. E., Hansen, J. A., Starr, R., Nicola, N. A., Hilton, D. J., and Billestrup, N. (1998). Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signaling. J. Biol. Chem. 273, 1285–1287. Akira, S., Takeda, K., and Kaisho, T. (2001). Toll-like receptors: Critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675–680. Alexander, W. S., and Hilton, D. J. (2004). The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu. Rev. Immunol. 22, 503–529. Alexander, W. S., Starr, R., Fenner, J. E., Scott, C. L., Handman, E., Sprigg, N. S., Corbin, J. E., Cornish, A. L., Darwiche, R., Owczarek, C. M., Kay, T. W., Nicola, N. A., Hertzog, P. J., Metcalf, D., and Hilton, D. J. (1999). SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell 98, 597–608. Alexopoulou, L., Holt, A. C., Medzhitov, R., and Flavell, R. A. (2001). Recognition of doublestranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732–738. Aliprantis, A. O., Yang, R.-B., Mark, M. R., Suggett, S., Devaux, B., Radolf, J. D., Klimpel, G. R., Godowski, P., and Zychlinsky, A. (1999). Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285, 736–739. Aman, M. J., Migone, T. S., Sasaki, A., Ascherman, D. P., Zhu, M., Soldaini, E., Imada, K., Miyajima, A., Yoshimura, A., and Leonard, W. J. (1999). CIS associates with the interleukin2 receptor beta chain and inhibits interleukin-2-dependent signaling. J. Biol. Chem. 274, 30266–30272. Applequist, S. E., Wallin, R. P. A., and Ljunggren, H.-G. (2001). Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines. Int. Immunol. 14, 1065–1074. Barsness, K. A., Arcaroli, J., Harken, A. H., Abraham, E., Banerjee, A., Reznikov, L., and McIntyre, R. C. J. (2004). Hemorrhage-induced acute lung injury is TLR-4 dependent. Am. J. Physiol. Regul. Integr. Comp. Physiol. 297, R592–R599. Berlato, C., Cassatella, M. A., Kinjyo, I., Gatto, L., Yoshimura, A., and Bazzoni, F. (2002). Involvement of suppressor of cytokine signaling-3 as a mediator of the inhibitory effects of IL-10 on lipopolysaccharide-induced macrophage activation. J. Immunol. 168, 6404–6411. Biragyn, A., Adelchi Ruffini, P., Leifer, C. A., Klyushnenkova, E., Shakhov, A., Chertov, O., Shirakawa, A. K., Farber, J. M., Segal, D. M., Oppenheim, J. J., and Kwak, L. W. (2002). Tolllike receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science 298, 1025–1029. Bjorbaek, C., Elmquist, J. K., Frantz, J. D., Shoelson, S. E., and Flier, J. S. (1998). Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol. Cell 1, 619–625. Bjorbak, C., Lavery, H. J., Bates, S. H., Olson, R. K., Davis, S. M., Flier, J. S., and Myers, M. G., Jr. (2000). SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985. J. Biol. Chem. 275, 40649–40657. Bjorkbacka, H., Kunjathoor, V. V., Moore, K. J., Koehn, S., Ordija, C. M., Lee, M. A., Means, T., Halmen, K., Luster, A. D., Golenbock, D. T., and Freeman, M. W. (2004). Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Nat. Med. 10, 416–421.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 105 Boonstra, A., Asselin-Paturel, C., Gilliet, M., Crain, C., Trinchieri, G., Liu, Y.-J., and O’Garra, A. (2003). Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: Dependency on antigen dose and differential toll-like receptor ligation. J. Exp. Med. 197, 101–109. Boule, M. W., Broughton, C., Mackay, F., Akira, S., Marshak-Rothstein, A., and Rifkin, I. R. (2004). Toll-like receptor 9-dependent and -independent dendritic cell activation by chromatinimmunoglobulin G complexes. J. Exp. Med. 199, 1631–1640. Bouma, G., and Strober, W. (2003). The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 3, 521–533. Brender, C., Columbus, R., Metcalf, D., Handman, E., Starr, R., Huntington, N., Tarlinton, D., Odum, N., Nicholson, S. E., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (2004). SOCS5 is expressed in primary B and T lymphoid cells but is dispensable for lymphocyte production and function. Mol. Cell. Biol. 24, 6094–6103. Burns, K., Janssens, S., Brissoni, B., Olivos, N., Beyaert, R., and Tschopp, J. (2003). Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4. J. Exp. Med. 197, 263–268. Callus, B. A., and Mathey-Prevot, B. (2002). SOCS36E, a novel Drosophila SOCS protein, suppresses JAK/STAT and EGF-R signaling in the imaginal wing disc. Oncogene 21, 4812–4821. Campos, M. A. S., Almeida, I. C., Takeuchi, O., Akira, S., Valente, E. P., Procopio, D. O., Travassos, L. R., Smith, J. A., Golenbock, D. T., and Gazzinelli, R. T. (2001). Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite. J. Immunol. 167, 416–423. Cao, Z., Henzel, W. J., and Gao, X. (1996). IRAK: a kinase associated with the interleukin-1 receptor. Science 271, 1128–1131. Cassatella, M. A., Gasperini, S., Bovolenta, C., Calzetti, F., Vollebregt, M., Scapini, P., Marchi, M., Suzuki, R., Suzuki, A., and Yoshimura, A. (1999). Interleukin-10 (IL-10) selectively enhances CIS3/SOCS3 mRNA expression in human neutrophils: Evidence for an IL-10-induced pathway that is independent of STAT protein activation. Blood 94, 2880–2889. Chen, X. P., Losman, J. A., Cowan, S., Donahue, E., Fay, S., Vuong, B. Q., Nawijn, M. C., Capece, D., Cohan, V. L., and Rothman, P. (2002). Pim serine/threonine kinases regulate the stability of Socs-1 protein. Proc. Natl. Acad. Sci. USA 99, 2175–2180. Chong, M. M., Cornish, A. L., Darwiche, R., Stanley, E. G., Purton, J. F., Godfrey, D. I., Hilton, D. J., Starr, R., Alexander, W. S., and Kay, T. W. (2003). Suppressor of cytokine signaling-1 is a critical regulator of interleukin-7-dependent CD8þ T cell differentiation. Immunity 18, 475–487. Chong, M. M., Thomas, H. E., and Kay, T. W. (2002). Suppressor of cytokine signaling-1 regulates the sensitivity of pancreatic beta cells to tumor necrosis factor. J. Biol. Chem. 277, 27945–27952. Chung, C. D., Liao, J., Liu, B., Rao, X., Jay, P., Berta, P., and Shuai, K. (1997). Specific inhibition of Stat3 signal transduction by PIAS3. Science 278, 1803–1805. Cohney, S. J., Sanden, D., Cacalano, N. A., Yoshimura, A., Mui, A., Migone, T. S., and Johnston, J. A. (1999). SOCS-3 is tyrosine phosphorylated in response to interleukin-2 and suppresses STAT5 phosphorylation and lymphocyte proliferation. Mol. Cell. Biol. 19, 4980–4988. Cornish, A. L., Chong, M. M., Davey, G. M., Darwiche, R., Nicola, N. A., Hilton, D. J., Kay, T. W., Starr, R., and Alexander, W. S. (2003a). Suppressor of cytokine signaling-1 regulates signaling in response to interleukin-2 and other gamma c-dependent cytokines in peripheral T cells. J. Biol. Chem. 278, 22755–22761. Cornish, A. L., Davey, G. M., Metcalf, D., Purton, J. F., Corbin, J. E., Greenhalgh, C. J., Darwiche, R., Wu, L., Nicola, N. A., Godfrey, D. I., Heath, W. R., Hilton, D. J., Alexander, W. S., and Starr, R. (2003b). Suppressor of cytokine signaling-1 has IFN-gamma-independent actions in T cell homeostasis. J. Immunol. 170, 878–886.

106

TETSUJI N AKA ET A L .

Crespo, A., Filla, M. B., and Murphy, W. J. (2002). Low responsiveness to IFN-gamma, after pretreatment of mouse macrophages with lipopolysaccharides, develops via diverse regulatory pathways. Eur. J. Immunol. 32, 710–719. Croker, B. A., Krebs, D. L., Zhang, J. G., Wormald, S., Willson, T. A., Stanley, E. G., Robb, L., Greenhalgh, C. J., Forster, I., Clausen, B. E., Nicola, N. A., Metcalf, D., Hilton, D. J., Roberts, A. W., and Alexander, W. S. (2003). SOCS3 negatively regulates IL-6 signaling in vivo. Nat. Immunol. 4, 540–545. Croker, B. A., Metcalf, D., Robb, L., Wei, W., Mifsud, S., DiRago, L., Cluse, L. A., Sutherland, K. D., Hartley, L., Williams, E., Zhang, J. G., Hilton, D. J., Nicola, N. A., Alexander, W. S., and Roberts, A. W. (2004). SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis. Immunity 20, 153–165. Day, N., Tangsinmankong, N., Ochs, H., Rucker, R., Picard, C., Casanova, J.-L., Haraguchi, S., and Good, R. (2004). Interleukin receptor-associated kinase (IRAK-4) deficiency associated with bacterial infections and failure to sustain antibody responses. J. Pediatr. 144, 524–526. De Sepulveda, P., Ilangumaran, S., and Rottapel, R. (2000). Suppressor of cytokine signaling-1 inhibits VAV function through protein degradation. J. Biol. Chem. 275, 14005–14008. De Sepulveda, P., Okkenhaug, K., Rose, J. L., Hawley, R. G., Dubreuil, P., and Rottapel, R. (1999). SOCS1 binds to multiple signaling proteins and suppresses steel factor-dependent proliferation. EMBO J. 18, 904–915. De Souza, D., Fabri, L. J., Nash, A., Hilton, D. J., Nicola, N. A., and Baca, M. (2002). SH2 domains from suppressor of cytokine signaling-3 and protein tyrosine phosphatase SHP-2 have similar binding specificities. Biochemistry 41, 9229–9236. Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., and Chen, Z. J. (2000). Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitinconjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361. Dey, B. R., Spence, S. L., Nissley, P., and Furlanetto, R. W. (1998). Interaction of human suppressor of cytokine signaling (SOCS)-2 with the insulin-like growth factor-I receptor. J. Biol. Chem. 273, 24095–24101. Edelson, B. T., and Unanue, E. R. (2002). MyD88-dependent but Toll-like receptor 2-independent innate immunity to Listeria: No role for either in macrophage listericidal activity. J. Immunol. 169, 3869–3875. Egan, P. J., Lawlor, K. E., Alexander, W. S., and Wicks, I. P. (2003). Suppressor of cytokine signaling-1 regulates acute inflammatory arthritis and T cell activation. J. Clin. Invest. 111, 915–924. Endo, T. A., Masuhara, M., Yokouchi, M., Suzuki, R., Sakamoto, H., Mitsui, K., Matsumoto, A., Tanimura, S., Ohtsubo, M., Misawa, H., Miyazaki, T., Leonor, N., Taniguchi, T., Fujita, T., Kanakura, Y., Komiya, S., and Yoshimura, A. (1997). A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387, 921–924. Eyles, J. L., Metcalf, D., Grusby, M. J., Hilton, D. J., and Starr, R. (2002). Negative regulation of interleukin-12 signaling by suppressor of cytokine signaling-1. J. Biol. Chem. 277, 43735–43740. Fausto, N. (2000). Liver regeneration. J. Hepatol. 32, 19–31. Favre, H., Benhamou, A., Finidori, J., Kelly, P. A., and Edery, M. (1999). Dual effects of suppressor of cytokine signaling (SOCS-2) on growth hormone signal transduction. FEBS Lett. 453, 63–66. Fitzgerald, K. A., McWhirter, S. M., Faia, K. L., Rowe, D. C., Latz, E., Golenbock, D. T., Goyle, A. J., Liao, S.-M., and Maniatis, T. (2003a). Nat. Immunol. 4, 491. Fitzgerald, K. A., Palsson-McDermott, E. M., Bowie, A. G., Jefferies, C. A., Mansell, A. S., Brady, G., Brint, E., Dunne, A., Gray, P., Harte, M. T., McMurray, D., Smith, D. E., Sims, J. E., Bird, T. A., and O’Neill, L. A. J. (2001). Mal (MyD88-adaptor-like) is required for Toll-like receptor-4 signal transduction. Nature 413, 78–83.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 107 Fitzgerald, K. A., Rowe, D. C., Barners, B. J., Caffrey, D. R., Visintin, A., Latz, E., Monks, B., Pitha, P. M., and Golenbock, D. T. (2003b). LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adaptors TRAM and TRIF. J. Exp. Med. 198, 1043–1055. Frantsve, J., Schwaller, J., Sternberg, D. W., Kutok, J., and Gilliland, D. G. (2001). Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation. Mol. Cell. Biol. 21, 3547–3557. Fujimoto, M., and Naka, T. (2003). Regulation of cytokine signaling by SOCS family molecules. Trends Immunol. 24, 659–666. Fujimoto, M., Naka, T., Nakagawa, R., Kawazoe, Y., Morita, Y., Tateishi, A., Okumura, K., Narazaki, M., and Kishimoto, T. (2000). Defective thymocyte development and perturbed homeostasis of T cells in STAT-induced STAT inhibitor-1/suppressors of cytokine signaling-1 transgenic mice. J. Immunol. 165, 1799–1806. Fujimoto, M., Tsutsui, H., Xinshou, O., Tokumoto, M., Watanabe, D., Shima, Y., Yoshimoto, T., Hirakata, H., Kawase, I., Nakanishi, K., Kishimoto, T., and Naka, T. (2004). Inadequate induction of suppressor of cytokine signaling-1 causes systemic autoimmune diseases. Int. Immunol. 16, 303–314. Fujimoto, M., Tsutsui, H., Yumikura-Futatsugi, S., Ueda, H., Xingshou, O., Abe, T., Kawase, I., Nakanishi, K., Kishimoto, T., and Naka, T. (2002). A regulatory role for suppressor of cytokine signaling-1 in T(h) polarization in vivo. Int. Immunol. 14, 1343–1350. Galm, O., Wilop, S., Reichelt, J., Jost, E., Gehbauer, G., Herman, J. G., and Osieka, R. (2004). DNA methylation changes in multiple myeloma. Leukemia 18, 1687–1692. Galm, O., Yoshikawa, H., Esteller, M., Osieka, R., and Herman, J. G. (2003). SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood 101, 2784–2788. Gewirtz, A. T., Navas, T. A., Lyons, S., Godowski, P. J., and Madara, J. L. (2001). Cutting edge: Bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J. Immunol. 167, 1882–1885. Gingras, S., Parganas, E., de Pauw, A., Ihle, J. N., and Murray, P. J. (2004). Re-examination of the role of suppressor of cytokine signaling 1 (SOCS1) in the regulation of toll-like receptor signaling. J. Biol. Chem. 279, 54702–54707. Gomez-Gomez, L., and Boller, T. (2002). Flagellin perception: A paradigm for innate immunity. Trends Plant Sci. 7, 251–256. Greenhalgh, C. J., Bertolino, P., Asa, S. L., Metcalf, D., Corbin, J. E., Adams, T. E., Davey, H. W., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (2002a). Growth enhancement in suppressor of cytokine signaling 2 (SOCS-2)-deficient mice is dependent on signal transducer and activator of transcription 5b (STAT5b). Mol. Endocrinol. 16, 1394–1406. Greenhalgh, C. J., Metcalf, D., Thaus, A. L., Corbin, J. E., Uren, R., Morgan, P. O., Fabri, L. J., Zhang, J. G., Martin, H. M., Willson, T. A., Billestrup, N., Nicola, N. A., Baca, M., Alexander, W. S., and Hilton, D. J. (2002b). Biological evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling. J. Biol. Chem. 277, 40181–40184. Gruber, A., Mancek, M., Wagner, H., Kirshcning, C. J., and Jerala, R. (2004). Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition. J. Biol. Chem. 279, 28475–28482. Hanada, T., Yoshida, H., Kato, S., Tanaka, K., Masutani, K., Tsukada, J., Nomura, Y., Mimata, H., Kubo, M., and Yoshimura, A. (2003). Suppressor of cytokine signaling-1 is essential for suppressing dendritic cell activation and systemic autoimmunity. Immunity 19, 437–450. Hanada, T., Yoshida, T., Kinjyo, I., Minoguchi, S., Yasukawa, H., Kato, S., Mimata, H., Nomura, Y., Seki, Y., Kubo, M., and Yoshimura, A. (2001). A mutant form of JAB/SOCS1 augments the

108

TETSUJI N AKA ET A L .

cytokine-induced JAK/STAT pathway by accelerating degradation of wild-type JAB/CIS family proteins through the SOCS-box. J. Biol. Chem. 276, 40746–40754. Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett, D. R., Enk, J. K., Akira, S., Underhill, D. M., and Aderem, A. (2001). The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103. He, B., You, L., Uematsu, K., Zang, K., Xu, Z., Lee, A. Y., Costello, J. F., McCormick, F., and Jablons, D. M. (2003). SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. Proc. Natl. Acad. Sci. USA 100, 14133–14138. Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschnning, C., Akira, S., Lipford, G., Wagner, H., and Bauer, S. (2004). Species-specific recognition of single-stranded RNA via tolllike receptor 7 and 8. Science 303, 1526–1529. Hemmi, H., Kaisho, T., Takeuchi, O., Sato, H., Sanjo, K., Hoshino, K., Horiuchi, H., Tomizawa, K., Takeda, K., and Akira, K. (2002). Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3, 196–200. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000). A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745. Hemmi, H., Takeuchi, O., Sato, S., Yamamoto, M., Kaisho, T., Sanjo, H., Kawai, T., Hoshino, K., Takeda, K., and Akira, S. (2004). The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection. J. Exp. Med. 199, 1641–1650. Hertz, C. J., Kiertscher, S. M., Godowski, P. J., Bouis, D. A., Norgard, M. V., Rooth, M. D., and Modlin, R. L. (2001). Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J. Immunol. 166, 2444–2450. Hilton, D. J., Richardson, R. T., Alexander, W. S., Viney, E. M., Willson, T. A., Sprigg, N. S., Starr, R., Nicholson, S. E., Metcalf, D., and Nicola, N. A. (1998). Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc. Natl. Acad. Sci. USA 95, 114–119. Hirschfeld, M., Weis, J. J., Toshchakov, V., Salkowski, C. A., Cody, M. J., Ward, D. C., Qureshi, N., Michalek, Z. M., and Vogel, S. N. (2001). Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69, 1477–1482. Hoebe, K., Du, X., Janssen, E., Tabeta, K., Kim, S. O., Soeda, J., Lin, P., Mann, N., Muda, S., Crozat, K., Sovath, S., Han, J., and Beutler, B. (2003). Identification of Lps2 as a key transducer of MyD88-independent TIR signaling. Nature 424, 743–748. Hoffmann, A., Levchenko, A., Scott, M. L., and Baltimore, D. (2002). The IkappaB-NF-kappaB signaling module: Temporal control and selective gene activation. Science 298, 1241–1245. Hornef, M. W., Frisan, T., Vandewalle, A., Normark, S., and Richter-Dahlfors, A. (2002). Toll-like receptor 4 resides in the Golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells. J. Exp. Med. 195, 559–570. Horng, T., Barton, G. M., Flavell, R. A., and Medzhitov, R. (2002). The adaptor molecule TIRAP provides signaling specificity for Toll-like receptors. Nature 420, 329–333. Horng, T., Barton, G. M., and Medzhitov, R. (2001). TIRAP: An adaptor molecule in the Toll signaling pathway. Nat. Immunol. 2, 835–841. Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, S., and Hartmann, G. (2002). Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168, 4531–4537. Hortner, M., Nielsch, U., Mayr, L. M., Heinrich, P. C., and Haan, S. (2002). A new high affinity binding site for suppressor of cytokine signaling-3 on the erythropoietin receptor. Eur. J. Biochem. 269, 2516–2526.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 109 Hortner, M., Nielsch, U., Mayr, L. M., Johnston, J. A., Heinrich, P. C., and Haan, S. (2002). Suppressor of cytokine signaling-3 is recruited to the activated granulocyte-colony stimulating factor receptor and modulates its signal transduction. J. Immunol. 169, 1219–1227. Horvat, S., and Medrano, J. F. (2001). Lack of Socs2 expression causes the high-growth phenotype in mice. Genomics 72, 209–212. Hoshino, K., Kaisho, T., Iwabe, T., Takeuchi, O., and Akira, S. (2002). Differential involvement of IFN-beta in Toll-like receptor-stimulated dendritic cell activation. Int. Immunol. 14, 1225–1231. Hoshino, K., Kashiwamura, S., Kuribayashi, K., Kodama, T., TSujimura, T., Nakanishi, K., Matsuyama, T., Takeda, K., and Akira, S. (1999a). The absence of interleukin 1 receptor-related T1/ST2 does not affect T helper cell type 2 development and its effector function. J. Exp. Med. 190, 1541–1548. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K., and Akira, S. (1999b). Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: Evidence for TLR4 as the Lps gene product. J. Immunol. 162, 3749–3752. Howard, J. K., Cave, B. J., Oksanen, L. J., Tzameli, I., Bjorbaek, C., and Flier, J. S. (2004). Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3. Nat. Med. 10, 734–738. Hsu, H., Huang, J., Shu, H.-B., Baichwal, V., and Goeddel, D. V. (1996). TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4, 387–396. Ilangumaran, S., Ramanathan, S., La Rose, J., Poussier, P., and Rottapel, R. (2003a). Suppressor of cytokine signaling 1 regulates IL-15 receptor signaling in CD8þCD44 high memory T lymphocytes. J. Immunol. 171, 2435–2445. Ilangumaran, S., Ramanathan, S., Ning, T., La Rose, J., Reinhart, B., Poussier, P., and Rottapel, R. (2003b). Suppressor of cytokine signaling 1 attenuates IL-15 receptor signaling in CD8þ thymocytes. Blood 102, 4115–4122. Ilangumaran, S., Ramanathan, S., and Rottapel, R. (2004). Regulation of the immune system by SOCS family adaptor proteins. Semin. Immunol. 16, 351–365. Inohara, N., and Nunez, G. (2001). The NOD: A signaling module that regulates apoptosis and host defense against pathogens. Oncogene 20, 6473–6481. Irie-Sasaki, J., Sasaki, T., Matsumoto, W., Opavsky, A., Cheng, M., Welstead, G., Griffiths, E., Krawczyk, C., Richardson, C. D., Aitken, K., Iscove, N., Koretzky, G., Johnson, P., Liu, P., Rothstein, D. M., and Penninger, J. M. (2001). CD45 is a JAK phosphatase and negatively regulates cytokine receptor signaling. Nature 409, 349–354. Ito, S., Ansari, P., Sakatsume, M., Dickensheets, H., Vazquez, N., Donnelly, R. P., Larner, A. C., and Finbloom, D. S. (1999). Interleukin-10 inhibits expression of both interferon alpha- and interferon gamma-induced genes by suppressing tyrosine phosphorylation of STAT1. Blood 93, 1456–1463. Ito, T., Amakawa, R., Kaisho, T., Hemmi, H. K. T., Uehira, K., Ozaki, Y., Tomizawa, H., Akira, S., and Fukuhara, S. (2002). Interferon-alpha and interleukin-12 are induced differentially by Tolllike receptor 7 ligands in human blood dendritic cell subsets. J. Exp. Med. 195, 1507–1512. Ivashkiv, L. B., and Tassiulas, I. (2003). Can SOCS make arthritis better? J. Clin. Invest. 111, 795–797. Janeway, C. A. J., and Medzhitov, R. (2002). Innate immune recognition. Annu. Rev. Immunol. 20, 197–216. Jegalian, A. G., and Wu, H. (2002). Regulation of Socs gene expression by the proto-oncoprotein GFI-1B: Two routes for STAT5 target gene induction by erythropoietin. J. Biol. Chem. 277, 2345–2352.

110

TETSUJI N AKA ET A L .

Jiang, Z., Mak, T. W., Sen, G., and Li, X. (2004). Toll-like receptor 3-mediated activation of NFkappaB and IRF3 diverges at Toll-IL-1 receptor domain-containing adaptor-inducing IFN-beta. Proc. Nat. Acad. Soci. USA 101, 3533–3538. Kadowaki, N., Ho, S., Antonenko, S., de Waal Malefyt, R., Kastelein, R. A., Bazan, F., and Liu, Y.-J. (2001). Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J. Exp. Med. 194, 863–869. Kaisho, T., and Akira, S. (2002). Toll-like receptors as adjuvant receptors. Biochem. Biophys. Acta 2002, 1–13. Kaisho, T., Hoshino, K., Iwabe, T., Takeuchi, O., Yasui, T., and Akira, S. (2002). Endotoxin can induce MyD88-deficient dendritic cells to support T(h)2 cell differentiation. Int. Immunol. 14, 695–700. Kaisho, T., Takeuchi, O., Kawai, T., Hoshino, K., and Akira, S. (2001). Endotoxin-induced maturation of MyD88-deficient dendritic cells. J. Immunol. 166, 5688–5694. Kamio, M., Yoshida, T., Ogata, H., Douchi, T., Nagata, Y., Inoue, M., Hasegawa, M., Yonemitsu, Y., and Yoshimura, A. (2004). SOC1 inhibits HPV-E7-mediated transformation by inducing degradation of E7 protein. Oncogene 23, 3107–3115. Kamizono, S., Hanada, T., Yasukawa, H., Minoguchi, S., Kato, R., Minoguchi, M., Hattori, K., Hatakeyama, S., Yada, M., Morita, S., Kitamura, T., Kato, H., Nakayama, K., and Yoshimura, A. (2001). The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2. J. Biol. Chem. 276, 12530–12538. Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004). VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes. Dev. 18, 3055–3065. Kamura, T., Sato, S., Haque, D., Liu, L., Kaelin, W. G., Jr., Conaway, R. C., and Conaway, J. W. (1998). The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes. Dev. 12, 3872–3881. Kanakaraj, P., Ngo, K., Wu, Y., Angulo, A., Ghazal, P., Harris, C. A., Siekierka, J. J., Peterson, P. A., and Fung-Leung, W.-P. (1999). Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice. J. Exp. Med. 189, 1129–1138. Kanakaraj, P., Schafer, P. H., Cavender, D. E., Wu, Y., Ngo, K., Grealish, P. F., Wadsworth, S. A., Peterson, P. A., Siekierka, J. J., Harris, C. A., and Fung-Leung, W.-P. (1998). Interleukin (IL)-1 receptor-associated kinase (IRAK) requirement for optimal induction of multiple IL-1 signaling pathways and IL-6 production. J. Exp. Med. 187, 2073–2079. Karin, M., and Ben-Neriah, Y. (2000). Phosphorylation meets ubiquitination: The control of NF[kappa]B activity. Annu. Rev. Immunol. 18, 621–663. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999). Unresponsiveness of MyD88deficient mice to endotoxin. Immunity 11, 115–122. Kawai, T., Takeuchi, O., Fujita, T., Inoue, J., Muhlradt, P. F., Sato, S., Hoshino, K., and Akira, S. (2001). Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167, 5887–5894. Kawasaki, K., Akashi, S., Shimazu, R., Yoshida, T., Miyake, K., and Nishijima, M. (2000). Mouse toll-like receptor 4.MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol. J. Biol. Chem. 275, 2251–2254. Kawazoe, Y., Naka, T., Fujimoto, M., Kohzaki, H., Morita, Y., Narazaki, M., Okumura, K., Saitoh, H., Nakagawa, R., Uchiyama, Y., Akira, S., and Kishimoto, T. (2001). Signal transducer and

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 111 activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1)/suppressor of cytokine signaling 1 (SOCS1) inhibits insulin signal transduction pathway through modulating insulin receptor substrate 1 (IRS-1) phosphorylation. J. Exp. Med. 193, 263–269. Kile, B. T., Schulman, B. A., Alexander, W. S., Nicola, N. A., Martin, H. M., and Hilton, D. J. (2002). The SOCS box: A tale of destruction and degradation. Trends Biochem. Sci. 27, 235–241. Kimura, A., Kinjyo, I., Matsumura, Y., Mori, H., Mashima, R., Harada, M., Chien, K. R., Yasukawa, H., and Yoshimura, A. (2004). SOCS3 is a physiological negative regulator for granulopoiesis and granulocyte colony-stimulating factor receptor signaling. J. Biol. Chem. 279, 6905–6910. Kinjyo, I., Hanada, T., Inagaki-Ohar, K., Mori, H., Aki, D., Ohishi, M., Yoshida, H., Kubo, M., and Yoshura, A. (2002). SOCS1/JAB is a negative regulator of LPS-induced macrophage activation. Immunity 17, 583–591. Kobayashi, K., Hernandez, l. D., Galan, J. E., Janeway, C. A. J., Medzhitov, R., and Flavell, R. A. (2002a). IRAK-M is a negative regulator of Toll-like receptor signaling. Cell 110, 191–202. Kobayashi, K., Inohara, N., Hernandez, L. C., Galan, J. E., Nunez, G., Janeway, C. A., Medzhitov, R., and Flavell, R. A. (2002b). RICK/Rip2/CARDIAK mediates signaling for receptors of the innate and adaptive immune systems. Nature 416, 194–199. Krebs, D. L., Metcalf, D., Merson, T. D., Voss, A. K., Thomas, T., Zhang, J. G., Rakar, S., O’Bryan, M. K., Willson, T. A., Viney, E. M., Mielke, L. A., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (2004). Development of hydrocephalus in mice lacking SOCS7. Proc. Natl. Acad. Sci. USA 101, 15446–15451. Krebs, D. L., Uren, R. T., Metcalf, D., Rakar, S., Zhang, J. G., Starr, R., De Souza, D. P., Hanzinikolas, K., Eyles, J., Connolly, L. M., Simpson, R. J., Nicola, N. A., Nicholson, S. E., Baca, M., Hilton, D. J., and Alexander, W. S. (2002). Mol. Cell. Biol. 22, 4567–4578. Krieg, A. M. (2002). CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20, 709–760. Kubo, M., Hanada, T., and Yoshimura, A. (2003). Suppressors of cytokine signaling and immunity. Nat. Immunol. 4, 1169–1176. Kurt-Jones, E. A., Mandell, L., Whitney, C., Padgett, A., Gosselin, K., Newburger, P. E., and Finberg, R. W. (2002). Role of toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils. Blood 100, 1860–1868. Lang, R., Pauleau, A. L., Parganas, E., Takahashi, Y., Mages, J., Ihle, J. N., Rutschman, R., and Murray, P. J. (2003). SOCS3 regulates the plasticity of gp130 signaling. Nat. Immunol. 4, 546–550. Leadbetter, E. A., Rifkin, I. R., Hohlbaum, A. M., Beaudette, B. C., Shlomchik, M. J., and Marshal-Robhstein, A. (2002). Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603–607. Lee, J. Y., Sohn, K. H., Rhee, S. H., and Gwang, D. (2001). Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4. J. Biol. Chem. 276, 16683–16689. Lehner, M. D., Morath, S., Michelsen, K. S., Schumann, R. R., and Hartung, T. (2001). Induction of cross-tolerance by lipopolysaccharide and highly purified lipoteichoic acid via different Tolllike receptors independent of paracrine mediators. J. Immunol. 166, 5161–5167. Leonard, W. J., and O’Shea, J. J. (1998). Jaks and STATs: Biological implications. Annu. Rev. Immunol. 16, 293–322. Li, L., Gronning, L. M., Anderson, P. O., Li, S., Edvardsen, K., Johnston, J., Kioussis, D., Shepherd, P. R., and Wang, P. (2004). Insulin induces SOCS-6 expression and its binding to the p85 monomer of phosphoinositide 3-kinase, resulting in improvement in glucose metabolism. J. Biol. Chem. 279, 34107–34114.

112

TETSUJI N AKA ET A L .

Li, S., Chen, S., Xu, X., Sundstedt, A., Paulsson, K. M., Anderson, P., Karlsson, S., Sjogren, H. O., and Wang, P. (2000). Cytokine-induced Src homology 2 protein (CIS) promotes T cell receptormediated proliferation and prolongs survival of activated T cells. J. Exp. Med. 191, 985–994. Li, S., Strelow, A., Fontana, E. J., and Weshe, H. (2002). IRAK-4: A novel member of the IRAK family with the properties of an IRAK-kinase. Proc. Natl. Acad. Sci. USA 99, 5567–5572. Lien, E., and Ingalls, R. R. (2002). Toll-like receptors. Crit. Care Med. 30, S1–S11. Lien, E., Sellati, T. J., Yoshimura, A., Flo, T. H., Rawadi, G., Finberg, R. W., Carroll, J. D., Espevik, T., Ingalls, R. R., Radolf, J. D., and Golenbock, D. T. (1999). Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J. Biol. Chem. 274, 33419–33425. Lindeman, G. J., Wittlin, S., Lada, H., Naylor, M. J., Santamaria, M., Zhang, J. G., Starr, R., Hilton, D. J., Alexander, W. S., Ormandy, C. J., and Visvader, J. (2001). SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice. Genes Dev. 15, 1631–1636. Liu, B., Mink, S., Wong, K. A., Stein, N., Getman, C., Dempsey, P. W., Wu, H., and Shuai, K. (2004). PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity. Nat. Immunol. 5, 891–898. Liu, T. C., Lin, S. F., Chang, J. G., Yang, M. Y., Hung, S. Y., and Chang, C. S. (2003). Epigenetic alteration of the SOCS1 gene in chronic myeloid leukaemia. Br. J. Haematol. 123, 654–661. Malley, R., Henneke, P., Morse, S. C., Cieslewicz, M. J., Lipsitch, M., Thompson, C. M., KurtJones, E., Paton, J. C., Wessels, M. R., and Golenbock, D. T. (2003). Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection. Proc. Natl. Acad. Sci. USA 100, 1966–1971. Mao, Y., Ling, P. R., Fitzgibbons, T. P., McCowen, K. C., Frick, G. P., Bistrian, B. R., and Smith, R. J. (1999). Endotoxin-induced inhibition of growth hormone receptor signaling in rat liver in vivo. Endocrinology 140, 5505–5515. Marine, J. C., McKay, C., Wang, D., Topham, D. J., Parganas, E., Nakajima, H., Pendeville, H., Yasukawa, H., Sasaki, A., Yoshimura, A., and Ihle, J. N. (1999a). SOCS3 is essential in the regulation of fetal liver erythropoiesis. Cell 98, 617–627. Marine, J. C., Topham, D. J., McKay, C., Wang, D., Parganas, E., Stravopodis, D., Yoshimura, A., and Ihle, J. N. (1999b). SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell 98, 609–616. Masuda, A., Yoshikai, Y., Aiba, K., and Matsuguchi, T. (2002). J. Immunol. 169, 3801. Matsumoto, A., Masuhara, M., Mitsui, K., Yokouchi, M., Ohtsubo, M., Misawa, H., Miyajima, A., and Yoshimura, A. (1997). Th2 cytokine production from mast cells is directly induced by lipopolysaccharide and distinctly regulated by c-Jun N-terminal kinase and p38 pathways. Blood 89, 3148–3810. Matsumoto, A., Seki, Y., Kubo, M., Ohtsuka, S., Suzuki, A., Hayashi, I., Tsuji, K., Nakahata, T., Okabe, M., Yamada, S., and Yoshimura, A. (1999). Suppression of STAT5 functions in liver, mammary glands, and T cells in cytokine-inducible SH2-containing protein 1 transgenic mice. Mol. Cell. Biol. 19, 6396–6407. Matsumoto, A., Seki, Y., Watanabe, R., Hayashi, K., Johnston, J. A., Harada, Y., Abe, R., Yoshimura, A., and Kubo, M. (2003). A role of suppressor of cytokine signaling 3 (SOCS3/CIS3/SSI3) in CD28-mediated interleukin 2 production. J. Exp. Med. 197, 425–436. Matsushima, H., Yamada, N., Matsue, H., and Shimada, S. (2004). TLR3-, TLR7-, and TLR9mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. J. Immunol. 173, 531–541.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 113 Matuoka, K., Miki, H., Takahashi, K., and Takenawa, T. (1997). A novel ligand for an SH3 domain of the adaptor protein Nck bears an SH2 domain and nuclear signaling motifs. Biochem. Biophys. Res. Commun. 239, 488–492. McCurdy, J. D., Olynych, T. J., Maher, L. H., and Marshall, J. S. (2003). Cutting edge: Distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells. J. Immunol. 170, 1625–1629. McWhirter, S. M., Fitzgerald, K. A., Rosains, J., Rowe, D. C., Golenbock, D. T., and Maniatis, T. (2004). IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc. Natl. Acad. Soci. USA 101, 233–238. Medvedev, A. E., Henneke, P., Schromm, A., Lien, E., Ingalls, R., Fenton, M. J., Golenbock, D. T., and Vogel, S. N. (2001). Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4. J. Immunol. 167, 2257–2267. Medvedev, A. E., Lentschat, A., Wahl, L. M., Golenbock, D. T., and Vogel, S. N. (2002). Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells. J. Immunol. 169, 5209–5216. Medzhitov, R., Preston-Hurburt, P., and Janeway, C. A. J. (1998). Innate immunity: The virtues of a nonclonal system of recognition. Cell 91, 295–298. Mengozzi, M., and Ghezzi, P. (1993). Cytokine down-regulation in endotoxin tolerance. Eur. Cytokine Netw. 4, 89–98. Metcalf, D., Greenhalgh, C. J., Viney, E., Willson, T. A., Starr, R., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (2000). Gigantism in mice lacking suppressor of cytokine signaling-2. Nature 405, 1069–1073. Metcalf, D., Mifsud, S., Di Rago, L., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (2002). Polycystic kidneys and chronic inflammatory lesions are the delayed consequences of loss of the suppressor of cytokine signaling-1 (SOCS-1). Proc. Natl. Acad. Sci. USA 99, 943–948. Meylan, E., Burns, K., Hofmann, K., Blanchesteau, V., Martinon, F., Kelliher, M., and Tschopp, J. (2004). RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation. Nat. Immunol. 5, 503–507. Michalopoulos, G. K., and DeFrances, M. C. (1997). Liver regeneration. Science 276, 60–66. Michelsen, K. S., Wong, M. H., Shah, P. K., Zhang, W., Yano, J., Doherty, T. M., Akira, S., Rajavashisth, T. B., and Arditi, M. (2004). Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Proc. Natl. Acad. Sci. USA 101, 10679–10684. Michelson, K. S., Aicher, A., Mohaupt, M., Hartung, T., Dimmeler, S., Kirschning, C. J., and Schumann, R. R. (2001). The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2. J. Biol. Chem. 276, 25680–25686. Miura, Y., Miyake, K., Yamashita, Y., Shimazu, R., and Copeland, N. G. (1996). Molecular cloning of a human RP105 homologue and chromosomal localization of the mouse and human RP105 genes (Ly64 and LY64). Genomics 38, 299–304. Miura, Y., Shimazu, R., Miyake, K., Akashi, S., Ogata, H., Yamashita, Y., Narisawa, Y., and Kimoto, M. (1998). RP105 is associated with MD-1 and transmits an activation signal in human B cells. Blood 92, 2815–2822. Miyake, K., Shimazu, R., Kondo, J., Niki, T., Akashi, S., Ogata, H., Yamashita, Y., Miura, Y., and Kimoto, M. (1998). Mouse MD-1, a molecule that is physically associated with RP105 and positively regulates its expression. J. Immunol. 161, 1348–1353.

114

TETSUJI N AKA ET A L .

Miyake, K., Yamashita, Y., Hitoshi, Y., Takatsu, K., and Kimoto, M. (1994). Murine B cell proliferation and protection from apoptosis with an antibody against a 105-kD molecule: Unresponsiveness of X-linked immunodeficient B cells. J. Exp. Med. 180, 1217–1224. Miyake, K., Yamashita, Y., Ogata, M., Sudo, T., and Kimoto, M. (1995). RP105, a novel B cell surface molecule implicated in B cell activation, is a member of the leucine-rich repeat protein family. J. Exp. Med. 154, 3333–3340. Mizel, S. B., and Snipes, J. A. (2002). Gram-negative flagellin-induced self-tolerance is associated with a block in interleukin-1 receptor-associated kinase release from toll-like receptor 5. J. Biol. Chem. 277, 22414–22420. Monick, M. M., Yarovinsky, T. O., Powers, L. S., Butler, N. S., Brent Carter, A., Gudmundsson, G., and Hunninghake, G. W. (2003). Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin. J. Biol. Chem. 278, 53035–53044. Monni, R., Santos, S. C., Mauchauffe, M., Berger, R., Ghysdael, J., Gouilleux, F., Gisselbrecht, S., Bernard, O., and Penard-Lacronique, V. (2001). The TEL-Jak2 oncoprotein induces Socs1 expression and altered cytokine response in Ba/F3 cells. Oncogene 20, 849–858. Mooney, R. A., Senn, J., Cameron, S., Inamdar, N., Boivin, L. M., Shang, Y., and Furlanetto, R. W. (2001). Suppressors of cytokine signaling-1 and -6 associate with and inhibit the insulin receptor. A potential mechanism for cytokine-mediated insulin resistance. J. Biol. Chem. 276, 25889–25893. Mori, H., Hanada, R., Hanada, T., Aki, D., Mashima, R., Nishinakamura, H., Torisu, T., Chien, K. R., Yasukawa, H., and Yoshimura, A. (2004). Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity. Nat. Med. 10, 739–743. Morita, Y., Naka, T., Kawazoe, Y., Fujimoto, M., Narazaki, M., Nakagawa, R., Fukuyama, H., Nagata, S., and Kishimoto, T. (2000). Signals transducers and activators of transcription (STAT)induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor alpha-induced cell death in fibroblasts. Proc. Natl. Acad. Sci. USA 97, 5405–5410. Muzio, M., Bosisio, D., Polentarutti, N., D’amico, G., Stoppacciaro, A., Mancinelli, R., van’t Veer, C., Penton-Rol, G., Ruco, L. P., Allavena, P., and Mantovani, A. (2000). Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: Selective expression of TLR3 in dendritic cells. J. Immunol. 164, 5998–6004. Myers, M. P., Andersen, J. N., Cheng, A., Tremblay, M. L., Horvath, C. M., Parisien, J. P., Salmeen, A., Barford, D., and Tonks, N. K. (2001). TYK2 and JAK2 are substrates of protein-tyrosine phosphatase 1B. J. Biol. Chem. 276, 47771–47774. Nagai, H., Kim, Y. S., Konishi, N., Baba, M., Kubota, T., Yoshimura, A., and Emi, M. (2002a). Combined hypermethylation and chromosome loss associated with inactivation of SSI-1/SOCS1/JAB gene in human hepatocellular carcinomas. Cancer Lett. 186, 59–65. Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y., Akira, S., Kitamura, T., Kosugi, A., Kimoto, M., and Mioyake, K. (2002b). Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat. Immunol. 3, 667–672. Nagai, Y., Shimazu, R., Ogata, H., Akashi, S., Sudo, K., Yamasaki, H., Hayashi, S., Iwakura, Y., Kimoto, M., and Miyake, K. (2002c). Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide. Blood 99, 1699–1705. Naka, T., Fujimoto, M., and Kishimoto, T. (1999). Negative regulation of cytokine signaling: STATinduced STAT inhibitor. Trends Biochem. Sci. 24, 394–398. Naka, T., Matsumoto, T., Narazaki, M., Fujimoto, M., Morita, Y., Ohsawa, Y., Saito, H., Nagasawa, T., Uchiyama, Y., and Kishimoto, T. (1998). Accelerated apoptosis of lymphocytes by augmented induction of Bax in SSI-1 (STAT-induced STAT inhibitor-1) deficient mice. Proc. Natl. Acad. Sci. USA 95, 15577–15582.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 115 Naka, T., Narazaki, M., Hirata, M., Matsumoto, T., Minamoto, S., Aono, A., Nishimoto, N., Kajita, T., Taga, T., Yoshizaki, K., Akira, S., and Kishimoto, T. (1997). Structure and function of a new STAT-induced STAT inhibitor. Nature 387, 924–929. Naka, T., Tsutsui, H., Fujimoto, M., Kawazoe, Y., Kohzaki, H., Morita, Y., Nakagawa, R., Narazaki, M., Adachi, K., Yoshimoto, T., Nakanishi, K., and Kishimoto, T. (2001). SOCS-1/SSI-1-deficient NKT cells participate in severe hepatitis through dysregulated cross-talk inhibition of IFNgamma and IL-4 signaling in vivo. Immunity 14, 535–545. Nakagawa, R., Naka, T., Tsutsui, H., Fujimoto, M., Kimura, A., Abe, T., Seki, E., Sato, S., Takeuchi, O., Takeda, K., Akira, S., Yamanishi, K., Kawase, I., Nakanishi, K., and Kishimoto, T. (2002a). SOCS-1 participates in negative regulation of LPS responses. Immunity 17, 677–687. Narazaki, M., Fujimoto, M., Matsumoto, T., Morita, Y., Saito, H., Kajita, T., Yoshizaki, K., Naka, T., and Kishimoto, T. (1998). Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling. Proc. Natl. Acad. Sci. USA 95, 13130–13134. Netea, M. G., Van der Graaf, C. A. A., Vonk, A. G., Berschueren, I., Van der Meer, J. W. M., and Kullberg, B. J. (2002). The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis. J. Infect. Dis. 185, 1483–1489. Nicholson, S. E., De Souza, D., Fabri, L. J., Corbin, J., Willson, T. A., Zhang, J. G., Silva, A., Asimakis, M., Farley, A., Nash, A. D., Metcalf, D., Hilton, D. J., Nicola, N. A., and Baca, M. (2000). Suppressor of cytokine signaling-3 preferentially binds to the SHP-2-binding site on the shared cytokine receptor subunit gp130. Proc. Natl. Acad. Sci. USA 97, 6493–6498. Nicholson, S. E., Willson, T. A., Farley, A., Starr, R., Zhang, J. G., Baca, M., Alexander, W. S., Metcalf, D., Hilton, D. J., and Nicola, N. A. (1999). Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. EMBO J. 18, 375–385. Nomura, F., Akashi, S., Sakao, Y., Sato, S., Kawai, T., Matsumoto, M., Nakanishi, K., Kimoto, M., Miyake, K., Takeda, K., and Akira, S. (2000). Cutting edge: Endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression. J. Immunol. 164, 3476–3479. O’Neill, L. A. J. (2000). The Toll/interleukin-1 receptor domain: A molecular switch for inflammation and host defence. Biochem. Sic. Transac. 28, 557–563. O’Neill, L. A. J. (2002). Toll-like receptor signal transduction and the tailoring of innate immunity: A role for Mal? Trends Immunol. 23, 296–300. O’Neill, L. A. J., and Dinarello, C. A. (2000). The IL-1 receptor/toll-like receptor superfamily: Crucial receptors for inflammation and host defense. Immunol. Today 21, 206–209. O’Neill, L. A. J., Fitzgerald, K. A., and Bowie, A. G. (2003). The Toll-IL-1 receptor adaptor family grows to five members. Trends Immunol. 2003, 286–290. O’Neill, L. A. J., and Greene, C. (1998). Signal transduction pathways activated by the IL-1 receptor family: Ancient signaling machinery in mammals, insects, and plants. J. Leukoc. Biol. 63, 650–657. O’Shea, J. J., Gadina, M., and Schreiber, R. D. (2002). Cytokine signaling in 2002: New surprises in the Jak/Stat pathway. Cell 109(Suppl.), S121–S131. Ogata, H., Su, I., Miyake, K., Nagai, T., Akashi, S., Mecklenbrauker, R., Rajewski, K., Kimoto, M., and Tarakhovsky, A. (2000). The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells. J. Exp. Med. 192, 23–29. Ogawa, T., Asai, Y., Hashimoto, M., Takeuchi, O., Kurita, T. Y. Y., Miyake, K., and Akira, S. (2002). Cell activation by Porphyromonas gingivalis lipid A molecule through Toll-like receptor 4- and myeloid differentiation factor 88-dependent signaling pathway. Int. Immunol. 14, 1325–1332.

116

TETSUJI N AKA ET A L .

Ogura, Y., Inohara, N., Benito, A., Chen, F. F., Yamaoka, S., and Nunez, G. (2001). Nod2, a Nod1/ Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J. Biol. Chem. 276, 4812–4818. Ohashi, K., Burkart, V., Flohe, S., and Kolb, H. (2000). Cutting edge: Heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 164, 558–561. Ohya, K., Kajigaya, S., Yamashita, Y., Miyazato, A., Hatake, K., Miura, Y., Ikeda, U., Shimada, K., Ozawa, K., and Mano, H. (1997). SOCS-1/JAB/SSI-1 can bind to and suppress Tec proteintyrosine kinase. J. Biol. Chem. 272, 27178–27182. Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T., and Seya, T. (2003). TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat. Immunol. 4, 161–167. Ozinsky, A., Underhill, D. M., Fontenot, J. D., Hajjar, A. M., Smith, K. D., Wilson, C. B., Schroeder, L., and A. A. (2000). The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc. Natl. Acad. Sci. USA 97, 13766–13771. Perera, P.-Y., Mayadas, T. N., Takeuchi, O., Akira, S., Zaks-Zilberman, M., Goyert, S. M., and Vogel, S. N. (2001). CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression. J. Immunol. 166, 574–581. Perry, A. K., Chow, E. K., Goodnough, J. B., Yeh, W.-C., and Cheng, G. (2004). Differential requirement for TANK-binding kinase-1 in type I interferon responses to toll-like receptor activation and viral infection. J. Exp. Med. 199, 1651–1658. Picard, C., Puel, A., Bonnet, M., Ku, C.-L., Bustamante, J., Yang, K., Soudais, C., Dupuis, S., Feinberg, J., Fieschi, C., Elbim, C., Hitchcock, R., Lammas, D., Dacies, G., Al-Ghonaium, A., Al-Rayes, H., Al-Jumaah, S., Al-Hijjar, S., Zaid Al-Mohsen, I., Frayha, H. H., Rucker, R., Hawn, T. R., A. A., Tufenkeji, H., Haraguchi, S., Day, N. K., Golld, R. A., Gougerot-Pocidalo, M.-A., Ozinsky, A., and Casanova, J.-L. (2003). Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 299, 2076–2079. Poltorak, A., He, X., Smirnova, I., Liu, M.-Y., Van Guffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galonos, C., Freudenberg, M., Ricciarde-Castagnoli, P., Layton, B., and Beutler, B. (1998). Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene. Science 282, 2085–2088. Qureshi, M. H., Zhang, T., Koguchi, Y., Nakashima, K., Okamura, H., Kurimoto, M., and Kawakami, K. (1999). Combined effects of IL-12 and IL-18 on the clinical course and local cytokine production in murine pulmonary infection with Cryptococcus neoformans. Eur. J. Immunol. 29, 643–649. Raetz, C. R., and Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S., and Medzhitov, R. (2004). Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241. Ram, P. A., and Waxman, D. J. (1999). SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms. J. Biol. Chem. 274, 35553–35561. Ram, P. A., and Waxman, D. J. (2000). Role of the cytokine-inducible SH2 protein CIS in desensitization of STAT5b signaling by continuous growth hormone. J. Biol. Chem. 275, 39487–39496. Reiling, N., Holscher, C., Fehrenbach, A., Kroger, S., Kirschning, C. J., Goyert, S., and Ehlers, S. (2002). Cutting edge: Toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne infection with Mycobacterium tuberculosis. J. Immunol. 169, 3480–3484.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 117 Rissoan, M.-C., Soumelis, V., Kadowaki, N., Grouard, G., Briere, F., de Waal Malefyt, R., and Liu, Y.-J. (1999). Reciprocal control of T helper cell and dendritic cell differentiation. Science 283, 1183–1186. Roberts, A. W., Robb, L., Rakar, S., Hartley, L., Cluse, L., Nicola, N. A., Metcalf, D., Hilton, D. J., and Alexander, W. S. (2001). Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3. Proc. Natl. Acad. Sci. USA 98, 9324–9329. Rottapel, R., Ilangumaran, S., Neale, C., La Rose, J., Ho, J. M., Nguyen, M. H., Barber, D., Dubreuil, P., and de Sepulveda, P. (2002). The tumor suppressor activity of SOCS-1. Oncogene 21, 4351–4362. Rui, L., Yuan, M., Frantz, D., Shoelson, S., and White, M. F. (2002). SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. J. Biol. Chem. 277, 42394–42398. Ryo, A., Suizu, F., Yoshida, Y., Perrem, K., Liou, Y. C., Wulf, G., Rottapel, R., Yamaoka, S., and Lu, K. P. (2003). Regulation of NF-kappaB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol. Cell. 12, 1413–1426. Sabroe, I., Jones, E. C., Usher, L. R., Whyte, M. K. B., and Dower, S. K. (2002). Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: A critical role for monocytes in leukocyte lipopolysaccharide responses. J. Immunol. 168, 4701–4710. Saito, H., Morita, Y., Fujimoto, M., Narazaki, M., Naka, T., and Kishimoto, T. (2000). IFN regulatory factor-1-mediated transcriptional activation of mouse STAT-induced STAT inhibitor1 gene promoter by IFN-gamma. J. Immunol. 164, 5833–5843. Sakai, I., Takeuchi, K., Yamauchi, H., Narumi, H., and Fujita, S. (2002). Constitutive expression of SOCS3 confers resistance to IFN-alpha in chronic myelogenous leukemia cells. Blood 100, 2926–2931. Sasaki, A., Yasukawa, H., Shouda, T., Kitamura, T., Dikic, I., and Yoshimura, A. (2000). CIS3/ SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2. J. Biol. Chem. 275, 29338–29347. Sato, S., Nomura, F., Kawai, T., Takeuchi, O., Muhlradt, P. F., Takeda, K., and Akira, S. (2000). Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways. J. Immunol. 165, 7096. Sato, S., Takeuchi, O., Fujita, T., Tomizawa, H., Takeda, K., and Akira, S. (2002). A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88dependent and -independent pathways. Int. Immunol. 14, 783–791. Scanga, C. A., Aliberti, J., Jankovic, D., Tilloy, F., Bennouna, S., Denkers, E. Y., Medzhitov, R., and Sher, A. (2002). Cutting edge: MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J. Immuinol. 168, 5997–6001. Schaefer, F., Chen, Y., Tsao, T., Nouri, P., and Rabkin, R. (2001). Impaired JAK-STAT signal transduction contributes to growth hormone resistance in chronic uremia. J. Clin. Invest. 108, 467–475. Schluter, G., Boinska, D., and Nieman-Seyde, S. C. (2000). Evidence for translational repression of the SOCS-1 major open reading frame by an upstream open reading frame. Biochem. Biophys. Res. Commun. 268, 255–261. Schmitz, J., Weissenbach, M., Haan, S., Heinrich, P. C., and Schaper, F. (2000). SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130. J. Biol. Chem. 275, 12848–12856. Schultheis, B., Carapeti-Marootian, M., Hochhaus, A., Weisser, A., Goldman, J. M., and Melo, J. V. (2002). Overexpression of SOCS-2 in advanced stages of chronic myeloid leukemia: Possible inadequacy of a negative feedback mechanism. Blood 99, 1766–1775.

118

TETSUJI N AKA ET A L .

Schwandner, R., Dziarski, R., Wesche, H., Rothe, M., and Kirschning, C. J. (1999). Peptidoglycanand lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J. Biol. Chem. 274, 17406–17409. Seki, E., Tsutsui, H., Iimuro, Y., Nak, T., Son, G., Akira, S., Kishimoto, T., Nakanishi, K., and Fujimoto, J. (2005). Contribution of Toll-like receptor/Myeloid differentiation factor 88 signaling of murine liver regeneration. Hepatology 41, 443–450. Seki, E., Tsutsui, H., Tsuji, N. M., Hayashi, N., Adachi, K., Nakano, H., Futatsugi-Yumikura, S., Takeuchi, O., Hoshino, K., Akira, S., Fujimoto, J., and Nakanishi, K. (2002a). Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice. J. Immunol. 169, 3863–3868. Seki, Y., Hayashi, K., Matsumoto, A., Seki, N., Tsukada, J., Ransom, J., Naka, T., Kishimoto, T., Yoshimura, A., and Kubo, M. (2002b). Expression of the suppressor of cytokine signaling-5 (SOCS5) negatively regulates IL-4-dependent STAT6 activation and Th2 differentiation. Proc. Natl. Acad. Sci. USA 99, 13003–13008. Seki, Y., Inoue, H., Nagata, N., Hayashi, K., Fukuyama, S., Matsumoto, K., Komine, O., Hamano, S., Himeno, K., Inagaki-Ohara, K., Cacalano, N., O’Garra, A., Oshida, T., Saito, H., Johnston, J. A., Yoshimura, A., and Kubo, M. (2003). SOCS-3 regulates onset and maintenance of T(H)2mediated allergic responses. Nat. Med. 9, 1047–1054. Senn, J. J., Klover, P. J., Nowak, I. A., Zimmers, T. A., Koniaris, L. G., Furlanetto, R. W., and Mooney, R. A. (2003). Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator of interleukin-6-dependent insulin resistance in hepatocytes. J. Biol. Chem. 278, 13740–13746. Sharma, S., ten Oever, B. R., Grandvaux, N., Shou, G.-P., Lin, R., and Hiscott, J. (2003). Triggering the interferon antiviral response through an IKK-related pathway. Science 300, 1148–1151. Shen, L., Evel-Kabler, K., Strube, R., and Chen, S. Y. (2004). Silencing of SOCS1 enhances antigen presentation by dendritic cells and antigen-specific anti-tumor immunity. Nat. Biotechnol. 22, 1546–1553. Shi, H., Tzameli, I., Bjorbaek, C., and Flier, J. S. (2004). Suppressor of cytokine signaling 3 is a physiological regulator of adipocyte insulin signaling. J. Biol. Chem. 279, 34733–34740. Shouda, T., Yoshida, T., Hanada, T., Wakioka, T., Oishi, M., Miyoshi, K., Komiya, S., Kosai, K., Hanakawa, Y., Hashimoto, K., Nagata, K., and Yoshimura, A. (2001). Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis. J. Clin. Invest. 108, 1781–1788. Shultz, L. D., Rajan, T. V., and Greiner, D. L. (1997). Severe defects in immunity and hematopoiesis caused by SHP-1 protein-tyrosine-phosphatase deficiency. Trends Biotechnol. 15, 302–307. Simoncic, P. D., Lee-Loy, A., Barber, D. L., Tremblay, M. L., and McGlade, C. J. (2002). The T cell protein tyrosine phosphatase is a negative regulator of janus family kinases 1 and 3. Curr. Biol. 12, 446–453. Smith, M. F. J., Mitchell, A., Li, G., Ding, S., Fitzmaurice, A. M., Ryan, K., Crowe, S., and Goldberg, J. B. (2003). Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pylori-induced NF-kappa B activation and chemokine expression by epithelial cells. J. Biol. Chem. 278, 32552–32560. Starr, R., Metcalf, D., Elefanty, A. G., Brysha, M., Willson, T. A., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (1998). Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1. Proc. Natl. Acad. Sci. USA 95, 14395–14399. Starr, R., Willson, T. A., Viney, E. M., Murray, L. J., Rayner, J. R., Jenkins, B. J., Gonda, T. J., Alexander, W. S., Metcalf, D., Nicola, N. A., and Hilton, D. J. (1997). A family of cytokineinducible inhibitors of signaling. Nature 387, 917–921.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 119 Stoiber, D., Kovarik, P., Cohney, S., Johnston, J. A., Steinlein, P., and Decker, T. (1999). Lipopolysaccharide induces in macrophages the synthesis of the suppressor of cytokine signaling 3 and suppresses signal transduction in response to the activating factor IFN-gamma. J. Immunol. 163, 2640–2647. Strober, W., Fuss, I. J., and Blumberg, R. S. (2002). The immunology of mucosal models of inflammation. Annu. Rev. Immunol. 20, 495–549. Supajatura, V., Ushio, H., Nakao, A., Akira, S., Okumura, K., Ra, C., and Ogawa, H. (2002). Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J. Clin. Invest. 109, 1351–1359. Supajatura, V., Ushio, H., Nakao, A., Okurura, K., Ra, C., and Ogawa, H. (2001). Protective roles of mast cells against enterobacterial infection are mediated by Toll-like receptor 4. J. Immunol. 167, 2250–2256. Suzuki, A., Hanada, T., Mitsuyama, K., Yoshida, T., Kamizono, S., Hoshino, T., Kubo, M., Yamashita, A., Okabe, M., Takeda, K., Akira, S., Matsumoto, S., Toyonaga, A., Sata, M., and Yoshimura, A. (2001). CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation. J. Exp. Med. 193, 471–481. Suzuki, N., Suzuki, S., Duncan, G. S., Millar, D. G., Wada, T., Mirtsos, C., Takada, H., Wakeham, A., Itie, A., Li, S., Penninger, J. M., Wesche, H., Ohashi, P. S., Mak, T. W., and Yen, W.-C. (2002a). Severe impairment of interleukin-1 and Toll-like receptor signaling in mice lacking IRAK-4. Nature 416, 750–756. Suzuki, N., Suzuki, S., and Yeh, W.-C. (2002b). IRAK-4 as the central TIR signaling mediator in innate immunity. Trends Immunol. 23, 503–506. Takahashi, Y., Carpino, N., Cross, J. C., Torres, M., Parganas, E., and Ihle, J. N. (2003). SOCS3: An essential regulator of LIF receptor signaling in trophoblast giant cell differentiation. EMBO J. 22, 372–384. Takami, M., Kim, N., Rho, J., and Choi, Y. (2002). Stimulation by toll-like receptors inhibits osteoclast differentiation. J. Immunol. 169, 1516–1523. Takeda, K., Clausen, B. E., Kaisho, T., Tsujimura, T., Terada, N., Forster, I., and Akira, S. (1999). Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10, 39–49. Takeda, K., Kaisho, T., and Akira, S. (2003). Toll-like receptors. Annu. Rev. Immunol. 21, 335–376. Takeuchi, O., Hoshino, K., and Akira, S. (2000). Cutting edge: TLR2-deficient and MyD88deficient mice are highly susceptible to Staphylococcus aureus infection. J. Immunol. 165, 5392–5396. Takeuchi, O., Kaufmann, A., Grote, K., Kawai, T., Hoshino, K., Morr, M., Muhlradt, P. F., and Akira, S. (2000). Cutting edge: Preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway. J. Immunol. 164, 554–557. Takeuchi, O., Kawai, T., Muhlradt, P. F., Morr, M., Radolf, J. D., Zychlinsky, A., Takeda, K., and Akira, K. (2001). Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13, 933–940. Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., Modlin, R., and Akira, S. (2002). Cutting edge: Role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169, 10–14. Taylor, K. R., Trowbridge, J. M., Rudisill, J. A., Termeer, C. C., Simon, J. C., and Gallo, R. L. (2004). Hyaluronan fragments stimulate endothelial recognition of injury through TLR4. J. Biol. Chem. 279, 17079–17084.

120

TETSUJI N AKA ET A L .

Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, K., Freudenberg, M., Galanos, C., and Simon, J. C. (2002). Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195, 99–111. Thomas, J. A., Allen, J. L., Tsen, M., Dubnicoff, T., Danao, J., Liao, X. C., Cao, Z., and Wasserman, S. A. (1999). Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase. J. Immunol. 163, 978–984. Tomassen, E., Renshaw, B. R., and Sims, J. E. (1999). Identification and characterization of SIGIRR, a molecule representing a novel subtype of the IL-1R superfamily. Cytokine 11, 389–399. Toniato, E., Chen, X. P., Losman, J., Flati, V., Donahue, L., and Rothman, P. (2002). TRIM8/GERP RING finger protein interacts with SOCS-1. J. Biol. Chem. 277, 37315–37322. Tschopp, J., Martinon, F., and Burns, K. (2003). NALPs: A novel protein family involved in inflammation. Nat. Rev. 4, 95–104. Tsuji, S., Matsumoto, M., Takeuchi, O., Akira, S., Azuma, I., Hayashi, A., Toyoshima, K., and Seya, T. (2000). Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guerin: Involvement of toll-like receptors. Infect. Immun. 68, 6883–6890. Turnley, A. M., Faux, C. H., Rietze, R. L., Coonan, J. R., and Bartlett, P. F. (2002). Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling. Nat. Neurosci. 5, 1155–1162. Ueki, K., Kondo, T., Tseng, Y. H., and Kahn, C. R. (2004). Central role of suppressors of cytokine signaling proteins in hepatic steatosis, insulin resistance, and the metabolic syndrome in the mouse. Proc. Natl. Acad. Sci. USA 101, 10422–10427. Vabulas, R. M., Ahmad-Nejad, P., da Costa, C., Miethke, T., Kirschning, C. J., Hacker, H., and Wagner, H. (2001). Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J. Biol. Chem. 276, 31332–31339. Vabulas, R. M., Ahmad-Nejad, P., Ghose, S., Kirschning, C. J., Issels, R. D., and Wagner, H. (2002). HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277, 15107–15112. Verdier, F., Chretien, S., Muller, O., Varlet, P., Yoshimura, A., Gisselbrecht, S., Lacombe, C., and Mayeux, P. (1998). Proteasomes regulate erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) activation. Possible involvement of the ubiquitinated Cis protein. J. Biol. Chem. 273, 28185–28190. Viglianti, G. A., Lau, C. M., Hanley, T. M., Miko, B. A., Shiomchik, M. J., and Marshak-Rothestein, A. (2003). Activation of autoreactive B cells by CpG dsDNA. Immunity. 19, 837–847. Wald, D., Qin, J., Zhao, Z., Qian, Y., Naramura, M., Tian, L., Towne, J., Sims, J. E., Stark, G. R., and Li, X. (2003). SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat. Immunol. 4, 920–927. Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J., and Chen, Z. J. (2001). TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351. Wang, J.-H., Doyle, M., Manning, B. J., Wu, Q.-D., Blankson, S., and Redmond, H. P. (2002). Induction of bacterial lipoprotein tolerance is associated with suppression of toll-like receptor 2 expression. J. Biol. Chem. 277, 36068–36075. Watanabe, T., Kitani, A., Murray, P. J., and Strober, W. (2004). NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nat. Immunol. 5, 800–808. Wesche, H., Gao, X., Li, X., Kirschning, C. J., Stark, G. R., and Gao, Z. (1999). IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family. J. Biol. Chem. 274, 19403–19410.

n eg at i v e r e gu l at i on of c yt o k i n e a n d t l r s i gnal i n g s 121 Wesche, H., Henzel, W. J., Shillinglaw, W., Li, S., and Cao, Z. (1997). MyD88: An adaptor that recruits IRAK to the IL-1 receptor complex. Immunity 7, 837–847. Wong, K. A., Kim, R., Christofk, H., Gao, J., Lawson, G., and Wu, H. (2004). Protein inhibitor of activated STAT Y (PIASy) and a splice variant lacking exon 6 enhance sumoylation but are not essential for embryogenesis and adult life. Mol. Cell. Biol. 24, 5577–5586. Wormald, S., and Hilton, D. J. (2004). Inhibitors of cytokine signal transduction. J. Biol. Chem. 279, 821–824. Xia, L., Wang, L., Chung, A. S., Ivanov, S. S., Ling, M. Y., Dragoi, A. M., Platt, A., Gilmer, T. M., Fu, X. Y., and Chin, Y. E. (2002). Identification of both positive and negative domains within the epidermal growth factor receptor COOH-terminal region for signal transducer and activator of transcription (STAT) activation. J. Biol. Chem. 277, 30716–30723. Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O., Sugiyama, H., Okabe, M., Takeda, K., and Akira, S. (2003a). Role of adaptor TRIF in the MyD88independent toll-like receptor signaling pathway. Science 301, 640–643. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M. T. F., Takeda, K., and Akira, S. (2002a). Essential role for TIRAP in activation of the signaling cascade shared by TLR2 and TLR4. Nature 420, 324–329. Yamamoto, M., Sato, S., Hemmi, H., Uematsu, S., Hoshino, K., Kaisho, T., Takeuchi, O., Takeda, K., and Akira, S. (2003b). TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat. Immunol. 4, 1144–1150. Yamamoto, M., Sato, S., Mori, K., Hoshino, K., Takeuchi, O., Takeda, K., and Akira, S. (2002b). Cutting edge: A novel Toll/IL-1 receptor domain-containing adaptor that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J. Immunol. 169, 6668–6672. Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Kuwata, H., Takeuchi, O., Takeshige, K., Saitoh, T., Yamaoka, S., Yamamoto, N., Yamamoto, S., Muta, T., Takeda, K., and Akira, S. (2004). Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IkappaBzeta. Nature 430, 218–222. Yang, D., Chertov, O., Bykovskaia, S. N., Chen, Q., Buffo, M. J., Shogan, J., Anderson, M., Schroder, J. M., Wang, J. M., Howard, O. M. Z., and Oppenheim, J. J. (1999). Beta-defensins: Linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286, 525–528. Yang, Y., Huang, C.-H., Huang, X., and Pardoll, D. M. (2004). Persistent Toll-like receptor signals are required for reversal of regulatory T cell-mediated CDB tolerance. Nat. Immunol. 5, 508–515. Yasukawa, H., Hoshijima, M., Gu, Y., Nakamura, T., Pradervand, S., Hanada, T., Hanakawa, Y., Yoshimura, A., Ross, J., Jr., and Chien, K. R. (2001). Suppressor of cytokine signaling-3 is a biomechanical stress-inducible gene that suppresses gp130-mediated cardiac myocyte hypertrophy and survival pathways. J. Clin. Invest. 108, 1459–1467. Yasukawa, H., Misawa, H., Sakamoto, H., Masuhara, M., Sasaki, A., Wakioka, T., Ohtsuka, S., Imaizumi, T., Matsuda, T., Ihle, J. N., and Yoshimura, A. (1999). The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J. 18, 1309–1320. Yasukawa, H., Ohishi, M., Mori, H., Murakami, M., Chinen, T., Aki, D., Hanada, T., Takeda, K., Akira, S., Hoshijima, M., Hirano, T., Chien, K. R., and Yoshimura, A. (2003). IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunol. 4, 551–556. Yoshida, T., Ogata, H., Kamio, M., Joo, A., Shiraishi, H., Tokunaga, Y., Sata, M., Nagai, H., and Yoshimura, A. (2004). SOCS1 is a suppressor of liver fibrosis and hepatitis-induced carcinogenesis. J. Exp. Med. 199, 1701–1707.

122

TETSUJI N AKA ET A L .

Yoshikawa, H., Matsubara, K., Qian, G. S., Jackson, P., Groopman, J. D., Manning, J. E., Harris, C. C., and Herman, J. G. (2001). SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat. Genet. 28, 29–35. Yoshimura, A., Ohkubo, T., Kiguchi, T., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Hara, T., and Miyajima, A. (1995). A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors. EMBO J. 14, 2816–2826. Zhang, D., Zhang, G., Hayden, M. S., Greenblatt, M. B., Bussey, C., Flavell, R. A., and Ghosh, S. (2004). A toll-like receptor that prevents infection by uropathogenic bacteria. Science 303, 1522–1526. Zhang, J., Somani, A. K., and Siminovitch, K. A. (2000). Roles of the SHP-1 tyrosine phosphatase in the negative regulation of cell signaling. Semin. Immunol. 12, 361–378. Zhang, J. G., Farley, A., Nicholson, S. E., Willson, T. A., Zugaro, L. M., Simpson, R. J., Moritz, R. L., Cary, D., Richardson, R., Hausmann, G., Kile, B. J., Kent, S. B., Alexander, W. S., Metcalf, D., Hilton, D. J., Nicola, N. A., and Baca, M. (1999). The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proc. Natl. Acad. Sci. USA 96, 2071–2076.

Pathogenic T-Cell Clones in Autoimmune Diabetes: More Lessons from the NOD Mouse Kathryn Haskins Department of Immunology and Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center and National Jewish Medical and Research Center, Denver, Colorado 80206

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

Abstract............................................................................................................. Introduction ....................................................................................................... Pathogenic T-Cell Clones—Effector Function .......................................................... Migration of Pathogenic T-Cell Clones .................................................................... T-Cell Clones in T-Cell Receptor Transgenic (TCR-Tg) Mice ...................................... Antigens for Pathogenic T-Cell Clones .................................................................... Tracking of Pathogenic T-Cell Clones with MHC Tetramers........................................ Concluding Remarks............................................................................................ References .........................................................................................................

123 123 126 133 135 140 149 152 154

Abstract T-cell clones that can efficiently transfer diabetes to prediabetic nonobese diabetic (NOD) mice provide a powerful approach to dissecting the autoimmune disease process and for investigating immunoregulation. Diabetogenic T-cell clones carried in culture allow for detailed analysis of T-cell effector function and in vivo activity, and thus the contribution of a single clonotype to pathogenesis can be studied. As T cells comprising most or all of the repertoire in T-cell receptor transgenic (TCR-Tg) mice, diabetogenic T-cell clones have led to new variations on the NOD mouse model of autoimmune disease. T-cell clones are being used to screen peptide libraries and proteomic arrays to identify the autoantigens that drive these clones in vivo and to extend our knowledge of the processes that give rise to these antigens. With the identification of peptide agonists and natural ligands, the development of MHC-peptide multimers has been possible. These reagents can track T cells in vivo and thus provide new approaches for disease diagnosis and therapy as well as a versatile set of tools for basic research on how T cells contribute to autoimmune disease. 1. Introduction The destruction of the insulin-producing pancreatic beta cells in type 1 diabetes (T1D) occurs through an autoimmune process, as evidenced by the inflammatory infiltrate in the islets (insulitis), the presence of anti-islet antibodies, and strong genetic associations with loci of the major histocompatibility

123 advances in immunology, vol. 87 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)87004-X

124

k at h r yn h a s k i n s

complex (MHC). The selective loss of beta cells and development of disease usually in young patients (hence the original name of juvenile or childhood diabetes) sets this disease apart from the more common form of type II diabetes (T2D), also known as adult-onset and noninsulin dependent. Along with multiple sclerosis and rheumatoid arthritis, T1D is considered one of the most severely debilitating autoimmune disorders (Delovitch and Singh, 1997), and with an incidence of up to 1% in some populations (Notkins and Lernmark, 2001), is also one of the most prevalent. In the efforts to find a cure for T1D, great emphasis is being placed on islet replacement. Since it is the only procedure in which patients can become insulin independent, transplantation of healthy functioning islets into diabetic recipients is considered the closest we have to a cure for this autoimmune disease. From an immunological standpoint, autoimmunity can be considered to be synonymous with loss of tolerance to self tissue and in the case of T1D, the target is the pancreatic beta cell. Thus, the replacement of islets must be accompanied by induction of tolerance for grafts to survive. Development of procedures that result in transplantation tolerance is one of the most important research goals in this field (Rossini, 2004). Unfortunately, most forms of immunosuppressive therapy are accompanied by serious side effects, so the ultimate goal is to find a way to provide islets without lifelong immunosuppressive therapy. The use of stem cells as a source of new islets is thought to be one avenue to islet transplantation that would lessen the requirement for immunosuppression. In conjunction with the impressive gains being made in transplantation and stem cell research, however, it is still critically important to understand the underlying immunological mechanisms of autoimmunity and how to regulate this process. T cells are primary mediators of the autoimmune disease process, and therefore, investigation into what governs the activity and regulation of autoreactive T cells continues to be a high priority. Fortunately, we have been aided in these research endeavors by the availability of rodents that spontaneously develop autoimmune diabetes, and since its discovery more than 20 years ago, the NOD mouse has become the most studied animal model of T1D (Delovitch and Singh, 1997). Islet pathogenesis in the NOD mouse closely resembles the disease process in humans, and as set forth in this review, the investigation of autoreactive T cells obtained from the NOD mouse has contributed substantially to our present knowledge of the immunological mechanisms of T1D. The isolation and characterization of diabetogenic T cells from the NOD mouse has been a major objective of investigators since reports from the midto-late 1980s demonstrated an important role for T cells in pathogenesis of diabetes in the NOD and implicated both CD4 and CD8 T cells (Bendelac et al., 1987; Miller et al., 1988; Wang et al., 1987). These studies showed that depletion of either subset abrogated spontaneous disease and inhibited

path o g e ni c t- c e l l c lo n e s

125

adoptive transfers with splenocytes from diabetic mice. In the last 3–4 years, there have been a number of excellent reviews on T cells in T1D covering, for example, the genetic control of autoreactive CD4 and CD8 T cells (Serreze and Leiter, 2003), the role and therapeutic potential of autoreactive CD8 T cells (Liblau et al., 2002), the status of research on autoreactive T cells in human diabetic patients (Roep, 2002), and autoantigens in both humans and mice (Lieberman and DiLorenzo, 2003). The particular focus of this chapter will be on how T-cell clones, either carried in culture or present in transgenic mice, have contributed to recent advances in our understanding of T cells in T1D. Since autoimmune diabetes in the NOD mouse develops spontaneously and is very similar to the disease in humans, this animal model for T1D is widely used. To analyze in more detail the role of CD4 and CD8 T cells in T1D, investigators began to isolate and characterize T-cell clones from the NOD mouse. In a previous review (Bergman and Haskins, 1997), we provided a table listing the T-cell clones that had been isolated before that time, beginning with the first publication of a pathogenic T-cell clone in 1988 (Haskins et al., 1988) and ending with the first report of a pathogenic CD8 T-cell clone that could cause disease in the absence of CD4 T cells (Wong et al., 1996). Most of the T-cell clones appeared to have some pathogenic activity in vivo, at least leading to pancreatic infiltration if not overt diabetes, but a few were noted to be protective. Unfortunately, most of these T-cell clones were shortlived and because follow-up studies were very limited, there are few details available as to the phenotypic and functional properties of many NOD-derived T-cell lines. The majority of T-cell clones listed in the table were CD4 T cells, and for awhile, CD4 T cells received most of the attention as the ‘‘initiators’’ of disease. In more recent years, with reports of CD8 T-cell clones and several Class I tetramers providing tracking reagents, the pendulum has swung in the other direction, and some investigators now maintain that CD8 T cells play a major role in both initiation and progression of diabetes. This issue has not been fully resolved, but the most likely consensus is that both CD4 and CD8 T cells are important contributors to disease initiation and progression. In this review, the emphasis will be on new developments in the use of T-cell clones as reagents; what they have taught us about T-cell effector function in vitro and in vivo; and how, as T cells dominating the repertoire in T cell receptor transgenic (TCR-Tg) mice, they function in new mouse models for T1D. In addition, efforts have been underway for some time to use T-cell clones as reagents to screen beta cell autoantigens that are relevant to T1D, and since the first diabetogenic T-cell clones were described, the identification of their ligands has been under intense investigation. With the generation of

126

k at h r yn h a s k i n s

peptide libraries and MHC-peptide multimer binding reagents, we are now able to probe T-cell activity in greater depth and ask questions about when and where the pathogenic T cells arise and what autoantigens are driving them. 2. Pathogenic T-Cell Clones—Effector Function 2.1. The BDC Panel of CD4 T-Cell Clones The first diabetogenic T-cell clones described were a panel of CD4 T-cell clones, all isolated from the spleen and lymph nodes of diabetic NOD mice (Haskins et al., 1988, 1989). The BDC panel was so named because these cell lines were derived and characterized at the Barbara Davis Center for Childhood Diabetes in Denver, Colorado. Some properties of the BDC clones, as well as a number of CD4 clones described by others, were summarized in previous reviews (Bergman and Haskins, 1997; Haskins and Wegmann, 1996), but salient features are reiterated here since these CD4 T-cell clones have been in existence for over 15 years and to our knowledge, comprise the largest panel of diabetogenic T-cell clones available. At the time the BDC T-cell clones were derived, no candidate autoantigens for T cells had been identified, so all of these clones were selected on the basis of their reactivity with whole cell suspensions made from NOD islets. Thus they are all reactive with NOD islet cells in the presence of NOD antigen-presenting cells (APC), and most— but not all—can react with islet cells from any mouse strain tested (Haskins et al., 1989; Peterson et al., 1994). Because the T-cell clones also respond to beta cell tumor lines and not to other non-islet endocrine tissues (Bergman and Haskins, 1994), it is likely that their antigen reactivity is b-cell specific. The BDC clones were found to have varying TCR a and b chain usage (Candeias et al., 1991, Haskins, unpublished results), but as indicated in Table 1, there appears to be a predominant use of Vb4 as over half of the clones have this Vb type. Early characterization studies established that upon stimulation with NOD islet cells and APC, the T-cell clones proliferate and produce IL-2, IFNg, TNFa, IL-3, and GM-CSF, but no IL-4 (Peterson and Haskins, 1996). The clones have been kept in continuous culture through biweekly restimulation with islet antigen and APC to facilitate their use in studies on function in vitro and in vivo. The T-cell clones in the BDC panel are by definition highly diabetogenic in young (<2-week-old) NOD recipients (Haskins and McDuffie, 1990; Peterson et al., 1995), and because their numbers are easily expanded and they consistently and rapidly cause fulminant disease upon transfer, they provide very useful tools for in vivo studies. The normal time frame for spontaneous

path o g e ni c t- c e l l c lo n e s

127

Table 1 BDC Diabetogenic CD4þ Th1 T-Cell Clones Diabetogenicity Clone BDC-2.5 BDC-4.12 BDC-5.2 BDC-5.10.3 BDC-6.3 BDC-6.9 BDC-9.3 BDC-10.1

TCR

Islet Ag reactivity

NOD (<14 day)

NOD.scid (<14 day)

Vb4Va1 Vb12Va(nd) Vb6Va12 Vb4Va(nd) Vb4Va3.1 Vb4Va13.1 Vb4Va(13.1) Vb15Va13

All mouse strains* All mouse strains Mouse, rat All mouse strains All mouse strains NOD, SWR NOD, SWR All mouse strains

þ þ þ þ þ þ þ þ

þ n.d. þ þ
þ þ þ

*All mouse strains ¼ all inbred mouse strains tested (NOD, NOR, BALB/c, CBA, C57BL/6, C57L/J, SWR, SJL). n.d., not determined.

development of diabetes in the female NOD mouse is around 4–6 months, wherein about 50% are diabetic, and by a year, incidence is almost 100%. Onset and incidence of spontaneous disease in male NOD mice lags behind, but the majority (70–80%) are diabetic within a year’s time. In contrast, using our adoptive transfer system, the time for disease development is greatly compressed, with 7–10-day-old recipient animals, male or female, becoming hyperglycemic within 1 to 2 weeks after a single injection of cloned T cells. Most of the BDC T-cell clones can also transfer disease into young NOD.scid mice, providing a clean system in which the specific contribution of a single T-cell type to the progression of disease can be analyzed, and demonstrating the ability of these clones to transfer disease in the absence of other T cells. One example of the value of this system was provided by studies using neonatal NOD.scid mice, in adoptive transfers with the CD4 diabetogenic T-cell clone BDC-2.5, to show that treatment of recipients with a nondepleting anti-CD4 antibody could prevent induction and development of diabetes through inhibition of pro-inflammatory cytokine production and elimination of the T-cell clone in the pancreas (Phillips et al., 2000). The fact that the anti-CD4 antibody treatment was effective even after the islets were infiltrated, preventing further disease development and promoting survival of recipient mice, provided a dramatic demonstration of how a reagent that acts upon activated T cells can inhibit an ongoing pathogenic process (Phillips et al., 2000). These results from adoptive transfer of T-cell clones into NOD.scid mice helped bolster the argument that CD4 T cells were the primary effector T cells in diabetes, a theory that has since been undermined by the isolation

k at h r yn h a s k i n s

128

of pathogenic CD8 T cells that transfer disease in the absence of CD4 T cells. Further studies, however, with both the BDC T-cell clones and TCR transgenic mice, have demonstrated the continued value of this panel in providing information about pathogenic CD4 T-cell clones. One example is the BDC-2.5 clone, the best known T cell of the panel, which was used to generate the 2.5 TCR-Tg mouse (Katz et al., 1993), an animal model that has been widely employed to investigate mechanisms of CD4 T-cell–governed pathogenicity and immunoregulation. Except in the case of transgenic T cells isolated from monoclonal TCR-Tg mice, T cells that we have found to be atypical at least in the 2.5 TCR-Tg, it is difficult to obtain purified T-cell populations of one clonotype. For that reason, we have used T-cell clones from the BDC panel to investigate effector mechanisms and to provide a systematic comparison of defined pathogenic CD4 T cells at the level of the single T cell. We have analyzed expression of cytokine and chemokine mRNAs of these CD4 T-cell clones by RNAse protection assay (RPA) and SuperArray, and at the protein level by ELISA and intracellular cytokine staining (Cantor and Haskins, manuscript in preparation). Our results indicate that cytokines produced as part of the functional activity of pathogenic Th1 T-cell clones, both in vitro and in the inflammatory site, go beyond the usual textbook description for Th1 T-cell subsets, a list that typically includes IFNg, IL-2, TNFa, IL-3, and GM-CSF. As summarized in Table 2, the data indicate that among individual BDC T-cell clones, there is a common pattern of cytokines produced, most of which are considered to be pro-inflammatory. Another important finding to come from this work was the discovery that cytokines like IFNg and TNFa can be detected at high levels intracellularly in diabetogenic T-cell clones recovered ex vivo. Even in the absence of a secondary stimulus with anti-CD3 or PMA upon recovery, these cytokines

Table 2 mRNA Expression of Proinflammatory Cytokines, Chemokines, and Their Receptors in Diabetogenic CD4 þ T-Cell Clones Cytokines IL-2 IFNg TNFa LT-a IL-17 B Mif

Chemokines

Cytokine receptors

Chemokine receptors

RANTES (CCL5)* MIP-1a (CCL3) MIP-1b (CCL4) MIP-1g (CCL9/10) MCP-3 (CCL7) Lymphotactin (XCL1) SLC (CCL21) C10 (CCL6)

IL-2R-a IL-2R-b IL-2R-g IL-1R2 IL-12Rb2 IL-15R

CCR1 CCR2 CCR5 CXCR3

*Newer CC versions of chemokine names are in parentheses.

path o g e ni c t- c e l l c lo n e s

129

are detectable in the cloned T cells recovered from the pancreas a week after they are injected into recipient mice. In addition to the expected Th1 cytokines such as IL-2, IFNg, and TNFa, the Th1 clones express at least one other important pro-inflammatory cytokine, lymphotoxin-a (LTa). It has been shown that LTa, like TNFa, is a pro-inflammatory cytokine, capable of inducing chemokines and adhesion molecules (Cuff et al., 1998; Sacca et al., 1998), and transgenic expression of LTa in the pancreas results in infiltration of the islets (Picarella et al., 1992). The cytokine IL-17B has been detected in proteins extracted from bovine cartilage, but it has not been reported as a factor secreted by T cells; however, another member of this family, IL-17A, is produced by activated T cells and has been shown to contribute to inflammation in several models of arthritis (Moseley et al., 2003; Stamp et al., 2004). In no case have we observed any expression of Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) in the Th1 T-cell clones, nor have we found conditions (e.g., antiCD3 or PMA/Ionomycin) under which the CD4 Th1 T-cell clones can be induced to secrete the regulatory factor, TGFb, although the mRNA for this cytokine is detected by SuperArray. Table 2 shows that there are also a large variety of chemoattractant cytokines, or chemokines, produced by pathogenic CD4þ T-cell clones. The production at the mRNA level of some of these factors by short-term lines of 2.5 TCR-Tg T cells cultured under Th1-promoting conditions, as well as their presence in islet infiltrates induced by 2.5 TCR-Tg T cells, has been previously described (Bradley et al., 1999). Recent work by Dorner et al. (Dorner et al., 2002, 2003) has established that MIP-1a, MIP-1b, RANTES, and lymphotactin are differentially expressed by naı¨ve and memory CD4 and CD8 T cells, as well as by NK cells. Their data showed that in CD4 and CD8 T cells, these chemokines are expressed primarily in activated CD8 T cells and only in a very small percentage (<5%) of memory (but not naı¨ve) CD4 Th1 (but not Th2) T cells. Although apparently rare in CD4 T cells, all of these chemokines are expressed by our Th1 T-cell clones. Thus these results provide a direct analysis of mediators made by pathogenic T-cell clones and suggest broad effector mechanisms for pathogenic Th1 T cells through the production of multiple inflammatory mediators (Cantor and Haskins, manuscript in preparation). The possibility is intriguing that some of these factors may contribute to T-cell autoreactivity, especially those such as IL-17B, C10, lymphotactin, and TCA-3, for which little or no information on function in T cells has been reported. Our results also indicate that cytokine/chemokine production by autoreactive CD4 Th1 T-cell clones is quite complex, especially since many of these factors—including the prototype pro-inflammatory cytokines, IL-2, IFNg, and TNFa—can have dual functions, promoting inflammation under some conditions and being immunosuppressive under others (O’Shea et al., 2002).

130

k at h r yn h a s k i n s

2.2. Other Pathogenic CD4 T-Cell Clones In an earlier review, the characteristics of diabetogenic clones reported up to 1996 were covered (Bergman and Haskins, 1997). Included was a description of pathogenic insulin-reactive CD4 T-cell clones (Wegmann et al., 1994), but for the most part, the CD4 T-cell clones isolated to that point were reactive to undefined antigens in islet cells. One of these clones, the NY4.1 CD4 T-cell clone, was isolated from the islets of diabetic NOD mice (Nagata and Yoon, 1992). Later studies on this clone in a transgenic mouse bearing the 4.1 TCR have demonstrated its pathogenic properties and are reviewed below under Section 4. Since our previous review, there have been at least three other reports of note on pathogenic CD4 T-cell clones, all involving isolation of T-cell clones from mice immunized with putative autoantigens. A pathogenic CD4 T cell line 5A was isolated from splenocytes of a NOD mouse immunized with GAD and in vitro, this line, consisting primarily of Vb2 and Vb12 TCR, was shown to proliferate and produce IFNg and TNFa in response to GAD in a I-Ag7-restricted manner (Zekzer et al., 1998). The line also showed cytotoxic activity toward a GAD-transfected target. Unlike most GAD-reactive T-cell clones, the 5A line was diabetogenic and could efficiently transfer diabetes in the absence of CD8 T cells to adult NOD.scid recipients. In a second report of pathogenic GAD-reactive CD4 T cells, clones were described that differed from other diabetogenic T-cell lines in that they were produced from a non-NOD mouse lacking endogenous mouse class II molecules (Wen et al., 1998). Upon immunization with GAD peptides, transgenic mice expressing the human HLA Class II DQ8 molecule (DQþ/classII0 mice) produced GAD-specific antibodies and T cells that proliferated in response to the immunizing GAD peptides. Two CD4 T-cell clones were isolated that could induce insulitis in recipient mice, but only after treatment with lowdose streptozotocin. In addition to demonstrating that human class II molecules could present GAD peptides to mouse CD4 T cells, this study showed that the GAD-specific T cells could induce islet pathology in the absence of either murine class II or other diabetes susceptibility genes in the numerous Idd loci of the NOD mouse. It was hypothesized that the use of streptozotocin in effect provided an environmental insult to the b-cells, thereby inducing an inflammatory environment that could attract the GAD-specific T-cell clones to the islets (Wen et al., 1998). Another report described CD4 T-cell clones reactive to a secretory granule protein, phogrin (Kelemen et al., 1999). These lines were obtained through immunization of NOD mice with recombinant protein containing the cytosolic region of rat phogrin, which had been determined to contain the major

path o g e ni c t- c e l l c lo n e s

131

serological epitopes from human patients. All of the lines and clones isolated were of the CD4 Th1 type, but they showed variable TCR Vb usage. Three clones were shown to be responsive to endogenous antigens in rat islets and two of these were able to cause destruction of rat islets in NOD.scid mice. As summarized in Section 5.1. of this review, these T-cell clones were used in later studies to map phogrin epitopes for T-cell reactivity. 2.3. CD8 T-Cell Clones For reasons that may have to do mostly with the in vitro culturing of T cells, the isolation of diabetogenic CD8 T-cell clones proved to be more elusive than that of CD4 T-cell clones. Pathogenic CD8 T-cell clones were described by Nagata and Yoon in 1992, but could only be isolated from NOD mice older than 10 weeks of age and could not transfer disease in the absence of CD4 T cells (Nagata and Yoon, 1992; Nagata et al., 1994). The authors concluded that CD4 T cells predominated in the early stages of insulitis, whereas CD8 T cells were effectors later in the disease process. One of their CD8 T-cell clones, NY8.3, was subsequently used to produce TCR transgenic mice (Verdaguer et al., 1996, 1997). Wong et al. were the first to describe a CD8 T-cell clone that was diabetogenic in the absence of any other T cells (Wong et al., 1996). The G9 clone, and others isolated from the islets of prediabetic NOD mice, was stimulated with islets containing B7-1 on the rat insulin promoter (RIP-B7). Expression of this costimulatory molecule on the beta cells appeared to bypass the need for CD4 T cells in the activation of CD8 T cells. The resulting CD8 T-cell clones were able to proliferate and express pro-inflammatory cytokines in response to NOD islets, and they could efficiently transfer diabetes to irradiated NOD or to 3-week-old NOD.scid recipients in the absence of any other T cells. The fact that these clones were isolated from 7-week-old prediabetic NOD mice, combined with findings reported by others that NOD mice deficient in b2-microglobulin do not develop diabetes (Serreze et al., 1994), was taken as evidence that CD8 T cells have an important role in the initiation of disease. This conclusion received support from another report of a diabetogenic CD8 T-cell clone, in which the authors concluded that optimally activated CD8 clones in sufficient numbers could transfer disease in the absence of CD4 T cells (Yoneda et al., 1997). The CD8 T-cell clone, YNK1.3, isolated from a diabetic NOD mouse, could cause disease in 7-day-old NOD or NOD.scid recipients, and the susceptibility of the islet cells to cytotoxic T cells at this early age was suggested to be an indicator of the role of CD8 T cells in disease initiation.

132

k at h r yn h a s k i n s

Further support for the role of CD8 T cells in the initiation of disease came from studies on a panel of CD8 T-cell clones isolated from young (5–6 week-old) NOD mice and cultured on islets from a RIP.B7 NOD.scid mouse. The clones in this panel showed restricted TCR alpha chain usage (e.g., Va17 and Ja42), a point thought to be significant because these gene segments are not commonly used by CD8 T cells in the NOD (DiLorenzo et al., 1998). From adoptive transfer experiments with spleen cells from NOD mice of different ages, the authors concluded that class I-restricted T cells were required for all but the final stages of disease development. A subsequent study focused on a TCR-transgenic mouse produced from one CD8 T-cell clone, AI4, from this panel, (Graser et al., 2000), and is described under Section 4.4 of this review. In another interesting study, pathogenic islet-reactive CD8 T-cell clones isolated from NOD spleen were used to investigate the interaction of CD8 T cells with b-cells and other cells in islets (Gurlo et al., 1999). In vitro assays on isolated islets showed that islet destruction and IFNg release by the CD8 T-cell clones increased with the degree of islet infiltration, and indicated that CD8 T cells not only interact with b-cells but also with inflammatory cells in islets. It was hypothesized that CD8 T cells activate macrophages, which then facilitate destruction by CD8 T cells through a NO synthesis-dependent pathway. Complementary to these results with CD8 T-cell clones are our findings on the contributions of activated macrophages to the effector function of CD4 Th1 T-cell clones. An immunization approach described by Videbaek et al. (2003) was used to derive a pathogenic CD8 T-cell clone with specificity for the putative islet cell antigen, GAD65. The CD8þ R1 clone was derived from a line established from a NOD mouse immunized with a peptide corresponding to the rat 515–524 sequence of GAD65. The R1 clone was found to proliferate and produce IFNg in response to GAD65-presenting APC and to kill GAD65presenting targets. Although this CD8 T-cell clone was unable to cause diabetes in recipient mice, it was demonstrated that the clone could migrate to the pancreas and cause peri-insulitis. It is interesting that the authors found that infiltrates were comprised primarily of CD4 T cells and a relative lack of CD8 T cells, an observation that led to the speculation that perhaps the CD8 T-cell clone induced beta cell damage through production of IFNg, leading to subsequent recruitment of other inflammatory cells. It was also interesting that of a number of GAD65-specific T-cell clones isolated, the R1 clone was the only one that could cause insulitis. This finding suggests that pathogenic T-cell clones specific for GAD may be rare, and in fact, this clone is only the second NOD-derived GAD-reactive T cell clone reported to be pathogenic.

path o g e ni c t- c e l l c lo n e s

133

3. Migration of Pathogenic T-Cell Clones 3.1. Homing of CD4 T-Cell Clones To address how T cells get to the pancreas, we have also used the BDC panel to investigate homing mechanisms of pathogenic CD4 T cells. Migration of T cells is controlled by their chemokine receptor expression and on the expression of the corresponding ligands in islets and lymph nodes. For example, CCR5 is a receptor for the inflammatory chemokines, RANTES, MIP-1a, and MIP-1b, and in addition to CXCR3 and CXCR6, it is preferentially expressed on Th1 effectors (Moser and Loetscher, 2001). We have found that CXCR3 is expressed on the T-cell clones both in vitro and ex vivo, whereas the chemokine receptor CCR5 appears on the surface of these T cells only after they have been injected into mice. Since the T cells produce the chemokine ligands for CCR5 in addition to expressing this receptor, they are also promoting migration of other cells to the site. The recruitment and activation of macrophages contribute to the effector function of these pathogenic clones, and macrophages recovered from the pancreas after clone transfer uniformly display high levels of CCR5 ex vivo (Cantor and Haskins, unpublished data). As indicated by their ability to induce diabetes in the NOD.scid, pre-existing inflammation in the islets is not required for the T cells to migrate to the pancreas, and CCR5 may appear on the surface of these cells only after they have initiated disease. Since CCR5 cannot be detected on the clones in vitro, this is a case in which the importance of ex vivo analysis of proinflammatory mediators and receptors expressed by the T-cell clones is demonstrated. Again, adoptive transfers into lymphocyte-deficient mice provide a useful model for assessing the specific contribution of a single T-cell clone. Homing of T cells is also dependent on the expression of integrins and addressins, cellular adhesion molecules that direct trafficking (Butcher et al., 1999; Pribila et al., 2004). Our interest in T-cell migration was stimulated through the work of others demonstrating that the expression of the integrin a4b7, or LPAM-1, on T cells in the NOD mouse defines the diabetogenic T-cell subset (Hanninen et al., 1998). The adhesion molecule a4b7 integrin helps direct migration of lymphocytes to the addressin, mucosal addressin cellular adhesion molecule-1 or MAdCAM-1 (Hynes, 1992). MAdCAM-1 is ordinarily an intestinal marker, but it can also be induced during inflammatory responses in the pancreas (Hynes, 2002). The interaction between MAdCAM1 and a4b7 integrin leads to recruitment of IFNg-producing Th1 T cells (Abramson et al., 2001) and has been demonstrated to have an important role in the development of T1D in the NOD mouse (Hanninen et al., 1998; McMurray, 1996; Yang et al., 1997). We have found that our CD4 T-cell clones express a4b7 integrin, and this may be another sign of their pathogenicity.

134

k at h r yn h a s k i n s

Treatment of NOD mice with antibodies to a4b7 integrin has been found to be protective, with combinations of antibodies to a4b7 and MAdCAM-1 being the most effective (Kommajosyula et al., 2001). Furthermore, it has been hypothesized that the gut-associated lymphoid tissue (GALT) is an important site for the initial priming of diabetogenic T cells (Hanninen et al., 1998) and that the expression of a4b7 integrin is a marker of T cells that have arisen in the GALT (Brandtzaeg et al., 1999). Thus the BDC T-cell clones may also be of mucosal origin since they express a4b7 integrin (Ward et al., 1998). Another molecule of interest with respect to homing mechanisms is Ly6C, a GPI-linked cell surface protein (Fleming and Malek, 1994). GPI-linked molecules are enriched in cholesterol-containing membrane rafts (Sharma et al., 2004) that are involved in the formation of the ‘‘immunological synapse’’ (Krawczyk et al., 2000; Tseng and Dustin, 2002). A possible new role for Ly6C in the migration of T cells has been proposed as a receptor whose ligation can induce activation of LFA-1 and a4 integrins on the cell surface (Hanninen et al., 1997; Jaakkola et al., 2003). Although one of the abnormalities of the NOD strain is that NOD T cells usually do not express Ly6C (Langmuir et al., 1993; Philbrick et al., 1990), its expression has been detected in some islet-infiltrating T cells and in vitro after TCR-crosslinking (Herold et al., 1990). We have recently found that Ly6C is expressed on the BDC CD4 T-cell clones at high levels, and we are currently investigating whether this molecule affects their migratory behavior (Cantor, Hanninen, and Haskins, unpublished results). 3.2. Homing of CD8 T-Cell Clones Migration of pathogenic CD8 T cells has also been studied through the use of a diabetogenic T-cell clone. The G9C8 clone isolated by Wong et al. (Wong et al., 1996) was used to investigate homing requirements of insulin-specific CD8 T cells, with the goal of determining whether cross-presentation of antigen by pancreatic endothelium was involved, since these clones, like the BDC CD4 T-cell clones, can cause disease in the absence of pre-existing inflammation in the islets (Savinov et al., 2003). The results indicated that homing of the G9C8 clone to the islets required expression of the specific peptide as well as of Class I. The authors presented several lines of evidence that the insulin peptide could be cross-presented by pancreatic endothelial cells, including demonstration of direct recognition by the CD8 T cells of endothelial cells in islet organ cultures. It was further found that TCR stimulation of the CD8 clone led to upregulation of integrins on their surface and that chemokines, SLC (or CCL21) in particular, were required for adhesion of T cells to pancreatic endothelium. The conclusion from this study was that

path o g e ni c t- c e l l c lo n e s

135

endothelial cells may play an important role in the trafficking of activated T cells and that they provide antigen-driven specificity of homing. 4. T-Cell Clones in T-Cell Receptor Transgenic (TCR-Tg) Mice 4.1. The 2.5 TCR Transgenic Mouse The production of TCR-Tg mice has been one of the most important developments to come from studies on pathogenic clones, and there is now extensive literature on TCR-Tg mice employed as animal models in autoimmunity, including a series of reviews published in 2004 by the Journal of Autoimmunity. The review by Yang and Santamaria (Yang and Santamaria, 2004) provides an excellent survey of the diabetogenic TCR transgenic mouse models, produced from both CD4 and CD8 T-cell clones, and the review by Lafaille (Lafaille, 2004) covers TCR transgenics that are models for other autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE) and rheumatoid arthritis, as well as for type 1 diabetes. Since the 2.5 TCR-Tg was the first to be made with the TCR genes from a diabetogenic T-cell clone, some of the characteristics of this mouse are briefly summarized here. One interesting feature of the 2.5 TCR-Tg was the fact that although the transgenic T cells were activated and migrated to the pancreas very early in disease, with insulitis evident by the age of 3 weeks (Katz et al., 1993), the mice did not exhibit any acceleration of diabetes onset, and with continued backcrossing to the NOD, turned out to have a much lower incidence of disease than nontransgenic NOD mice (Gonzalez et al., 1997). This lower incidence was later attributed to development of an inhibitory T-cell population characterized as CD4 T cells bearing endogenous (i.e., non-BDC-2.5) TCR (Gonzalez et al., 2001). Although there is extensive pancreatic infiltration in 2.5 TCR-Tg mice, the infiltrate is not aggressive unless diabetes is precipitated with a reagent such as cyclophosphamide (Andre-Schmutz et al., 1999). In a study of macrophages and dendritic cells recruited to the pancreas, the distinctive pattern of infiltration in the 2.5 TCR-Tg was further investigated and compared to that induced by the parent BDC-2.5 in clone transfers (Rosmalen et al., 2000). One interesting outcome was that the type of macrophages (BM8þ) associated with islet destruction and prevalent in the islets of NOD females developing spontaneous disease (Jansen et al., 1994), were also abundant in islets following BDC-2.5 clone transfers, but were virtually absent from the largely periislet infiltrate in 2.5 TCR-Tg mice. Overt diabetes in 2.5 TCR-Tg mice could be readily induced through antibody blockade of CTLA4 (Luhder et al., 1998) or by treatment with cyclophosphamide (Andre-Schmutz et al., 1999). By breeding the 2.5 TCR-Tg onto the NOD.scid background, a rapid spontaneous

136

k at h r yn h a s k i n s

onset model was obtained, with mice developing diabetes by 3–6 weeks of age (Dobbs and Haskins, 2001; Kurrer et al., 1997). Beta cell destruction in these monoclonal T-cell mice was observed to be due to apoptosis and to be a TNFa-dependent process (Pakala et al., 1999; Suri and Katz, 1999). Valuable insights into the role of CD4 T cells in pathogenesis of NOD diabetes have been gained from the 2.5 TCR-Tg mouse, but we found that there were significant differences in activity and phenotype between the BDC2.5 parent clone and transgenic T cells, particularly when transgenic T cells were isolated from the monoclonal 2.5 TCR-Tg NOD.scid mouse. Differences between T-cell clones carried in culture and those arising in a mouse are predicted and provide some of the rationale for turning T-cell clones into TCR transgenics. That reasoning is based on the premise that transgenic T cells are naı¨ve and thus more physiologically relevant, but when TCR-Tg mice are generated on an immunodeficient background in which there are no other T cells, the situation may be quite different. For example, our studies (Dobbs and Haskins, 2001) showed that upon phenotypic analysis with antibodies to surface markers, the BDC-2.5 clone has all the expected characteristics of a memory/effector T cell, and transgenic T cells from prediabetic 2.5 TCR-Tg/NOD.scid mice appeared to be ‘‘naı¨ve’’ T cells, as predicted. However, tyrosine phosphorylation patterns of T cell lysates and the demonstration that thymocytes from 10-day-old transgenic donors could rapidly transfer disease suggested that in fact these monoclonal transgenic T cells were not naı¨ve, despite their surface phenotype (Dobbs and Haskins, 2001). From these data, as well as from studies on other TCR transgenics (Yang and Santamaria, 2004), it would appear that normal mechanisms of T-cell regulation are bypassed or overcome in monoclonal TCR-Tg mice. In another study, we explored whether T cells could be cloned from TCR-Tg mice, with the goal of determining whether T cells could be induced in culture to develop a Th2 phenotype. T-cell lines from the 2.5 TCR-Tg/NOD mouse were not readily obtained as most of the splenic T cells die upon encountering stimulus through the TCR with islet antigen. Ultimately, however, after repeated manipulations involving variation of IL-2 concentration and antigen stimulation, several lines were isolated, with only those cultured under Th2-promoting conditions (IL-4, anti-IFNg) being retained for further study. Although previous reports had indicated that short-term lines from 2.5 TCR-Tg mice could be obtained with a Th2-producing cytokine profile, the in vivo activities of such lines were inconsistent (Bradley et al., 1999; Katz et al., 1995), and our goal was to establish stable Th2 clones to determine whether 2.5 TCR-Tg T cells could be demonstrated to be protective. Instead, we found that almost without exception, the cloned Th2 lines were

path o g e ni c t- c e l l c lo n e s

137

diabetogenic in young NOD mice, despite the fact that they consistently produced Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) and no IFNg (Poulin and Haskins, 2000). On the other hand, these clones, unlike the BDC-2.5 parent Th1 T-cell clone, could not cause diabetes in young NOD.scid recipients, unless cotransferred with BDC-6.3, a Th1 T-cell clone that is not diabetogenic in NOD.scid (Table 1). Induction of diabetes in NOD.scid mice upon cotransfer of the Th2 clone with a Th1 clone suggested that the diabetogenic Th2 clones work in young NOD recipients through recruitment of other T cells into the site (Haskins, 2004; Poulin and Haskins, 2000). Subsequent studies have shown that although these 2.5 Tg-T2 T-cell clones do not make IFNg, they do produce TNFa in vitro and ex vivo, and this may be enough to initiate inflammation in the pancreas of young NOD recipients (He and Haskins, unpublished results). 4.2. The 4.1 TCR-Tg Mouse The low incidence of diabetes observed in the 2.5 TCR-Tg is not always a characteristic of TCR-Tg mice produced from diabetogenic CD4 T-cell clones, as is apparent from the accelerated diabetes observed in the 4.1 TCR ab Tg, the second transgenic mouse to be made from a pathogenic CD4þ T-cell clone (Schmidt et al., 1997). Like the 2.5 TCR-Tg mouse, the T cells in the 4.1 TCR-Tg NOD mouse primarily expressed the transgenic Vb chain, were skewed to the CD4 subset, and proliferated in response to islet cells as antigen. The high expression of the 4.1 TCR in pancreatic infiltrates and the ability of purified CD4 T cells from this mouse to transfer diabetes to NOD.scid recipients were further evidence that highly diabetogenic CD4 T cells were being selected in the 4.1 TCR-Tg. In contrast to the 2.5 TCR-Tg, the 4.1 TCR-Tg on the NOD background led to accelerated onset of disease, and a high incidence of diabetes in this mouse line has been maintained (Santamaria, personal communication). One interesting application of the 4.1 TCR-Tg was in studies to probe the influence of MHC on selection of autoreactive T cells. It was found that in diabetes-resistant NOD F1 mice, the transgenic TCR was deleted during thymic development by engaging ‘‘antidiabetogenic’’ class II molecules in a process that was peptide-specific, suggesting how expression of a protective haplotype might overcome genetic susceptibility to autoimmune disease (Schmidt et al., 1997, 1999; Yang and Santamaria, 2004). Another important contribution of the 4.1 TCR-Tg mouse came from a study in which the 4.1 transgenic T cells were cloned from spleen cells and then tested in chromium release assays for cytotoxicity. The results showed that the CD4þ 4.1 T cells differentiate into CTL and kill b cells in a

138

k at h r yn h a s k i n s

Fas-dependent, perforin-independent manner (Amrani et al., 2000b). It was also demonstrated that treatment of NOD islets with diabetogenic cytokines such as IFNg or IL-1a increased the expression of Fas on the surface of islet cells, and that this was necessary for promoting the CTL activity of the 4.1 T cells. Based on these findings, the authors proposed that diabetes in the NOD mouse is initiated by CD8 and CD4 clones that can directly lyse b cells via Fas and that the disease process is later amplified by clonotypes that kill by other pathways. The 4.1 TCR was also studied in the context of a monoclonal mouse, and on a NOD.RAG-2
/
background, the transgenic mouse developed diabetes as early and frequently as on the NOD background (Verdaguer et al., 1997). Thus in both the 2.5 and the 4.1 monoclonal TCR-Tg models, it was evident that CD4 T cells could efficiently home, differentiate into effectors, and cause disease in the absence of B cells or other T cells. 4.3. The 6.9 TCR Transgenic Mouse Further possibilities for investigating T-cell activation in TCR-Tg mice may come from a new TCR-Tg model that was derived from another clone in the BDC panel, BDC-6.9. This clone was chosen for its aggressive diabetogenic properties, being the only clone in the panel observed to cause disease in adult NOD.scid recipients (Peterson and Haskins, 1996), and due to its restricted reactivity to NOD islets, we were able to carry out a genetic mapping study that identified a locus on mouse chromosome six that encoded or controlled the antigen for BDC-6.9 (Dallas-Pedretti et al., 1995). We hypothesized that since the BDC-6.9 T-cell clone does not respond to BALB/c islets as antigen, a NOD strain carrying the chromosome six locus from BALB/c mice in place of the NOD sequence would not have the islet antigen for BDC-6.9. We generated a NOD congenic mouse, the NOD.C6, and although the NOD.C6 congenics have the same disease incidence as NOD, their islets are not antigenic for the BDC-6.9 clone. The 6.9 TCR-Tg mouse showed a markedly accelerated disease incidence in early backcrosses to NOD, but breeding of the 6.9 TCR-Tg onto the NOD.C6 background resulted in a mouse that has had no incidence of diabetes, a low level of insulitis, and a large population of T cells with an autoreactive TCR but having had no exposure to their antigen (Pauza et al., 2004). T cells expressing endogenous Va chains in this 6.9 TCR-Tg/ NOD.C6 mouse may be present in the pancreatic infiltrate and may also contain regulatory T cells, as described for the 2.5 TCR-Tg/NOD mouse (Gonzalez et al., 2001). Studies have not yet been conducted with monoclonal 6.9 TCR-Tg mice, and it should be interesting to explore questions of T-cell activation and tolerance in these animals.

path o g e ni c t- c e l l c lo n e s

139

4.4. TCR-Tg Mice from CD8 T-Cell Clones The TCR of the NY8.3 T-cell clone was the first TCR from a pathogenic CD8 T-cell clone to be expressed in a transgenic mouse. One mouse line was produced using only the b chain of the TCR, pairing with endogenous a chains (Verdaguer et al., 1996), and a second TCR-Tg mouse was made with both the a and b chains (Verdaguer et al., 1997). Studies from these mice indicated that the CTL clone NY8.3 represented a prevalent clonotype in the pancreatic infiltrate and provided the first direct evidence that b-cell-specific CTL are major effectors in diabetes (Verdaguer et al., 1996). To further investigate the relative contributions of CD8 T cells, monoclonal T-cell mice were made on the NOD.Rag-2
/
background with the NY8.3 TCR, and it was found that diabetes and insulitis were much reduced in the 8.3 NOD.Rag-2
/
mouse compared to the TCR transgenic in the NOD, but could be rapidly precipitated by the addition of CD4 T cells (Verdaguer et al., 1997). As in earlier studies with the NY8.3 clone, it was concluded that CD4 T cells initiate diabetes and that CD8 cells appear later. Another important transgenic mouse bearing the TCR from a CD8 T-cell clone is the NOD.AI4ab TCR-Tg (Graser et al., 2000). In NOD mice expressing the AI4 TCR, diabetes was accelerated, but it was found that about half of the transgenic T cells were CD4-positive. When crossed to the NOD.scid background, none of the TCR-Tg T cells were CD4þ, and disease still developed in an accelerated fashion, providing evidence that this pathogenic MHC Class I-restricted CD8 T-cell clone could cause diabetes independent of CD4 help. The authors also determined that the AI4 clone did not respond to either of the antigenic epitopes to which the pathogenic CD8 T-cell clones, G9 and NY8.3, respond. As described later, the most recent work by this group has led to the identification of both the peptide ligand and the protein antigen for the AI4 T-cell clone. 4.5. Retrogenic TCR A most interesting advance on the TCR-Tg mouse front has been the recent development of transgenics made through retroviral-mediated stem cell gene transfer, or ‘‘retrogenic’’ (Rg) mice. Retrogenic mice expressing the TCR from either of the diabetogenic clones, BDC-2.5 or 4.1, were found to have greater than a 60% incidence of diabetes by 80 days following transfer of retrovirally transduced bone marrow, confirming the power of this method to generate diabetogenic T cells in mice without the need for lengthy breeding programs (Arnold et al., 2004). One important finding from this study that has significant implications for studying T-cell autoantigen specificity was the result that

140

k at h r yn h a s k i n s

T cells bearing TCR from non-diabetogenic T-cell clones were invariably naı¨ve and inactive in vivo. TCR cloned from CD4þ T cell hybridomas specific for peptides of HEL or GAD were efficiently expressed in reconstituted recipients, and Rg T cells from these mice were responsive to challenge with peptide, but diabetes incidence in NOD Rg mice with HEL- or GAD-specific TCR was neither accelerated nor inhibited compared to wild-type NOD mice. Although retrogenic mice cannot be bred, this new approach to transgenic mice offers some definite advantages in terms of being able to analyze in mice the contributions of different TCR, from both pathogenic and non-pathogenic T-cell clones. 5. Antigens for Pathogenic T-Cell Clones The large body of literature on candidate autoantigens in T1D, targets for both antibodies and T cells, in mouse and in man, has been recently reviewed (Lieberman and DiLorenzo, 2003). The intent here is to provide a summary on the current status of autoantigens, defined and undefined, including peptide mimotopes, for pathogenic T-cell clones. 5.1. CD4 T-Cell Clones with Specificity for Defined Antigens To identify autoantigens for T cells, investigators first looked to antigens that had been identified as the targets of autoantibodies in T1D. Insulin and glutamic acid decarboxylase (GAD) are major candidate autoantigens, and the presence of antibodies to both are used to diagnose risk in humans (Verge et al., 1996). Up to about 70% of new onset patients have antibodies to insulin (Yu et al., 2000) and GAD (Hagopian et al., 1993). Although several GAD peptide epitopes—versus one epitope for insulin—have been identified in T1D patients (Lieberman and DiLorenzo, 2003), the conclusion of one recent review on these two proteins is that insulin is the more dominant antigen in the pathogenic T-cell response (Wegmann and Eisenbarth, 2000). The majority of attempts to isolate autoreactive T-cell clones with known antigen specificity have focused on these two antigens. The first such clones to be isolated were diabetogenic CD4 T-cell clones to insulin, and an immunogenic epitope in the insulin B chain, B9-23, was identified (Daniel et al., 1995; Wegmann et al., 1994). The potential importance of insulin as an autoantigen was underscored by demonstrating that it could be successfully used to induce tolerance and inhibit disease in NOD mice (Daniel and Wegmann, 1996a,b). In addition, insulin has been found to be expressed in the thymus, and quantitative RT-PCR studies indicated that expression in NOD thymus is at only about half the levels found in thymus tissue of normal strains (Brimnes et al., 2002). Insulin was the first protein

path o g e ni c t- c e l l c lo n e s

141

defined as an antigen for CD4 T-cell clones (Wegmann et al., 1994), and there are a number of studies that have been conducted on insulin peptides as T-cell ligands. Simone et al. found that there was restricted use of TCR Va (but not Vb) gene segments in a panel of CD4 T-cell clones reactive with insulin peptide B9-23, with 10 of 13 clones using Va13, and most of these segments were found to be a novel NOD sequence termed Va13.3 (Simone et al., 1997). The use of Va13 gene segments was found to be particularly high in NOD mice when compared to normal strains such as BALB/c and C57BL/6, leading to the hypothesis that the frequency of these T cells is due to targeting of the insulin B9-23 peptide (Simone et al., 1997). Since TCR from at least two of the BDC pathogenic T-cell clones (none of which react to insulin) also use segments from the Va13 gene family, insulin may not be the only autoantigen that is targeted by Va13 TCR. Further studies to characterize immunogenic insulin epitopes have been carried out on peptide libraries spanning the entire sequence of preproinsulin I and II, and T cells reactive with at least four epitopes of insulin were identified in islet cell infiltrates of prediabetic NOD mice (Halbout et al., 2002). In light of the high frequency of T cells reactive with the insulin B9-23 epitope reported in earlier studies (Wegmann et al., 1994), it is interesting that in the study by Halbout et al., using T cell hybridomas derived from islet-infiltrating T cells of 14-week-old mice, the frequency of T cells reacting to the B9-23 peptide was not higher than that of T cells reacting to other epitopes. Attempts to demonstrate reactivity to GAD in pathogenic CD4 T-cell clones have been less successful, despite initial reports (Kaufman et al., 1993; Tisch et al., 1993) that NOD T cells react to GAD early in the disease process. GAD-reactive T-cell clones derived from peripheral lymphoid organs of NOD mice were found to be unresponsive to islets and were non-pathogenic (Schloot et al., 1996), and no GAD reactivity could be detected in HLA-DRrestricted T-cell lines from newly diagnosed T1D patients (Huang et al., 1995). As described previously (Section 2.2.), an exception was the pathogenic CD4 T-cell line isolated from a NOD mouse that had been immunized with GAD and was shown to be diabetogenic in NOD.scid recipients (Zekzer et al., 1998). In addition, there were GAD-reactive CD4 T-cell clones, isolated from transgenic mice expressing the human HLA Class II DQ8 molecule after immunization with GAD peptides, that could induce insulitis in recipient mice if the animals were treated with low-dose streptozotocin (Wen et al., 1998). Since these clones were derived from a T-cell response obtained after immunization with GAD in non–diabetes-prone mice, it is not clear that they would be representative of spontaneously arising T-cell clones in NOD mice. Compared to the isolation of diabetogenic GAD-reactive T-cell clones, the attempts to induce tolerance with GAD have been somewhat more successful

142

k at h r yn h a s k i n s

(Kaufman et al., 1993; Tisch et al., 1993), and include the isolation of a GADreactive Th2 clone that could protect against disease (Tisch et al., 2001). Further evidence for the non-pathogenic nature of GAD-reactive T-cell clones was obtained in the recent ‘‘retrogenic’’ NOD mouse studies, describing retroviral-mediated expression of various TCR, in which mice with T cells reactive to GAD peptides failed to develop disease (Arnold et al., 2004). In the effort to further define the status of GAD as an autoantigen in T1D, there have been many studies to identify GAD epitopes for T-cell clones. Initial mapping studies identified GAD amino acid sequences 509–528 and 524–543 as early epitopes of GAD, eliciting responses in mice that are 4 weeks old (Kaufman et al., 1993), and other immunogenic epitopes were reported in later studies, including some that were shared between humans and mice (Lieberman and DiLorenzo, 2003). In an extensive set of studies on T-cell responses to GAD in the NOD mouse, Quinn et al. have mapped T-cell reactivity to overlapping GAD peptides spanning the 509–543 region. T cell responses were surveyed from mice immunized with GAD 524–543 (Quinn and Sercarz, 1996), and in a follow-up study, it was shown that there are two different regions within the 524–543 region that elicit different T-cell repertoires, each showing a predominant TCR Vb gene usage (Quinn et al., 2001a). A Vb12 T-cell subset responsive to 524–538 was dominant in immunized mice and appeared to have regulatory activity, whereas a Vb4 T-cell subset arising spontaneously in NOD mice responded to the GAD 530–543 region. As these authors point out, it is interesting that the diabetogenic clone BDC-2.5 is also a CD4 Vb4 T-cell clone and that a GAD 16-mer epitope has been found to stimulate weak responses in T cells from the 2.5 TCR-Tg mouse (Judkowski et al., 2001). As discussed later (Section 5.3.), we have been unable to detect responses to peptides in the 524–543 region with the original BDC2.5 clone (Yoshida et al., 2002). On the other hand, it is also of interest to note that the diabetogenic CD4 T-cell line containing Vb12 T cells described by Zekzer et al. (1998) was specific for GAD 524–543. In addition to insulin and GAD, another target of autoantibodies in both NOD mice and humans is phogrin (IA-2b), a molecule structurally related to (ICA)512 (IA-2), and both proteins are part of a group of tyrosine phosphatases localized to neuroendocrine secretory granules (Lan et al., 1996; Wasmeier and Hutton, 1996). Phogrin was found to be a target for autoreactive T cells, and phogrin-specific CD4 T-cell clones were generated from NOD mice that were capable of destroying islet transplants (Kelemen et al., 1999). It was subsequently shown that reactivity of T cells to phogrin could be detected in peripheral T cells of NOD mice as young as 4 weeks of age (Achenbach et al., 2002). Mapping of T-cell reactivity led to identification of two peptide epitopes that were targeted by diabetogenic T-cell clones (Kelemen et al.,

path o g e ni c t- c e l l c lo n e s

143

2001) and that were found to be highly conserved in sequence between rat, mouse, and human (Kelemen et al., 2004). These two peptides were also found to elicit T-cell responses in transgenic mice expressing the diabetespredisposing human MHC molecule HLA-DQ8, and in a group of prediabetic humans that included both HLA-DQ8 and HLA-DR-restricted reactivity (Kelemen et al., 2004), suggesting the potential importance of phogrin as an autoantigen in human disease. One of the more recent candidate autoantigens for CD4 T-cell clones is a retroviral envelope protein discovered through activity of a monoclonal antibody from a prediabetic NOD mouse (Levisetti et al., 2003). Although a similar approach to detecting beta cell autoantigens had been reported earlier by us, the only antigen specificity determined for the monoclonal antibodies we isolated was to insulin (Supon et al., 1990). In the study by Levisetti et al., immunoprecipitation of a b-cell tumor line lysate with the antibody yielded three bands corresponding to proteins of 70, 50, and 20 kDa. Sequence analysis of peptides extracted from these bands revealed that they were from a large family of mouse retroviral envelope glycoproteins, and the cDNA for one of these proteins termed EAD1 was cloned into an I-Ag7-expressing B cell lymphoma line. It was established that NOD islets contained the sequence and subsequently, T-cell clones were isolated, from unimmunized as well as immunized NOD mice, that were reactive with a peptide, EAD61-80, demonstrated to bind to soluble I-Ag7. Several of the T-cell clones were tested in transfers into NOD.scid recipients, but none were diabetogenic. Several possibilities were considered as to why the EAD-reactive T cells were not diabetogenic, but because these investigators performed adoptive transfers in adult NOD.scid mice, it cannot be determined from this report whether the clones are diabetogenic in young recipients. This is an important point from the standpoint of our experience, in which one injection of CD4 T-cell clones cultured in vitro can rarely transfer disease in NOD or NOD.scid recipients past 3 weeks of age. Although the role of the EAD1 antigen in the pathogenesis of diabetes is not yet defined, it is important to take note of this protein since it confirms the possibility of retroviral gene products contributing to the spectrum of b-cell autoantigens. 5.2. CD4 T-Cell Clones with Undefined Antigen Specificity In general, T-cell clones have been generated through a process of T-cell immunization, using defined proteins or peptides, and there has been little precedent for using T-cell clones to detect unknown autoantigens and natural ligands. This lack of precedent, however, has not been due to a lack of effort. One approach to identifying T-cell autoantigens has been to biochemically

144

k at h r yn h a s k i n s

isolate cellular fractions and proteins that can stimulate islet-reactive T-cell clones. The BDC panel of clones falls into this category as these T cells were selected for reactivity to whole islet cell preparations. Reactivity of the BDC clones to candidate autoantigens such as insulin and GAD was ruled out early in our investigations on the antigen specificity of these clones (Bergman and Haskins, 1997), and we therefore turned to a protein isolation approach. We used a procedure designed for the isolation of islet fractions highly enriched in the insulin secretory granules, which by SDS-PAGE were shown to contain over 150 protein species (Hutton, 1989). Our results indicated that the granule fraction contained the antigenic activity for BDC T-cell clones and that this activity was most likely membrane-associated; we could not detect antigen in cytosolic fractions (Bergman and Haskins, 1994). Biochemical studies in which membrane lysates were separated on HPLC ion exchange and size exclusion columns identified an active fraction containing proteins in a 50–80 kD molecular weight range (Bergman et al., 2000). Unfortunately, our attempts to detect antigenicity in subsequent fractions produced by SDS-PAGE were not successful (Bergman et al., 2000). Reports by other investigators also have provided evidence for membrane proteins as a source of T-cell autoantigens. In studies on antigen presentation to CD4 T-cell clones isolated from islets of diabetic NOD mice, Shimizu et al. concluded that since antigenic activity was not detected in secreted fractions from islet cells, the antigenicity of islet cells was likely to be membrane-associated (Shimizu et al., 1993). In another study, Ellerman and Like found that crude islet cell membrane fractions were antigenic for CD4 T-cell clones, isolated in this case from the BB rat (Ellerman and Like, 1999). These T-cell clones were unreactive with peripheral membrane protein extracts, cytosolic fractions, and recombinant human GAD65. Although the membrane antigens for these various CD4 T-cell clones are still undefined, we can anticipate that newer proteomic methods and other technologies now available will lead to identification of antigenic proteins for a number of pathogenic CD4 clones. 5.3. Peptide Mimotopes for CD4 T-Cell Clones Perhaps the most promising approach for identifying ligands for diabetogenic CD4 T-cell clones with undefined antigen specificity comes from the use of peptide libraries. Peptide libraries have been used for a number of years to identify ligands for CD8 T-cell clones, but progress with CD4 T-cell clones lagged behind since purification and structural analysis of class II molecules proved more intractable than that with class I. One of the first uses of peptide libraries to identify epitopes for CD4 T cells was described for a human T-cell clone, 1c10, that had been shown to be reactive with an antigen identified by

path o g e ni c t- c e l l c lo n e s

145

expression cloning (Roep et al., 1990). In this system, the synthetic peptide library was designed to bind the class II restriction molecule of the CD4 T-cell clone being screened, and the authors were able to identify two peptides that could stimulate the 1c10 clone (Hiemstra et al., 1997). A more recent system using a decapeptide combinatorial library arranged in a positional scanning format for screening with CD4 T-cell clones led not only to identification of immunogenic peptides, but also revealed new information with regard to degeneracy of T-cell receptors, the extent to which peptide specificity can be retained despite multiple amino acid substitutions, and the contribution of individual amino acid positions in a given peptide as contact residues for MHC and TCR binding (Wilson et al., 1999). The possibility that GAD is an important autoantigen in T1D received new impetus in a study in which T cells from the 2.5 TCR-Tg mouse were used to screen a large combinatorial decamer peptide library (Judkowski et al., 2004; Wilson, 2003). More than 100 different decapeptides were identified that could stimulate the transgenic T cells in culture and render them highly diabetogenic in vivo. The surprising finding was that some of the peptides contained sequences that were very similar to a 12-amino acid fragment (528–539) of GAD65. This fragment had very low stimulatory activity for the transgenic T cells, but a larger 16-mer fragment, 526–541, had activity in the low micromolar range. Because of this remarkable similarity to a fragment of GAD65, it was suggested that an epitope within this region might contain the native autoantigen for the BDC-2.5 T-cell clone. In a separate study, we identified a peptide motif through screening of a peptide library with two panels of diabetogenic CD4 T-cell clones, the BDC panel and a set of clones described by Nakano et al. (Nakano et al., 1991). We screened peptide mixtures in which anchor residues for I-Ag7 were fixed and then synthesized several peptides based on favored amino acid motifs. At least two of these synthetic peptides were able to stimulate most of the T-cell clones tested despite their variable TCR gene usage, results that suggested that T-cell clones from the two panels may recognize a similar natural ligand in islet b cells (Yoshida et al., 2002). In the process of testing the synthetic peptides, we identified several, varying by only one or two amino acids, that were highly stimulatory for the BDC-2.5 T-cell clone, and when compared to islet cells as antigen, could be termed super-agonists. We were interested to find that the peptide reactivity pattern for BDC-2.5 was shared by another clone in the panel, BDC-5.10.3, a clone that was obtained from a different mouse and that was subsequently found to have a CDR3 region identical to that of the BDC-2.5 clone (Huffman, Sercarz, and Haskins, unpublished results). It was also interesting that within the nine amino acid peptide motifs, the residues critical for activity, P3, W5, R7, and M8, corresponded exactly to

146

k at h r yn h a s k i n s

the positions P4, W6, R8, and M9 identified to be important residues in the decapeptides that could stimulate the 2.5 TCR transgenic T cells (Judkowski et al., 2001). We tested the GAD peptide amino acid sequence 521–543 (the whole peptide and overlapping 15-mers spanning this sequence) which is the region originally identified as immunogenic for CD4 T cells in NOD mice (Kaufman et al., 1993). As previously described, the 524–543 sequence of GAD65 had been shown to contain two overlapping epitopes that stimulate two functionally different sets of T cells with different TCR b-chains (Quinn et al., 2001a). This region also contains the 528–539 fragment of GAD65 suggested to be a possible source of the natural ligand for BDC-2.5 (Judkowski et al., 2001). Using both the BDC-2.5 clone and transgenic T cells from 2.5 TCR-Tg mice, we were unable to detect any reactivity to the GAD peptides (Yoshida et al., 2002), a result that is in agreement with our previous attempts to test GAD65 as an antigen (Bergman and Haskins, 1994). The discrepancy between our results and those of Judkowski et al. may be due to lack of precise overlap between the 15-mer peptides we tested and the GAD 526–541 16-mer they described. However, in their hands, one of the peptides we tested (524–538) was weakly stimulatory for transgenic T cells. With the exception of the GAD sequences reported by Judkowski et al., mimotopes for BDC-2.5 or other T-cell clones in the BDC panel have not led to matches in the database with any naturally occurring protein sequences. It could be that the natural ligand recognized by a BDC clone is a weak binder to MHC and differs from the mimotopes in amino acids used as MHC anchors (Yoshida et al., 2002). Most peptide library screening strategies involve the testing of peptide pools and are limited by the need for a very sensitive T-cell bioassay, as the responder T cells must detect the correct peptide among competing peptides. The advent of new peptide libraries may provide improvements on these methods. For example, baculovirus-based peptide libraries are now available in which the sequences encoding the peptides are embedded within the MHC genes in the viral DNA, such that in insect cells infected with virus encoding a library of peptides, each cell displays a unique peptide-MHC complex on its surface. In one instance, a library of peptides was attached to the displayed murine MHC class II molecule I-Ab and using fluorescently labeled multimeric soluble TCRs to ‘‘fish’’ for the insect cells, peptide mimotopes for two abTCRs were readily identified (Crawford et al., 2004). 5.4. Antigens for CD8 T-Cell clones First as antigens targeted by autoantibodies and then as candidate autoantigens for CD4 T cells, insulin and GAD have also been investigated as targets of CD8 T-cell responses. The first antigen specificity defined for a pathogenic

path o g e ni c t- c e l l c lo n e s

147

CD8 T cell was obtained through an expression cloning approach to screen a pancreatic islet cDNA library and was identified to be a peptide of insulin, B chain 15-23, presented by H-2Kd to the T-cell clone G9C8 (Wong et al., 1999). Twenty of 264 cDNA clones tested were found to stimulate IFNg production by G9C8 and of these, 85% were either preproinsulin 1 or 2; subsequent testing showed that the stimulating peptide was insulin B chain 15-23. This study was also the first to identify an antigen recognized by both CD4 and CD8 T cells, thus clearly confirming the importance of insulin as an autoantigen in T1D. Although the B15-23 peptide is a poor binder to the Class I molecule H-2Kd and high concentrations of peptide are needed to activate the T-cell clone, structural modeling studies indicated that T-cell recognition of this complex is very sensitive to most amino acid substitutions (Wong et al., 2002). It was hypothesized in this study that the poor binding capacity of the peptide to MHC could conceivably explain failure to delete such insulin-specific T-cell clones from the thymus. GAD65 has also been identified as an antigen for CD8 T cells, and two epitopes have been found that are outside the 524–543 region that is immunogenic for CD4 T cells. The GAD amino acid sequences 206–220 and 546–554 were found to stimulate NOD spleen cells to produce IFNg and exhibit cytotoxic activity against peptide-pulsed targets (Quinn et al., 2001b). It is interesting that another putative Kd binding determinant, 515–524, was used in the immunization of NOD mice to produce a GAD-reactive pathogenic CD8 T–cell clone (Videbaek et al., 2003). The discrepancies in GAD peptide immunogenicity, in immunized versus unmanipulated mice, and by CD4 T cells or CD8 T cells may well be due to differences in how this antigen is processed and presented by different antigen-presenting cells (Quinn, 2003). A new autoantigen for CD8 T-cell clones was recently identified by Panagiotopoulos et al. who found that a peptide of islet amyloid polypeptide (IAPP) was the natural ligand for a human CTL clone (Panagiotopoulos et al., 2003). An epitope from the leader sequence of human IAPP, preproIAPP 5-13, was found to be antigenic for a significant proportion of cytotoxic T cells from HLA-A*0201 patients with recent-onset T1D. Although not yet identified as the target of a pathogenic T-cell clone, IAPP is a very intriguing candidate antigen for a number of reasons. First, like insulin, this protein is beta cellspecific. Second, as the amyloid part of the name implies, this protein is found in aggregate forms and is a primary constituent of the pancreatic amyloid deposits found in type 2 diabetes patients (Nicolls, 2004). Although similar IAPP aggregates have not been reported in T1D patients, the fact that this protein may be a target for intracellular stress-induced alteration and can activate pro-inflammatory responses (Nicolls, 2004) makes it an interesting candidate for an autoantigen. A third reason it is of interest to us is that in a

148

k at h r yn h a s k i n s

previous study, we obtained results suggesting that IAPP might be a candidate antigen for BDC-6.9, one of our diabetogenic CD4 T-cell clones isolated from the NOD mouse (Dallas-Pedretti et al., 1995). The iapp gene maps to the same locus as the antigen gene for BDC-6.9 and is polymorphic since in the NOD mouse, the nucleotide sequence has three bases that differ from the published BALB/c sequence (Dallas-Pedretti et al., manuscript in preparation). As peptides from the IAPP prepropeptide do not stimulate the BDC-6.9 clone, we do not have direct evidence that IAPP is its antigen. Nevertheless, in light of the genetic data and the lack of antigenicity of islets from BALB/c mice, we are continuing to investigate the possibility of a link between this protein and the BDC-6.9 antigen. It would be of great interest if peptides from this protein, like those from insulin, could stimulate both CD4 and CD8 T-cell clones. The diabetogenic CD8 T-cell clone 8.3 (described in Section 2.2.) has also been used to screen combinatorial peptide libraries, leading to the identification of two major peptide ligands, NRP and NRP-A7 (Anderson et al., 1999). Investigations with these peptides indicated that the 8.3 TCR was prevalent in the islets of prediabetic NOD mice. The subsequent identification of the protein antigen for the 8.3 CD8 T-cell clone through a proteomics approach represents a considerable achievement and suggests that the technology is now at hand for this means of antigen discovery (Lieberman et al., 2003). To identify the 8.3 antigen, peptides were eluted from H-2Kd molecules purified from the NOD-derived pancreatic b-cell line NIT-1, and upon chromatographic separation, fractions were obtained for testing with the 8.3 T-cell clone. Analysis of the components that comprised the major peak of bioactivity by mass spectrometry led to the sequencing of a peptide that turned out to be the ligand for the T-cell clone. Subsequent protein database searches led to an exact match between the peptide and a sequence found in murine islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), a protein first identified through expression cloning of a pancreatic b-cellderived cDNA library (Arden et al., 1999). A peptide corresponding to residues 206–214 of murine IGRP was determined to be the antigenic epitope for the CD8 T-cell clone 8.3 and also for two other clones from a set of CD8 T-cell clones isolated from early insulitic lesions of young NOD mice. This peptide was also shown to be a good binder of H-2Kd, demonstrating that the poor MHC binding observed for the insulin B9-23 clone G9C8 (Wong et al., 2002) is not a universal property of self-peptides recognized by CD8 T-cell clones. In the most recent work by this group, AI4, the third pathogenic CD8 T-cell clone to be isolated from the NOD mouse (Graser et al., 2000) was used to screen a combinatorial peptide library in an effort to identify the antigen for

path o g e ni c t- c e l l c lo n e s

149

this clone. A peptide that binds to H-2Db and that is derived from the protein dystrophia myotonica kinase (DMK) was identified as the natural ligand for AI4 (Lieberman et al., 2004). DMK is a serine/threonine protein kinase expressed in most tissues (Jansen et al., 1992) and can be localized to different sites intracellularly (Wansink et al., 2003). Its exact function is not known, although it may have a variety of roles depending on the cell type, and it may be involved in myotonic dystrophy (Groenen and Wieringa, 1998). However, DMK has not previously been implicated as an autoantigen in T1D. The authors speculate on potential mechanisms by which this protein might elicit an autoimmune response, including the possibility that myositis observed in transgenic Th1 cytokine-deficient NOD mice (Serreze et al., 2003) could be due to AI4-like cells since DMK is a protein highly expressed in muscle. 6. Tracking of Pathogenic T-Cell Clones with MHC Tetramers 6.1. Tetramers for Pathogenic CD8 T-Cell Clones The development in recent years of MHC tetramers has provided a whole new set of tools for analysis of T-cell dynamics, distribution, and function (Klenerman et al., 2002). Since MHC tetramer technology was originally developed for analysis of class I restricted CD8 T cells, in which the TCR recognizes a MHC class I-antigen complex (Altman et al., 1996), in this section the discussion will begin with reagents for CD8 T cells. One of the first examples of a Class I tetramer used to stain T cells in the NOD mouse was that in which a G9 tetramer was made from the H-2Kd molecule and insulin peptide B15-23, and used to detect T cells recognizing this epitope. Cells were tested from spleen, pancreatic lymph nodes, and islets of NOD mice ranging in age from 4 weeks to diabetic (i.e., 4 months). Cells stained with the tetramer were found mostly in the islets and were the predominant population in islets from 4-week-old mice, but they comprised a much-reduced percentage of CD3þ T cells in older mice and/or diabetic mice. Few tetramer staining cells were found in spleen or pancreatic lymph nodes (Wong et al., 1999). In studies with Class I tetramers containing the NRP-A7 mimotope, an agonist peptide for the CD8 T cell 8.3 clone (Anderson et al., 1999), progression of disease in NOD mice was followed. The NRP-reactive CD8 T cells accumulate with age in the islets of prediabetic NOD mice and also in agedependent fashion, these cells were found to bind NRP-A7/H-2Kd tetramers with increased specificity, increased avidity, and longer half-lives (Amrani et al., 2000a). This result led to the hypothesis that progression of islet inflammation to overt diabetes is driven by avidity maturation of a prevailing CD8 T-cell population. An even higher affinity ligand (NRP-V7) of the 8.3

150

k at h r yn h a s k i n s

TCR made possible the development of high-avidity NRP/Class I MHC tetramers that could be used to detect NRP-reactive CD8 T cells in peripheral blood of NOD mice ex vivo, and quantification of this population in peripheral blood was found to be predictive of diabetes (Trudeau et al., 2003). The recent identification of IGRP as the protein antigen, and of an immunogenic peptide sequence from IGRP, has made possible the use of a Class I tetramer containing a natural T-cell ligand to track pathogenic CD8 T cells. The IGRP206-214 sequence differs from the peptide agonist NRP-V7 sequence by only three amino acids, and an IGRP peptide/H-2Kd tetramer was very comparable to the NRP-V7 tetramer in staining of the 8.3-like CD8 T cells in islets and peripheral blood of NOD mice (Lieberman et al., 2003). These results have implications of particular importance for human disease since better tools for diagnosis of T1D have been long sought. In one of the most recent reports on tracking diabetogenic T cells, peptide/ MHC tetramers were used to analyze at one time the distribution of the different pathogenic CD8 T-cell populations represented by G9C8, 8.3, and AI4, the three CD8 T-cell clones described under Section 2.2 of this review. Tetramers for each of these clones were used to detect and quantify all three CD8 populations among b cell-reactive T cells cultured from islets of individual NOD mice of different ages. One interesting result from this study was that even within age-matched groups, each mouse showed a unique distribution of CD8 b cell-reactive T cells in terms of numbers of tetramerstaining cells and in relative proportions of each population in islet infiltrates (Lieberman et al., 2004). These findings provide further evidence that disease progression varies considerably among individuals, even in the inbred NOD strain, and the data may also point toward the involvement of several antigen specificities in the development of diabetes. 6.2. Tetramers for Pathogenic CD4 T-Cell Clones Due to technical problems, the production of MHC class II tetramers proved to be more challenging than that of class I tetramers. However, the recent development of tetramers with the NOD I-Ag7 MHC class II molecule has provided new reagents for tracking autoreactive CD4 T-cell clones, as described in three reports published in 2003 within a few months of each other. All of these studies focus on the presence and distributions of the BDC2.5 clone in NOD mice or in the 2.5 TCR-Tg mouse. The first I-Ag7 tetramer contained a peptide (p79) with a sequence similar to a GAD peptide (Judkowski et al., 2001) and was found to be reactive with CD4 T cells from spleens and islets of 2.5 TCR-Tg mice (You et al., 2003). This tetramer could stain a small population of CD4 T cells in NOD mice also, but when the

path o g e ni c t- c e l l c lo n e s

151

tetramer was used to isolate T cells from NOD, the cultured T cells secreted more IL-10 than IFNg, less than 3% of this population expressed the Vb4 of the BDC-2.5 clone, and the T cells were unable to transfer diabetes. These results suggested that although the I-Ag7/p79 tetramer can detect 2.5-like T cells, only a small portion of p79-stimulated cells from NOD represent BDC-2.5 T cells. The second report (Stratmann et al., 2003) described a tetramer consisting of a peptide, chosen for its agonistic activity with BDC-2.5 (Yoshida et al., 2002), complexed to the NOD I-Ag7 molecule. When coated onto plates this tetramer could stimulate IL-2 production from a hybridoma made from the BDC-2.5 clone. It was also very specific for binding to BDC-2.5 and to BDC-5.10.3, a second clone from the BDC panel that shows the same peptide reactivity as BDC-2.5; no other clones in the panel were stained by the tetramer. When splenocytes from the 2.5 TCR-Tg mouse were stained, nearly 80% of the CD4 T cells were positive for the I-Ag7/2.5 mimotope tetramers, whereas there was no staining of T cells from the KRN mouse (Kouskoff et al., 1996), used as a negative control. A small population of CD4 T cells in wildtype NOD mice could also be observed to stain with the 2.5 tetramer, but not by I-Ag7 tetramers containing control peptides, and the number of cells in NOD mice reactive with the 2.5 tetramer could be increased through immunization with the mimotope peptide. Immunocytochemical staining of tissue in situ had not been previously reported with class II tetramers, and the ability to stain frozen tissue sections with the 2.5 tetramer proved to be another valuable feature of this new reagent. Extensive staining with the tetramer could be observed in 2.5 TCR-Tg spleen and pancreas, but tetramer staining could also be detected in prediabetic non-transgenic NOD mice, particularly in pancreatic lymph nodes and in islets. As had been previously observed with 2.5 TCR-Tg T cells (Hoglund et al., 1999), the in vivo activation of the 2.5-like T cells, signified by an increase in CD44hi T cells, was confined to the pancreatic lymph nodes, and suggested that the CD4 T cells detected by the tetramer in the NOD mouse were like the original BDC-2.5 T-cell clone in their antigen reactivity. This observation was strengthened by the fact that in a series of T-cell hybridomas generated from unimmunized NOD mice and selected for reactivity to the 2.5 tetramer, several reacted also to islet cells. Another notable result of this study was that the tetramer could detect 2.5-like T cells in I-Ag7 strains that do not develop diabetes, such as NOR and B6.H2g7 mice. This finding was interpreted by the authors to suggest that selection of BDC-2.5 T cells was dependent only on the NOD MHC class II haplotype and that other non-MHC genes were not required (Stratmann et al., 2003). However, this conclusion may not hold for all autoreactive CD4 T cells (Serreze and Leiter, 2003).

152

k at h r yn h a s k i n s

In the third study, Jang et al., generated I-Ag7/class II-associated invariant chain peptide (CLIP) precursors by peptide exchange, a novel approach that allows for investigation of different peptides with the same MHC classII/CLIP precursor (Jang et al., 2003). These investigators found that whereas no CD4 T-cell populations could be identified for either of two GAD65 peptides, tetramers containing BDC-2.5 peptide mimetopes (Judkowski et al., 2001; Yoshida et al., 2002) could detect CD4 T cells in NOD mice. An important result from this study was that cells staining with this BDC-2.5 tetramer could be detected in the thymus of young NOD mice as early as 2 weeks of age, before the onset of insulitis; in addition, the 2.5 tetramer labeled thymocytes in B10.H-2g7 but not in B10 mice. Based on these findings, the authors proposed that the T-cell repertoire in NOD mice is biased due to efficient positive selection of autoreactive T cells. 7. Concluding Remarks The purpose of this review has been to provide a comprehensive summary of recent advances in our understanding of the immunology of autoimmune diabetes made possible through the study of T-cell clones isolated from the NOD mouse. Relatively few laboratories have managed to maintain long-term cloned lines with stable phenotypic and functional properties, and thus the development of transgenic mice bearing the TCR of diabetogenic T-cell clones has greatly extended the availability of the reagents and the scope of the investigations. Nevertheless, established T-cell clones in culture provide the most unambiguous way to study at the single cell level the effector function of diabetogenic T cells in vitro and in vivo. The ability to reproducibly stimulate, expand, assay, and transfer cloned cell lines illustrates the great convenience and versatility of these reagents. Because they remain highly viable and diabetogenic for several days after expansion in IL-2, large numbers of the T-cell clones can be shipped to other laboratories, ready for analysis and/or injection into mice, omitting the need for others to establish the laborintensive process of maintaining the cell lines on a weekly basis. The T-cell clones covered in this review are those involved in the destructive pathology of T1D and do not include cells involved in the regulation of the autoimmune response. Regulatory T cells are a hot topic, and there are many recent reviews on the subject. However, with a focus on T-cell clones from the NOD mouse, there is little to say about regulatory T cells since very few cloned lines have been made from these mice. In the rare cases in which T-cell clones or lines have been reported to have regulatory activity, there is little if any follow-up, and so drawing any conclusions about regulatory T cells from cloned lines at this point is not a practical exercise. With the great interest in these

path o g e ni c t- c e l l c lo n e s

153

cells, along with the considerable confusion as to what phenotypes actually constitute regulatory T cells, there is bound to be good progress in this area in the near future, perhaps including production of some new T-cell clones with regulatory properties. The development of transgenic mice bearing the T-cell receptors of diabetogenic T-cell clones has increased the availability of these reagents and provided genetically altered versions of the NOD mouse for study. The TCR-Tg mouse made from the BDC-2.5 clone has been widely used, and this mouse has provided new insights into the progression and regulation of autoimmune diabetes, as has the 4.1 mouse made from another highly diabetogenic NOD CD4 T-cell clone. The new 6.9 TCR-Tg mouse, described in this review, has been bred onto an antigen-deficient background and may thus provide new opportunities to investigate naı¨ve versus activated diabetogenic T cells. There are also transgenic mice produced with TCR from diabetogenic CD8 T-cell clones such as the TCR-Tg mice made from the 8.3 and AI4 clones, both of which have revealed important new findings about CD8 T cell contributions to pathogenesis. These five TCR-Tg mice, however, represent a rather small yield of such animals, considering the numerous attempts to produce transgenic mice that exhibit spontaneous islet pathology. Thus the new retrogenic approach may extend the numbers and types of animals that can be used to study T-cell contributions to diabetes. The section of this review devoted to autoantigens for T-cell clones reflects in part the large number of studies on this subject and the controversy that has emerged from them. One of the reasons that there are so many candidate autoantigens for T cells, and why so many investigators are attempting to define which ones may be important in pathogenesis and regulation of disease, is that T1D is unusual among organ-specific autoimmune diseases in that no one antigen has been identified that can induce disease. (A notable exception to this rule is the report by Zekzer et al. (1998) that immunization of a 3-weekold NOD mouse with an affinity-purified preparation of brain GAD led to development of diabetes in that mouse at 6 weeks of age.) Interest in T-cell autoantigens in T1D remains high as new candidate antigens are identified for both CD4 and CD8 T-cell clones. With improvements in the development of peptide libraries and in approaches based on proteomics continuing at a rapid pace, the progress in this field can only be expected to increase. In the last section of this review, an effort has been made to highlight the newest developments in the use of MHC tetramers, reagents that allow for studies on tracking and distribution of diabetogenic T-cell clones. Several of these reagents have now been used to follow the appearance and migration of T cells in both TCR-transgenic and NOD mice. As MHC multimers have a high potential for diagnosing and following disease in human patients, these

154

k at h r yn h a s k i n s

reagents constitute one of the more important advances on the diabetes research front. Although applying multimer technology to the monitoring of human T-cell responses is still a great challenge due to the large number of HLA alleles in human populations, autoimmune diseases provide a special case in that for most, such as T1D, HLA genetic associations with disease are high, and a majority of patients express one or two alleles (Nepom, 2003). For example, class II/GAD tetramers were used to probe the TCR specificity and avidity of GAD65-reactive T-cell clones isolated from T1D patients, leading to the identification of both high-avidity and low-avidity CD4 T cells to the same GAD65 epitope in peripheral blood (Reijonen et al., 2004). These studies have demonstrated that is possible to identify, recover, and study isletantigen-specific T cells from human blood, and thus, the opportunities for using MHC multimers are rapidly expanding, including new possibilities for ex vivo cell expansion, immunotherapy, and improved patient monitoring (Nepom, 2003). References Abramson, O., Qiu, S., and Erle, D. J. (2001). Preferential production of interferon-gamma by CD4þ T cells expressing the homing receptor integrin alpha4/beta7. Immunology 103, 155–163. Achenbach, P., Kelemen, K., Wegmann, D. R., and Hutton, J. C. (2002). Spontaneous peripheral T-cell responses to the IA-2beta (phogrin) autoantigen in young nonobese diabetic mice. J. Autoimmun. 19, 111–116. Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J., and Davis, M. M. (1996). Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96. Amrani, A., Verdaguer, J., Serra, P., Tafuro, S., Tan, R., and Santamaria, P. (2000a). Progression of autoimmune diabetes driven by avidity maturation of a T-cell population. Nature 406, 739–742. Amrani, A., Verdaguer, J., Thiessen, S., Bou, S., and Santamaria, P. (2000b). IL-1alpha, IL-1beta, and IFN-gamma mark beta cells for Fas-dependent destruction by diabetogenic CD4(þ) T lymphocytes. J. Clin. Invest. 105, 459–468. Anderson, B., Park, B. J., Verdaguer, J., Amrani, A., and Santamaria, P. (1999). Prevalent CD8(þ) T cell response against one peptide/MHC complex in autoimmune diabetes. Proc. Natl. Acad. Sci. USA 96, 9311–9316. Andre-Schmutz, I., Hindelang, C., Benoist, C., and Mathis, D. (1999). Cellular and molecular changes accompanying the progression from insulitis to diabetes. Eur. J. Immunol. 29, 245–255. Arden, S. D., Zahn, T., Steegers, S., Webb, S., Bergman, B., O’Brien, R. M., and Hutton, J. C. (1999). Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunitrelated protein. Diabetes 48, 531–542. Arnold, P. Y., Burton, A. R., and Vignali, D. A. (2004). Diabetes incidence is unaltered in glutamate decarboxylase 65-specific TCR retrogenic nonobese diabetic mice: Generation by retroviralmediated stem cell gene transfer. J. Immunol. 173, 3103–3111. Bendelac, A., Carnaud, C., Boitard, C., and Bach, J. F. (1987). Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4þ and Lyt-2þ T cells. J. Exp. Med. 166, 823–832.

path o g e ni c t- c e l l c lo n e s

155

Bergman, B., and Haskins, K. (1994). Islet-specific T-cell clones from the NOD mouse respond to beta-granule antigen. Diabetes 43, 197–203. Bergman, B., and Haskins, K. (1997). Autoreactive T-cell clones from the nonobese diabetic mouse. Proc. Soc. Exp. Biol. Med. 214, 41–48. Bergman, B., McManaman, J. L., and Haskins, K. (2000). Biochemical characterization of a beta cell membrane fraction antigenic for autoreactive T-cell clones. J. Autoimmun. 14, 343–351. Bradley, L. M., Asensio, V. C., Schioetz, L. K., Harbertson, J., Krahl, T., Patstone, G., Woolf, N., Campbell, I. L., and Sarvetnick, N. (1999). Islet-specific Th1, but not Th2, cells secrete multiple chemokines and promote rapid induction of autoimmune diabetes. J. Immunol. 162, 2511–2520. Brandtzaeg, P., Farstad, I. N., and Haraldsen, G. (1999). Regional specialization in the mucosal immune system: Primed cells do not always home along the same track. Immunol. Today 20, 267–277. Brimnes, M. K., Jensen, T., Jorgensen, T. N., Michelsen, B. K., Troelsen, J., and Werdelin, O. (2002). Low expression of insulin in the thymus of non-obese diabetic mice. J. Autoimmun. 19, 203–213. Butcher, E. C., Williams, M., Youngman, K., Rott, L., and Briskin, M. (1999). Lymphocyte trafficking and regional immunity. Adv. Immunol. 72, 209–253. Candeias, S., Katz, J., Benoist, C., Mathis, D., and Haskins, K. (1991). Islet-specific T-cell clones from nonobese diabetic mice express heterogeneous T-cell receptors. Proc. Natl. Acad. Sci. USA 88, 6167–6170. Crawford, F., Huseby, E., White, J., Marrack, P., and Kappler, J. W. (2004). Mimotopes for Alloreactive and Conventional T Cells in a Peptide-MHC Display Library. PLoS Biol. 2, E90. Cuff, C. A., Schwartz, J., Bergman, C. M., Russell, K. S., Bender, J. R., and Ruddle, N. H. (1998). Lymphotoxin alpha3 induces chemokines and adhesion molecules: Insight into the role of LT alpha in inflammation and lymphoid organ development. J. Immunol. 161, 6853–6860. Dallas-Pedretti, A., McDuffie, M., and Haskins, K. (1995). A diabetes-associated T-cell autoantigen maps to a telomeric locus on mouse chromosome 6. Proc. Natl. Acad. Sci. USA 92, 1386–1390. Daniel, D., Gill, R. G., Schloot, N., and Wegmann, D. (1995). Epitope specificity, cytokine production profile and diabetogenic activity of insulin-specific T-cell clones isolated from NOD mice. Eur. J. Immunol. 25, 1056–1062. Daniel, D., and Wegmann, D. R. (1996a). Intranasal administration of insulin peptide B: 9-23 protects NOD mice from diabetes. Ann. N. Y. Acad. Sci. 778, 371–372. Daniel, D., and Wegmann, D. R. (1996b). Protection of nonobese diabetic mice from diabetes by intranasal or subcutaneous administration of insulin peptide B-(9-23). Proc. Natl. Acad. Sci. USA 93, 956–960. Delovitch, T. L., and Singh, B. (1997). The nonobese diabetic mouse as a model of autoimmune diabetes: Immune dysregulation gets the NOD. Immunity 7, 727–738. DiLorenzo, T. P., Graser, R. T., Ono, T., Christianson, G. J., Chapman, H. D., Roopenian, D. C., Nathenson, S. G., and Serreze, D. V. (1998). Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T-cell receptor alpha chain gene rearrangement. Proc. Natl. Acad. Sci. USA 95, 12538–12543. Dobbs, C., and Haskins, K. (2001). Comparison of a T-cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical. J. Immunol. 166, 2495–2504. Dorner, B. G., Scheffold, A., Rolph, M. S., Huser, M. B., Kaufmann, S. H., Radbruch, A., Flesch, I. E., and Kroczek, R. A. (2002). MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines. Proc. Natl. Acad. Sci. USA 99, 6181–6186.

156

k at h r yn h a s k i n s

Dorner, B. G., Steinbach, S., Huser, M. B., Kroczek, R. A., and Scheffold, A. (2003). Single-cell analysis of the murine chemokines MIP-1alpha, MIP-1beta, RANTES and ATAC/lymphotactin by flow cytometry. J. Immunol. Methods 274, 83–91. Ellerman, K. E., and Like, A. A. (1999). Islet cell membrane antigens activate diabetogenic CD4þ T-cells in the BB/Wor rat. Diabetes 48, 975–982. Fleming, T. J., and Malek, T. R. (1994). Multiple glycosylphosphatidylinositol-anchored Ly-6 molecules and transmembrane Ly-6E mediate inhibition of IL-2 production. J. Immunol. 153, 1955–1962. Gonzalez, A., Andre-Schmutz, I., Carnaud, C., Mathis, D., and Benoist, C. (2001). Damage control, rather than unresponsiveness, effected by protective DX5þ T cells in autoimmune diabetes. Nat. Immunol. 2, 1117–1125. Gonzalez, A., Katz, J. D., Mattei, M. G., Kikutani, H., Benoist, C., and Mathis, D. (1997). Genetic control of diabetes progression. Immunity 7, 873–883. Graser, R. T., DiLorenzo, T. P., Wang, F., Christianson, G. J., Chapman, H. D., Roopenian, D. C., Nathenson, S. G., and Serreze, D. V. (2000). Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions. J. Immunol. 164, 3913–3918. Groenen, P., and Wieringa, B. (1998). Expanding complexity in myotonic dystrophy. Bioessays 20, 901–912. Gurlo, T., Kawamura, K., and von Grafenstein, H. (1999). Role of inflammatory infiltrate in activation and effector function of cloned islet reactive nonobese diabetic CD8þ T cells: Involvement of a nitric oxide-dependent pathway. J. Immunol. 163, 5770–5780. Hagopian, W. A., Michelsen, B., Karlsen, A. E., Larsen, F., Moody, A., Grubin, C. E., Rowe, R., Petersen, J., McEvoy, R., and Lernmark, A. (1993). Autoantibodies in IDDM primarily recognize the 65,000-M(r) rather than the 67,000-M(r) isoform of glutamic acid decarboxylase. Diabetes 42, 631–636. Halbout, P., Briand, J. P., Becourt, C., Muller, S., and Boitard, C. (2002). T cell response to preproinsulin I and II in the nonobese diabetic mouse. J. Immunol. 169, 2436–2443. Hanninen, A., Jaakkola, I., and Jalkanen, S. (1998). Mucosal addressin is required for the development of diabetes in nonobese diabetic mice. J. Immunol. 160, 6018–6025. Hanninen, A., Jaakkola, I., Salmi, M., Simell, O., and Jalkanen, S. (1997). Ly-6C regulates endothelial adhesion and homing of CD8(þ) T cells by activating integrin-dependent adhesion pathways. Proc. Natl. Acad. Sci. USA 94, 6898–6903. Haskins, K. (2004). T-cell receptor transgenic (TCR-Tg) mice from two diabetogenic CD4þ islet-antigen-specific T-cell clones. J. Autoimmun. 22, 107–109. Haskins, K., and McDuffie, M. (1990). Acceleration of diabetes in young NOD mice with a CD4þ islet-specific T cell clone. Science 249, 1433–1436. Haskins, K., Portas, M., Bergman, B., Lafferty, K., and Bradley, B. (1989). Pancreatic isletspecific T-cell clones from nonobese diabetic mice. Proc. Natl. Acad. Sci. USA 86, 8000–8004. Haskins, K., Portas, M., Bradley, B., Wegmann, D., and Lafferty, K. (1988). T-lymphocyte clone specific for pancreatic islet antigen. Diabetes 37, 1444–1448. Haskins, K., and Wegmann, D. (1996). Diabetogenic T-cell clones. Diabetes 45, 1299–1305. Herold, K. C., Montag, A. G., Meyer, S. M., Wojcikowski, C., and Fitch, F. W. (1990). Expression of Ly-6C by T lymphocytes of NOD mice after CD3-complex stimulation. Identification of activated cells during insulitis of prediabetic mice. Diabetes 39, 815–820. Hiemstra, H. S., Duinkerken, G., Benckhuijsen, W. E., Amons, R., de Vries, R. R., Roep, B. O., and Drijfhout, J. W. (1997). The identification of CD4þ T-cell epitopes with dedicated synthetic peptide libraries. Proc. Natl. Acad. Sci. USA 94, 10313–10318.

path o g e ni c t- c e l l c lo n e s

157

Hoglund, P., Mintern, J., Waltzinger, C., Heath, W., Benoist, C., and Mathis, D. (1999). Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes. J. Exp. Med. 189, 331–339. Huang, G. C., Tremble, J., Bailyes, E., Arden, S. D., Kaye, T., McGregor, A. M., and Banga, J. P. (1995). HLA-DR-restricted T-cell lines from newly diagnosed type 1 diabetic patients specific for insulinoma and normal islet beta cell proteins: Lack of reactivity to glutamic acid decarboxylase. Clin. Exp. Immunol. 102, 152–158. Hutton, J. C. (1989). The insulin secretory granule. Diabetologia 32, 271–281. Hynes, R. O. (1992). Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11–25. Hynes, R. O. (2002). Integrins: Bidirectional, allosteric signaling machines. Cell 110, 673–687. Jaakkola, I., Merinen, M., Jalkanen, S., and Hanninen, A. (2003). Ly6C induces clustering of LFA1 (CD11a/CD18) and is involved in subtype-specific adhesion of CD8 T cells. J. Immunol. 170, 1283–1290. Jang, M. H., Seth, N. P., and Wucherpfennig, K. W. (2003). Ex vivo analysis of thymic CD4 T cells in nonobese diabetic mice with tetramers generated from I-A(g7)/class II-associated invariant chain peptide precursors. J. Immunol. 171, 4175–4186. Jansen, A., Homo-Delarche, F., Hooijkaas, H., Leenen, P. J., Dardenne, M., and Drexhage, H. A. (1994). Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and beta-cell destruction in NOD mice. Diabetes 43, 667–675. Jansen, G., Mahadevan, M., Amemiya, C., Wormskamp, N., Segers, B., Hendriks, W., O’Hoy, K., Baird, S., Sabourin, L., Lennon, G., et al (1992). Characterization of the myotonic dystrophy region predicts multiple protein isoform-encoding mRNAs. Nat. Genet. 1, 261–266. Judkowski, V., Krakowski, M., Rodriguez, E., Mocnick, L., Santamaria, P., and Sarvetnick, N. (2004). Increased islet antigen presentation leads to type-1 diabetes in mice with autoimmune susceptibility. Eur. J. Immunol. 34, 1031–1040. Judkowski, V., Pinilla, C., Schroder, K., Tucker, L., Sarvetnick, N., and Wilson, D. B. (2001). Identification of MHC class II-restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice. J. Immunol. 166, 908–917. Katz, J. D., Benoist, C., and Mathis, D. (1995). T helper cell subsets in insulin-dependent diabetes. Science 268, 1185–1188. Katz, J. D., Wang, B., Haskins, K., Benoist, C., and Mathis, D. (1993). Following a diabetogenic T cell from genesis through pathogenesis. Cell 74, 1089–1100. Kaufman, D. L., Clare-Salzler, M., Tian, J., Forsthuber, T., Ting, G. S., Robinson, P., Atkinson, M. A., Sercarz, E. E., Tobin, A. J., and Lehmann, P. V. (1993). Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 366, 69–72. Kelemen, K., Crawford, M. L., Gill, R. G., Hutton, J. C., and Wegmann, D. (1999). Cellular immune response to phogrin in the NOD mouse: Cloned T-cells cause destruction of islet transplants. Diabetes 48, 1529–1534. Kelemen, K., Gottlieb, P. A., Putnam, A. L., Davidson, H. W., Wegmann, D. R., and Hutton, J. C. (2004). HLA-DQ8-associated T cell responses to the diabetes autoantigen phogrin (IA-2 beta) in human prediabetes. J. Immunol. 172, 3955–3962. Kelemen, K., Wegmann, D. R., and Hutton, J. C. (2001). T-cell epitope analysis on the autoantigen phogrin (IA-2beta) in the nonobese diabetic mouse. Diabetes 50, 1729–1734. Klenerman, P., Cerundolo, V., and Dunbar, P. R. (2002). Tracking T cells with tetramers: New tales from new tools. Nat. Rev. Immunol. 2, 263–272.

158

k at h r yn h a s k i n s

Kommajosyula, S., Reddy, S., Nitschke, K., Kanwar, J. R., Karanam, M., and Krissansen, G. W. (2001). Leukocytes infiltrating the pancreatic islets of nonobese diabetic mice are transformed into inactive exiles by combinational anti-cell adhesion therapy. J. Leukoc. Biol. 70, 510–517. Kouskoff, V., Korganow, A. S., Duchatelle, V., Degott, C., Benoist, C., and Mathis, D. (1996). Organ-specific disease provoked by systemic autoimmunity. Cell 87, 811–822. Krawczyk, C., Bachmaier, K., Sasaki, T., Jones, G. R., Snapper, B. S., Bouchard, D., Kozieradzki, I., Ohashi, S. P., Alt, W. F., and Penninger, M. J. (2000). Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells. Immunity 13, 463–473. Kurrer, M. O., Pakala, S. V., Hanson, H. L., and Katz, J. D. (1997). Beta cell apoptosis in T cell-mediated autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94, 213–218. Lafaille, J. J. (2004). T-cell receptor transgenic mice in the study of autoimmune diseases. J. Autoimmun. 22, 95–106. Lan, M. S., Wasserfall, C., Maclaren, N. K., and Notkins, A. L. (1996). IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus. Proc. Natl. Acad. Sci. USA 93, 6367–6370. Langmuir, P. B., Bridgett, M. M., Bothwell, A. L., and Crispe, I. N. (1993). Bone marrow abnormalities in the non-obese diabetic mouse. Int. Immunol. 5, 169–177. Levisetti, M. G., Suri, A., Vidavsky, I., Gross, M. L., Kanagawa, O., and Unanue, E. R. (2003). Autoantibodies and CD4 T cells target a beta cell retroviral envelope protein in non-obese diabetic mice. Int. Immunol. 15, 1473–1483. Liblau, R. S., Wong, F. S., Mars, L. T., and Santamaria, P. (2002). Autoreactive CD8 T cells in organ-specific autoimmunity: Emerging targets for therapeutic intervention. Immunity 17, 1–6. Lieberman, S. M., and DiLorenzo, T. P. (2003). A comprehensive guide to antibody and T-cell responses in type 1 diabetes. Tissue Antigens 62, 359–377. Lieberman, S. M., Evans, A. M., Han, B., Takaki, T., Vinnitskaya, Y., Caldwell, J. A., Serreze, D. V., Shabanowitz, J., Hunt, D. F., Nathenson, S. G., Santamaria, P., and DiLorenzo, T. P. (2003). Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8þ T cells in autoimmune diabetes. Proc. Natl. Acad. Sci. USA 100(14), 8384–8388. Lieberman, S. M., Takaki, T., Han, B., Santamaria, P., Serreze, D. V., and DiLorenzo, T. P. (2004). Individual Nonobese Diabetic Mice Exhibit Unique Patterns of CD8þ T Cell reactivity to three islet antigens, including the newly identified widely expressed Dystrophia myotonica kinase. J. Immunol. 173, 6727–6734. Luhder, F., Hoglund, P., Allison, J. P., Benoist, C., and Mathis, D. (1998). Cytotoxic T lymphocyteassociated antigen 4 (CTLA-4) regulates the unfolding of autoimmune diabetes. J. Exp. Med. 187, 427–432. McMurray, R. W. (1996). Adhesion molecules in autoimmune disease. Semin. Arthritis Rheum. 25, 215–233. Miller, B. J., Appel, M. C., O’Neil, J. J., and Wicker, L. S. (1988). Both the Lyt-2þ and L3T4þ T-cell subsets are required for the transfer of diabetes in nonobese diabetic mice. J. Immunol. 140, 52–58. Moseley, T. A., Haudenschild, D. R., Rose, L., and Reddi, A. H. (2003). Interleukin-17 family and IL-17 receptors. Cytokine. Growth Factor. Rev. 14, 155–174. Moser, B., and Loetscher, D. R. (2001). Lymphocyte traffic control by chemokines. Nat. Immunol. 2, 123–128. Nagata, M., Santamaria, P., Kawamura, T., Utsugi, T., and Yoon, J. W. (1994). Evidence for the role of CD8þ cytotoxic T cells in the destruction of pancreatic beta-cells in nonobese diabetic mice. J. Immunol. 152, 2042–2050.

path o g e ni c t- c e l l c lo n e s

159

Nagata, M., and Yoon, J. W. (1992). Studies on autoimmunity for T-cell-mediated b-cell destruction. Distinct difference in b-cell destruction between CD4þ and CD8þ T-cell clones derived from lymphocytes infiltrating the islets of NOD mice. Diabetes 41, 998–1008. Nakano, N., Kikutani, H., Nishimoto, H., and Kishimoto, T. (1991). T cell receptor V gene usage of islet beta cell-reactive T cells is not restricted in non-obese diabetic mice. J. Exp. Med. 173, 1091–1097. Nepom, G. T. (2003). MHC multimers: Expanding the clinical toolkit. Clin. Immunol. 106, 1–4. Nicolls, M. (2004). The clinical and biological relationship between Type II Diabetes Mellitus and Alzheimer’s Disease. Curr. Alzheimer Res. 1, 47–54. Notkins, A. L., and Lernmark, A. (2001). Autoimmune type 1 diabetes: Resolved and unresolved issues. J. Clin. Invest. 108, 1247–1252. O’Shea, J. J., Ma, A., Banatvala, J. E., and Shepherd, W. M. (2002). Cytokines and autoimmunity. Nat. Rev. 2, 37–45. Pakala, S. V., Chivetta, M., Kelly, C. B., and Katz, J. D. (1999). In autoimmune diabetes the transition from benign to pernicious insulitis requires an islet cell response to tumor necrosis factor alpha. J. Exp. Med. 189, 1053–1062. Panagiotopoulos, C., Qin, H., Tan, R., and Verchere, C. B. (2003). Identification of a beta-cellspecific HLA class I restricted epitope in type 1 diabetes. Diabetes 52, 2647–2651. Pauza, M. E., Dobbs, C. M., He, J., Patterson, T., Wagner, S., Anobile, B. S., Bradley, B. J., Lo, D., and Haskins, K. (2004). T-Cell Receptor Transgenic Response to an Endogenous Polymorphic Autoantigen Determines Susceptibility to Diabetes. Diabetes 53, 978–988. Peterson, J. D., and Haskins, K. (1996). Transfer of diabetes in the NOD-scid mouse by CD4 T-cell clones. Differential requirement for CD8 T-cells. Diabetes 45, 328–336. Peterson, J. D., Pike, B., Dallas-Pedretti, A., and Haskins, K. (1995). Induction of diabetes with islet-specific T-cell clones is age dependent. Immunology 85, 455–460. Peterson, J. D., Pike, B., McDuffie, M., and Haskins, K. (1994). Islet-specific T-cell clones transfer diabetes to nonobese diabetic (NOD) F1 mice. J. Immunol. 153, 2800–2806. Philbrick, W. M., Maher, S. E., Bridgett, M. M., and Bothwell, A. L. (1990). A recombination event in the 50 flanking region of the Ly-6C gene correlates with impaired expression in the NOD, NZB and ST strains of mice. EMBO J. 9, 2485–2492. Phillips, J. M., Harach, S. Z., Parish, N. M., Fehervari, Z., Haskins, K., and Cooke, A. (2000). Nondepleting anti-CD4 has an immediate action on diabetogenic effector cells, halting their destruction of pancreatic beta cells. J. Immunol. 165, 1949–1955. Picarella, D. E., Kratz, A., Li, C. B., Ruddle, N. H., and Flavell, R. A. (1992). Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas. Proc. Natl. Acad. Sci. USA 89, 10036–10040. Poulin, M., and Haskins, K. (2000). Induction of diabetes in nonobese diabetic mice by Th2 T-cell clones from a TCR transgenic mouse. J. Immunol. 164, 3072–3078. Pribila, J. T., Quale, A. C., Mueller, K. L., and Shimizu, Y. (2004). Integrins and T cell-mediated immunity. Annu. Rev. Immunol. 22, 157–180. Quinn, A. (2003). Antigen-induced T1D in NOD mice. J. Autoimmun. 20, 207–210. Quinn, A., McInerney, B., Reich, E. P., Kim, O., Jensen, K. P., and Sercarz, E. E. (2001a). Regulatory and effector CD4 T cells in nonobese diabetic mice recognize overlapping determinants on glutamic acid decarboxylase and use distinct V beta genes. J. Immunol. 166, 2982–2991. Quinn, A., McInerney, M. F., and Sercarz, E. E. (2001b). MHC class I-restricted determinants on the glutamic acid decarboxylase 65 molecule induce spontaneous CTL activity. J. Immunol. 167, 1748–1757.

160

k at h r yn h a s k i n s

Quinn, A., and Sercarz, E. E. (1996). T cells with multiple fine specificities are used by non-obese diabetic (NOD) mice in the response to GAD(524-543). J. Autoimmun. 9, 365–370. Reijonen, H., Mallone, R., Heninger, A. K., Laughlin, E. M., Kochik, S. A., Falk, B., Kwok, W. W., Greenbaum, C., and Nepom, G. T. (2004). GAD65-specific CD4þ T-cells with high antigen avidity are prevalent in peripheral blood of patients with type 1 diabetes. Diabetes 53, 1987–1994. Roep, B. O. (2002). Autoreactive T cells in endocrine/organ-specific autoimmunity: Why has progress been so slow? Springer Semin. Immunopathol. 24, 261–271. Roep, B. O., Arden, S. D., de Vries, R. R., and Hutton, J. C. (1990). T-cell clones from a type-1 diabetes patient respond to insulin secretory granule proteins. Nature 345, 632–634. Rosmalen, J. G., Martin, T., Dobbs, C., Voerman, J. S., Drexhage, H. A., Haskins, K., and Leenen, P. J. (2000). Subsets of macrophages and dendritic cells in nonobese diabetic mouse pancreatic inflammatory infiltrates: Correlation with the development of diabetes. Lab. Invest. 80, 23–30. Rossini, A. A. (2004). Autoimmune diabetes and the circle of tolerance. Diabetes 53, 267–275. Sacca, R., Cuff, C. A., Lesslauer, W., and Ruddle, N. H. (1998). Differential activities of secreted lymphotoxin-alpha3 and membrane lymphotoxin-alpha1beta2 in lymphotoxin-induced inflammation: Critical role of TNF receptor 1 signaling. J. Immunol. 160, 485–491. Savinov, A. Y., Wong, F. S., Stonebraker, A. C., and Chervonsky, A. V. (2003). Presentation of antigen by endothelial cells and chemoattraction are required for homing of insulin-specific CD8þ T cells. J. Exp. Med. 197, 643–656. Schloot, N. C., Daniel, D., Norbury-Glaser, M., and Wegmann, D. R. (1996). Peripheral T-cell clones from NOD mice specific for GAD65 peptides: Lack of islet responsiveness or diabetogenicity. J. Autoimmun. 9, 357–363. Schmidt, D., Amrani, A., Verdaguer, J., Bou, S., and Santamaria, P. (1999). Autoantigen-independent deletion of diabetogenic CD4þ thymocytes by protective MHC class II molecules. J. Immunol. 162, 4627–4636. Schmidt, D., Verdaguer, J., Averill, N., and Santamaria, P. (1997). A mechanism for the major histocompatibility complex-linked resistance to autoimmunity. J. Exp. Med. 186, 1059–1075. Serreze, D. V., and Leiter, E. H. (2003). Tracking autoimmune T cells in diabetes. J. Clin. Invest. 112, 826–828. Serreze, D. V., Leiter, E. H., Christianson, G. J., Greiner, D., and Roopenian, D. C. (1994). Major histocompatibility complex class I-deficient NOD-B2mnull mice are diabetes and insulitis resistant. Diabetes 43, 505–509. Serreze, D. V., Pierce, M. A., Post, C. M., Chapman, H. D., Savage, H., Bronson, R. T., Rothman, P. B., and Cox, G. A. (2003). Paralytic autoimmune myositis develops in nonobese diabetic mice made Th1 cytokine-deficient by expression of an IFN-gamma receptor beta-chain transgene. J. Immunol. 170, 2742–2749. Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004). Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 116, 577–589. Shimizu, J., Kanagawa, O., and Unanue, E. R. (1993). Presentation of beta-cell antigens to CD4þ and CD8þ T cells of non-obese diabetic mice. J. Immunol. 151, 1723–1730. Simone, E., Daniel, D., Schloot, N., Gottlieb, P., Babu, S., Kawasaki, E., Wegmann, D., and Eisenbarth, G. S. (1997). T-cell receptor restriction of diabetogenic autoimmune NOD T cells. Proc. Natl. Acad. Sci. USA 94, 2518–2521. Simone, E. A., Yu, L., Wegmann, D. R., and Eisenbarth, G. S. (1997). T cell receptor gene polymorphisms associated with anti-insulin, autoimmune T cells in diabetes-prone NOD mice. J. Autoimmun. 10, 317–321.

path o g e ni c t- c e l l c lo n e s

161

Stamp, L. K., James, M. J., and Cleland, L. G. (2004). Interleukin-17: The missing link between T-cell accumulation and effector cell actions in rheumatoid arthritis? Immunol. Cell. Biol. 82, 1–9. Stratmann, T., Martin-Orozco, N., Mallet-Designe, V., Poirot, L., McGavern, D., Losyev, G., Dobbs, C. M., Oldstone, M. B., Yoshida, K., Kikutani, H., Mathis, D., Benoist, C., Haskins, K., and Teyton, L. (2003). Susceptible MHC alleles, not background genes, select an autoimmune T cell reactivity. J. Clin. Invest. 112, 902–914. Supon, P., Stecha, P., and Haskins, K. (1990). Anti-islet cell antibodies from NOD mice. Diabetes 39, 1366–1372. Suri, A., and Katz, J. D. (1999). Dissecting the role of CD4þ T cells in autoimmune diabetes through the use of TCR transgenic mice. Immunol. Rev. 169, 55–65. Tisch, R., Wang, B., Atkinson, M. A., Serreze, D. V., and Friedline, R. (2001). A glutamic acid decarboxylase 65-specific Th2 cell clone immunoregulates autoimmune diabetes in nonobese diabetic mice. J. Immunol. 166, 6925–6936. Tisch, R., Yang, X. D., Singer, S. M., Liblau, R. S., Fugger, L., and McDevitt, H. O. (1993). Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 366, 72–75. Trudeau, J. D., Kelly-Smith, C., Verchere, C. B., Elliott, J. F., Dutz, J. P., Finegood, D. T., Santamaria, P., and Tan, R. (2003). Prediction of spontaneous autoimmune diabetes in NOD mice by quantification of autoreactive T cells in peripheral blood. J. Clin. Invest. 111, 217–223. Tseng, S. Y., and Dustin, M. L. (2002). T-cell activation: A multidimensional signaling network. Curr. Opin. Cell. Biol. 14, 575–580. Verdaguer, J., Schmidt, D., Amrani, A., Anderson, B., Averill, N., and Santamaria, P. (1997). Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice. J. Exp. Med. 186, 1663–1676. Verdaguer, J., Yoon, J. W., Anderson, B., Averill, N., Utsugi, T., Park, B. J., and Santamaria, P. (1996). Acceleration of spontaneous diabetes in TCR-beta-transgenic nonobese diabetic mice by beta-cell cytotoxic CD8þ T cells expressing identical endogenous TCR-alpha chains. J. Immunol. 157, 4726–4735. Verge, C. F., Gianani, R., Kawasaki, E., Yu, L., Pietropaolo, M., Jackson, R. A., Chase, H. P., and Eisenbarth, G. S. (1996). Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies. Diabetes 45, 926–933. Videbaek, N., Harach, S., Phillips, J., Hutchings, P., Ozegbe, P., Michelsen, B. K., and Cooke, A. (2003). An islet-homing NOD CD8þ cytotoxic T cell clone recognizes GAD65 and causes insulitis. J. Autoimmun. 20, 97–109. Wang, Y., Hao, L., Gill, R. G., and Lafferty, K. J. (1987). Autoimmune diabetes in NOD mouse is L3T4 T-lymphocyte dependent. Diabetes 36, 535–538. Wansink, D. G., van Herpen, R. E., Coerwinkel-Driessen, M. M., Groenen, P. J., Hemmings, B. A., and Wieringa, B. (2003). Alternative splicing controls myotonic dystrophy protein kinase structure, enzymatic activity, and subcellular localization. Mol. Cell. Biol. 23, 5489–5501. Ward, S. G., Bacon, K., and Westwick, J. (1998). Chemokines and T lymphocytes: More than an attraction. Immunity 9, 1–11. Wasmeier, C., and Hutton, J. C. (1996). Molecular cloning of phogrin, a protein-tyrosine phosphatase homologue localized to insulin secretory granule membranes. J. Biol. Chem. 271, 18161–18170. Wegmann, D. R., and Eisenbarth, G. S. (2000). It’s insulin. J. Autoimmun. 15, 286–291. Wegmann, D. R., Norbury-Glaser, M., and Daniel, D. (1994). Insulin-specific T cells are a predominant component of islet infiltrates in prediabetic NOD mice. Eur. J. Immunol. 24, 1853–1857.

162

k at h r yn h a s k i n s

Wen, L., Wong, F. S., Burkly, L., Altieri, M., Mamalaki, C., Kioussis, D., Flavell, R. A., and Sherwin, R. S. (1998). Induction of insulitis by glutamic acid decarboxylase peptide-specific and HLA-DQ8-restricted CD4(þ) T cells from human DQ transgenic mice. J. Clin. Invest. 102, 947–957. Wilson, D. B. (2003). GAD-about BDC2.5: peptides that stimulate BDC2.5 T cells and inhibit IDDM. J. Autoimmun. 20, 199–201. Wilson, D. B., Pinilla, C., Wilson, D. H., Schroder, K., Boggiano, C., Judkowski, V., Kaye, J., Hemmer, B., Martin, R., and Houghten, R. A. (1999). Immunogenicity. I. Use of peptide libraries to identify epitopes that activate clonotypic CD4þ T cells and induce T cell responses to native peptide ligands. J. Immunol. 163, 6424–6434. Wong, F. S., Karttunen, J., Dumont, C., Wen, L., Visintin, I., Pilip, I. M., Shastri, N., Pamer, E. G., and Janeway, C. A., Jr. (1999). Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library. Nat. Med. 5, 1026–1031. Wong, F. S., Moustakas, A. K., Wen, L., Papadopoulos, G. K., and Janeway, C. A., Jr. (2002). Analysis of structure and function relationships of an autoantigenic peptide of insulin bound to H-2K(d) that stimulates CD8 T cells in insulin-dependent diabetes mellitus. Proc. Natl. Acad. Sci. USA 99, 5551–5556. Wong, F. S., Visintin, I., Wen, L., Flavell, R. A., and Janeway, C. A., Jr. (1996). CD8 T-cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells. J. Exp. Med. 183, 67–76. Yang, X. D., Sytwu, H. K., McDevitt, H. O., and Michie, S. A. (1997). Involvement of beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the development of diabetes in obese diabetic mice. Diabetes 46, 1542–1547. Yang, Y., and Santamaria, P. (2004). T-cell receptor-transgenic NOD mice: A reductionist approach to understand autoimmune diabetes. J. Autoimmun. 22, 121–129. Yoneda, R., Yokono, K., Nagata, M., Tominaga, Y., Moriyama, H., Tsukamoto, K., Miki, M., Okamoto, N., Yasuda, H., Amano, K., and Kasuga, M. (1997). CD8 cytotoxic T-cell clone rapidly transfers autoimmune diabetes in very young NOD and MHC class I-compatible scid mice. Diabetologia 40, 1044–1052. Yoshida, K., Martin, T., Yamamoto, K., Dobbs, C., Munz, C., Kamikawaji, N., Nakano, N., Rammensee, H. G., Sasazuki, T., Haskins, K., and Kikutani, H. (2002). Evidence for shared recognition of a peptide ligand by a diverse panel of non-obese diabetic mice-derived, isletspecific, diabetogenic T-cell clones. Int. Immunol. 14, 1439–1447. You, S., Chen, C., Lee, W. H., Wu, C. H., Judkowski, V., Pinilla, C., Wilson, D. B., and Liu, C. P. (2003). Detection and characterization of T cells specific for BDC2.5 T cell-stimulating peptides. J. Immunol. 170, 4011–4020. Yu, L., Robles, D. T., Abiru, N., Kaur, P., Rewers, M., Kelemen, K., and Eisenbarth, G. S. (2000). Early expression of antiinsulin autoantibodies of humans and the NOD mouse: Evidence for early determination of subsequent diabetes. Proc. Natl. Acad. Sci. USA 97, 1701–1706. Zekzer, D., Wong, F. S., Ayalon, O., Millet, I., Altieri, M., Shintani, S., Solimena, M., and Sherwin, R. S. (1998). GAD-reactive CD4þ Th1 cells induce diabetes in NOD/SCID mice. J. Clin. Invest. 101, 68–73.

The Biology of Human Lymphoid Malignancies Revealed by Gene Expression Profiling Louis M. Staudt and Sandeep Dave Metabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892

1. 2. 3. 4.

Abstract............................................................................................................. Introduction ....................................................................................................... Molecular Diagnosis of Lymphoid Malignancies........................................................ Gene Expression-Based Survival Predictors.............................................................. Concluding Remarks............................................................................................ References .........................................................................................................

163 164 165 177 197 198

Abstract Gene expression profiling provides a quantitative molecular framework for the study of human lymphomas. This genomic technology has revealed that existing diagnostic categories are comprised of multiple molecularly and clinically distinct diseases. Diffuse large B-cell lymphoma (DLBCL), for example, consists of three gene expression subgroups, termed germinal center B-cell–like (GCB) DLBCL, activated B-cell–like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). These DLBCL subgroups arise from different stages of normal B-cell differentiation, utilize distinct oncogenic mechanisms, and differ in their ability to be cured by chemotherapy. Key regulatory factors and their target genes are differentially expressed among these subgroups, including BCL-6, Blimp-1, and XBP1. ABC DLBCL and PMBL depend upon constitutive activation of the NF-kB pathway for their survival but GCB DLBCL does not, demonstrating that this pathway is a potential therapeutic target for certain DLBCL subgroups. In DLBCL, mantle cell lymphoma, and follicular lymphoma, gene expression profiling has also been used to create gene expression-based models of survival, which have identified the biological characteristics of the tumors that influence their clinical behavior. In mantle cell lymphoma, the length of survival following diagnosis is primarily influenced by the tumor proliferation rate, which can be quantitatively measured by a proliferation gene expression ‘‘signature.’’ Based on this accurate measure, the proliferation rate can now be viewed as an integration of several oncogenic lesions that each increase progression from the G1 to the S phase of the cell cycle. In DLBCL and follicular lymphoma, gene expression profiling has revealed that the molecular characteristics of non-malignant tumorinfiltrating immune cells have a major influence on the length of survival.

163 advances in immunology, vol. 87 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)87005-1

164

L O UIS M. STA UDT A ND SA NDE EP DAVE

The implications of these insights for the diagnosis and treatment of non-Hodgkin lymphomas are discussed. 1. Introduction The study of human diseases presents a unique challenge to the experimental biologist. Traditional modes of biological inquiry often require the manipulation of a biological system, which may be difficult or impossible to achieve when studying a human disease. Therefore, initial insights into human diseases are often observational in nature, and the depth of these insights relies on the precision and breadth of the observational tools at hand. A traditional and successful approach to the study of human cancer begins with the identification of genes that are mutated and/or misexpressed in one or more cancer specimens. These potential oncogenes and tumor suppressor genes are then assessed for their ability to create or modify a transformed phenotype in cultured cells or in mice. By assessing such genes one by one, an edifice of knowledge has been created that provides a framework for understanding cancer biology (Hanahan and Weinberg, 2000). Two key methodological advances have provided an orthogonal approach to human cancer: the completion of the human genome sequence and the development of DNA microarrays to measure the activity of the genome. A genome-wide measurement of mRNA levels in a cancer specimen, known as its gene expression profile, can reveal aspects of the cancer phenotype that are difficult to perceive by studying individual oncogenes and tumor suppressor genes. Even more powerful insights can be derived when gene expression profiles are obtained from a large number of cancer specimens for which clinical data are available. Clinical data such as response to treatment and length of survival can be used to tease biological insights out of the large data sets that DNA microarrays generate. The beauty of gene expression profiling data is that they are quantitative and highly reproducible. Because of this, these data can be used to generate multivariate statistical models of the clinical behavior of cancer that have great predictive power. From this new perspective, human cancer is found to be mathematically tractable. Various biological phenotypes of a tumor, such as the proliferation rate, the activity of survival pathways, and the nature of non-malignant tumorinfiltrating cells, can now be accurately measured because they are reflected in the gene expression profile. Each biological phenotype can be associated with the expression of a characteristic set of genes, known as its gene expression signature, and the expression of these signature genes serves as a quantitative measure of the phenotype (Shaffer et al., 2001). The length of survival of cancer patients and their response to therapy can be traced to biological

b io l og y of h um an ly mp ho mas

165

differences in their tumors that are reflected quantitatively in gene expression signatures. Often, several gene expression signatures are needed to adequately model the complex clinical behavior of cancer, thus revealing cancer as the integration of multiple abnormal biological mechanisms. By combining gene expression profiling with traditional analysis of oncogenes and tumor suppressors, it becomes clear that the multiple oncogenic abnormalities in a single tumor act synergistically and quantitatively to alter discrete tumor phenotypes. This mathematical view of the cancer process will ultimately change our approach to cancer therapy, particularly as agents that target discrete biological pathways become available. This review will provide examples from studies of lymphoid malignancies that illustrate the ability of gene expression profiling to illuminate cancer biology. The first section demonstrates how gene expression profiling can reveal that current diagnostic categories are comprised of several molecularly and clinically distinct diseases. The second section demonstrates how mathematical models of survival in cancer can be created by gene expression profiling, thereby revealing biological attributes of the tumors that influence their clinical behavior. 2. Molecular Diagnosis of Lymphoid Malignancies 2.1. Molecularly and Clinically Distinct Diseases Within Diffuse Large B-Cell Lymphoma Diffuse large B-cell lymphoma (DLBCL) is the largest diagnostic category among the non-Hodgkin lymphomas, accounting for roughly 40% of all cases. Within the United States, this translates into more than 23,000 new cases of DLBCL diagnosed each year. Multi-agent chemotherapy can cure approximately 40% of these patients, and this represents one of the successes of modern cancer therapy. Unfortunately, DLBCL still accounts for approximately 10,000 deaths per year in the United States. Why some patients with DLBCL can be cured and others cannot has been a longstanding and frustrating puzzle. The first clue to this puzzle came when gene expression profiling studies revealed that DLBCL is comprised of at least two molecularly and clinically distinct diseases (Alizadeh et al., 2000; Rosenwald et al., 2002; Wright et al., 2003). One subgroup of DLBCL, termed germinal center B-cell–like (GCB) DLBCL, expresses genes that are hallmarks of normal germinal center B cells (Fig. 1A). By contrast, another DLBCL subgroup, termed activated B-cell–like (ABC) DLBCL, lacks expression of germinal center B-cell–restricted genes and instead expresses genes that are induced during mitogenic stimulation of

166

b io l og y of h um an ly mp ho mas

167

blood B cells. These two subgroups of DLBCL differ in the expression of thousands of genes, and in this respect they are as different as acute myelogenous leukemia and acute lymphoblastic leukemia. More recently, a third subgroup of DLBCL has been defined by gene expression profiling, termed primary mediastinal B-cell lymphoma (PMBL) (Rosenwald et al., 2003a; Savage et al., 2003). These three DLBCL subgroups should be considered separate disease entities since they arise from B cells at different stages of differentiation, utilize different oncogenic pathways, and have distinct clinical behaviors. 2.2. DLBCL Subgroups and the Regulatory Biology of B-Cell Differentiation Insight into the normal cellular counterparts of the DLBCL subgroups has been provided by an analysis of regulatory factors that control the differentiation of germinal center B cells to plasma cells (Fig. 2). BCL-6 is a transcriptional repressor that is required for mature B cells to differentiate into germinal center B cells during an immune response (Dent et al., 1997; Ye et al., 1997). Normal germinal center B cells express BCL-6 at high levels, but BCL-6 expression is silenced during plasmacytic differentiation (Allman et al., 1996; Cattoretti et al., 1995; Onizuka et al., 1995). DLBCLs belonging to the GCB subgroup express BCL-6 mRNA at significantly higher levels than DLBCLs belonging to the ABC subgroup (Alizadeh et al., 2000; Rosenwald et al., 2002; Wright et al., 2003). BCL-6 is deregulated by chromosomal translocations in roughly 20% of DLBCLs (Pasqualucci et al., 2003), but the high expression of BCL-6 in GCB DLBCLs is not accounted for by these translocations. Rather, BCL-6 is expressed in GCB DLBCLs along with a host of other germinal center B-cell–restricted-genes because these DLBCLs are derived from normal germinal center B cells and retain much of their biology. In keeping with this notion, GCB DLBCLs have ongoing somatic hypermutation of their immunoglobulin genes, a characteristic feature of normal germinal center B cells (Lossos et al., 2000). Figure 1 (A) Genes characteristically expressed by three subgroups diffuse large B‐cell lymphoma (DLBCL): Primary mediastinal B‐cell lymphoma (PMBL), germinal center B‐cell–like (GCB) DLBCL, and the activated B‐cell–like (ABC) DLBCL (Rosenwald et al., 2002, 2003a; Wright et al., 2003). Each of the 201 columns represents gene expression data from a single DLBCL biopsy sample, and each row represents expression of a single gene. Relative gene expression is indicated according to the color scale shown. (B) Kaplan‐Meier plot of overall survival for the different DLBCL subgroups. (C) Distinct oncogenic mechanisms in the DLBCL subgroups. (D) Selective toxicity of a small molecule IkB kinase inhibitor for ABC DLBCL and PMBL cell lines (Lam et al., 2004).

168

L O UIS M. STA UDT A ND SA NDE EP DAVE

Figure 2 Schematic of the regulatory network governing differentiation of a germinal center B cell to a plasma cell.

To elucidate the mechanisms by which BCL-6 regulates germinal center B-cell differentiation, gene expression profiling was used to identify the target genes of BCL-6 repression (Shaffer et al., 2000). DNA microarrays were used to identify genes that were downregulated when BCL-6 was introduced into cells lacking endogenous BCL-6 expression and that were upregulated when a dominant negative form of BCL-6 was introduced into cells that have endogenous BCL-6 expression. The BCL-6 target genes identified in this fashion were found to be expressed at much lower levels in germinal center B cells than in resting or activated blood B cells, in keeping with the germinal center B-cell–restricted expression of BCL-6 (Shaffer et al., 2000). One group of BCL-6 target genes are genes that are induced when B cells are activated through the antigen receptor, including cyclin D2, CD69, CD44, and MIP-1a. By blocking expression of B-cell activation genes, BCL-6 may guide an antigen-stimulated B cell towards germinal center differentiation and away from the alternate fate of rapid plasmacytic differentiation (Shaffer et al., 2000). Another important BCL-6 target gene is p27kip1, which encodes a negative regulator of cell cycle progression. By repressing p27kip1, BCL-6 may contribute to the extraordinarily high proliferation rate of germinal center B cells.

b io l og y of h um an ly mp ho mas

169

One particularly illuminating BCL-6 target gene is Blimp-1 (Shaffer et al., 2000), which encodes a transcriptional repressor that is required for plasma cell differentiation (Shapiro-Shelef et al., 2003). BCL-6 binds to a motif in intron 5 of the Blimp-1 gene that is conserved between the human and mouse genes (Tunyaplin et al., 2004). Since BCL-6 is bound to this site in vivo, as judged by chromatin immunoprecipitation, Blimp-1 appears to be a direct target of BCL-6 repression. Dominant negative inhibition of BCL-6 in a Burkitt lymphoma cell line (which is of germinal center origin) caused the endogenous Blimp-1 gene to be derepressed, and this was followed by partial plasmacytic differentiation: multiple mature B-cell markers were silenced while plasma cell markers such as CD38 were induced (Shaffer et al., 2000). In addition, c-myc and a host of other proliferation associated genes were turned off (Shaffer et al., 2000), which is consistent with the fact that c-myc is a direct target of Blimp-1 repression (Lin et al., 1997). These results led to the hypothesis that one role for BCL-6 in normal germinal center B cells and in non-Hodgkin lymphomas is to block differentiation into plasma cells (Shaffer et al., 2000). In keeping with this notion, mice in which the BCL-6 gene is disrupted generate plasma cells more readily in response to antigenic stimulation (Tunyaplin et al., 2004). In non-Hodgkin lymphomas with BCL-6 translocations, the promoter of BCL-6 is substituted with the promoter from the translocation partner gene, which may prevent the physiological downregulation of BCL-6 mRNA expression that occurs during plasmacytic differentiation. This deregulation of BCL-6 may contribute to malignant transformation by keeping Blimp-1 repressed, thereby preventing plasmacytic differentiation and the attendant cell cycle arrest. Gene expression profiling has also been used to elucidate the mechanisms by which Blimp-1 promotes plasmacytic differentiation (Shaffer et al., 2002a, 2004). Ectopic expression of Blimp-1 in B cells silences the expression of virtually all mature B-cell–restricted genes (Shaffer et al., 2002a). Blimp-1 exerts its powerful effect by directly repressing genes encoding key B-cell transcription factors, including Pax5, Spi-B, Id3, and CIITA, thereby indirectly blocking the expression of the genes that they transactivate (Lin et al., 2002; Piskurich et al., 2000; Shaffer et al., 2002a). One of the genes silenced by Blimp-1, directly or indirectly, is BCL-6 (Shaffer et al., 2002a). Blimp-1 and BCL-6 therefore create a double negative regulatory loop (Shaffer et al., 2002a), a form of regulation first described by Monod and Jacob four decades ago (Monod and Jacob, 1961). An important feature of this regulatory design is its plasticity; a change in the expression or activity of either repressor, even transiently, can swing the system towards one of two cellular states. In germinal center B cells, strong stimulation by antigen through the immunoglobulin receptor or by activated T cells through CD40

170

L O UIS M. STA UDT A ND SA NDE EP DAVE

downregulates BCL-6 mRNA and/or protein expression (Allman et al., 1996; Niu et al., 1998), which would tip the regulatory balance in favor of Blimp-1 and plasmacytic differentiation. Strong support for this double negative model has recently been provided by the analysis of MTA3, a germinal center B cell-restricted subunit of the Mi-2/NuRD corepressor complex (Fujita et al., 2003). MTA3 tethers the Mi-2/ NuRD complex to BCL-6 by binding to a central region of BCL-6 that is required for full transcriptional repression activity (Albagli et al., 1996; Chang et al., 1996; Seyfert et al., 1996). The Mi-2/NuRD complex has histone deacetylase activity, which BCL-6 uses to create a repressive chromatin structure at its target genes. Knockdown of MTA3 by RNA interference in germinal center B-cell–derived lymphoma cell lines caused Blimp-1 to be derepressed and partial plasmacytic differentiation to ensue (Fujita et al., 2003). Remarkably, coexpression of BCL-6 and MTA3 in a plasmacytic cell line derived from a patient with multiple myeloma caused the cells to ‘‘dedifferentiate’’ to a mature B-cell phenotype: the plasma cell genes Blimp-1, XBP1, and syndecan-1 were silenced, while the B-cell genes CD19, CD20, BLNK, syk, Igb, and MHC class II were expressed. Although this dedifferentiation is surprising, it is an inherent feature of the plasticity built into a double negative regulatory circuit (Monod and Jacob, 1961). In addition to Blimp-1, plasmacytic differentiation requires the transcriptional activator XBP1 (Reimold et al., 2001). XBP1 lies downstream of Blimp-1 in the regulatory hierarchy: XBP1 mRNA expression is induced during plasmacytic differentiation, in part, because Blimp-1 represses Pax5, which encodes a repressor of XBP1 (Lin et al., 2002; Reimold et al., 1996; Shaffer et al., 2002a). Recent experiments demonstrated that XBP1 is the master regulator of the secretory phenotype of plasma cells (Shaffer et al., 2004). Gene expression profiling revealed that XBP1 induces the expression of a large set of genes encoding components of the endoplasmic reticulum and golgi, leading to a dramatic expansion of the secretory apparatus (Shaffer et al., 2004). Unexpectedly, XBP1 also increases cell size, mitochondrial and lysosomal biogenesis, mitochondrial respiration, total cellular protein content, and the overall rate of protein synthesis (Shaffer et al., 2004). XBP1 is a mammalian homologue of the yeast transcription factor Hac1, which controls the unfolded protein response that is triggered by cellular events that disturb the processing of proteins in the endoplasmic reticulum. In mammalian cells, XBP1 coordinates a ‘‘physiological’’ unfolded protein response that allows permanent differentiation to a secretory state characterized by a continuous, high burden of proteins entering the secretory pathway (Shaffer et al., 2004). The pleiotropic effects of XBP1 on cellular structure, function, and energetics are all likely to

b io l og y of h um an ly mp ho mas

171

contribute to the high-level secretion of immunoglobulin that characterizes plasma cells. Based on the regulatory biology of plasmacytic differentiation, it is possible to speculate that the cell of origin of ABC DLBCL may be a plasmablastic B cell that is poised to exit the germinal center. In comparison to GCB DLBCLs, ABC DLBCLs express higher levels of many plasma cell genes (Wright et al., 2003). Among these are XBP1 and a variety of its target genes, which encode endoplasmic reticulum and golgi proteins. Furthermore, ABC DLBCLs express Blimp-1 mRNA at levels comparable to those in multiple myeloma cells. This phenotype is similar to that of a rare subpopulation of plasmablasts in the germinal center, which are thought to be in the process of migrating to the bone marrow where they differentiate fully into plasma cells (Angelin-Duclos et al., 2000; Falini et al., 2000). ABC DLBCLs do not express a variety of other genes that characterize normal plasma cells and multiple myeloma, suggesting that they are derived from a cell that is intermediate between a germinal center B cell and a plasma cell. In support of this notion, ABC DLBCLs have somatically mutated immunoglobulin genes, and therefore are derived from a B cell that has likely traversed the germinal center (Lossos et al., 2000). However, in contrast to GCB DLBCLs, ABC DLBCLs have a fixed complement of immunoglobulin gene mutations, suggesting that the somatic hypermutation machinery has been inactivated as occurs normally during plasmacytic differentiation. The increased understanding of the regulatory biology of DLBCLs may provide avenues for therapy. BCL-6 itself could be a therapeutic target, given that inhibition of BCL-6 function in a Burkitt lymphoma cell line caused cell cycle arrest by derepressing the cell cycle inhibitor p27kip1 and by derepressing Blimp-1, thereby downregulating c-myc expression (Shaffer et al., 2000). The activity of BCL-6 as a transcriptional repressor could be inhibited by blocking its interaction with corepressor complexes containing MTA3 or those containing SMRT, NCoR, or BCoR that bind to its aminoterminal POZ/BTB domain (Dhordain et al., 1997; Huynh et al., 2000; Melnick et al., 2002; Muscat et al., 1998; Wong and Privalsky, 1998). The function of BCL-6 is also inhibited by acetylation, and therefore drugs that inhibit deacetylation of BCL-6 might prove useful (Bereshchenko et al., 2002). Further work is needed to understand which DLBCLs might be susceptible to BCL-6 inhibition. ABC DLBCLs may be less susceptible than GCB DLBCLs since they have lower BCL-6 expression. Curiously, ABC DLBCLs have high Blimp-1 mRNA expression and yet are highly proliferative and express c-myc at high levels. It is therefore conceivable that ABC DLBCLs may have a means to inactivate Blimp-1 function, and understanding

172

L O UIS M. STA UDT A ND SA NDE EP DAVE

this mechanism might provide additional molecular targets for this DLBCL subgroup. 2.3. Primary Mediastinal B-Cell Lymphoma: A DLBCL Subgroup Related to Hodgkin Lymphoma Primary mediastinal B-cell lymphoma (PMBL) was initially defined as a subgroup of DLBCL based primarily on its unusual clinical presentation: these patients are often young and have an aggressive lymphoma originating in the mediastinum. Since this lymphoma subgroup was solely defined by its clinical characteristics, it was likely to be heterogeneous and include other lymphoma types that happened by chance to present in the mediastinum. Gene expression profiling was able to clarify this diagnostic category by identifying a clear gene expression signature that distinguishes this type of DLBCL from the other DLBCL subgroups (Fig. 1A) (Rosenwald et al., 2003a; Savage et al., 2003). Importantly, one quarter of the samples submitted with the clinical diagnosis of PMBL were found to lack the PMBL gene expression signature, and are therefore likely to be cases of GCB or ABC DLBCL with predominant mediastinal involvement (Rosenwald et al., 2003a). Patients with a molecular diagnosis of PMBL were found to be young (median age 33), as compared to patients with GCB and ABC DLBCL (median age over 60), and the majority of the young patients were women (Rosenwald et al., 2003a). PMBL tumors often involved other thoracic sites, such as the pleura, pericardium, lung, and breast, and these sites were infrequently involved in cases of GCB or ABC DLBCL (Rosenwald et al., 2003a). Conversely, sites of frequent extranodal involvement in GCB and ABC DLBCL are the gastrointestinal tract, bone marrow, liver, and muscle, and PMBL tumors never involved these sites. Thus, PMBL tumors appear to spread by local extension from the mediastinum, whereas GCB and ABC DLBCL may spread through the bloodstream. Gene expression profiling uncovered a striking and unexpected relationship between PMBL and Hodgkin lymphoma (Rosenwald et al., 2003a; Savage et al., 2003). More than one third of the genes that distinguish PMBL from the other DLBCL subgroups were also highly expressed in Hodgkin lymphoma cell lines as compared with GCB DLBCL cell lines. Several of the PMBL signature genes (e.g., MAL, IL4I) were found to be expressed in primary Hodgkin Reed-Sternberg cells microdissected from cases of nodularsclerosing Hodgkin lymphoma (Rosenwald et al., 2003a). Likewise, the PMBL signature includes several genes that are known to be characteristically expressed in Hodgkin Reed-Sternberg cells, such as CD30, IL-13 receptor a chain, and TARC (Peh et al., 2001; Schwab et al., 1982; Skinnider et al., 2001).

b io l og y of h um an ly mp ho mas

173

However, the gene expression programs of PMBL and Hodgkin lymphoma are not identical. A subset of the PMBL signature genes is not expressed in Hodgkin lymphoma cells (Rosenwald et al., 2003a). Moreover, Hodgkin lymphoma cells characteristically lack expression of many mature B-cell genes, but these are expressed by PMBLs. Once realized, it is evident that PMBL and Hodgkin lymphoma have many clinical and pathological features in common (Jaffe and Muller-Hermelink, 1999). Both lymphoma types are common in young individuals, especially women. Furthermore, Hodgkin lymphoma often originates in the mediastinum, like PMBL, and upon pathological examination both lymphoma types can be associated with thymic remnants. PMBL is characterized frequently by prominent sclerotic reactions histologically as is also the case in nodularsclerosing Hodgkin lymphoma. Interestingly, case reports have documented that patients with PMBL can relapse after treatment with Hodgkin lymphoma (Gonzalez et al., 1991; Zarate-Osorno et al., 1992). This spectrum of molecular, pathological, and clinical similarities suggests that PMBL and Hodgkin lymphoma are pathogenetically related. One explanation for these similarities could be that both lymphoma types may originate from a rare B-cell population in the thymus (Addis and Isaacson, 1986). In support of this notion, a gene that is characteristically expressed in PMBL and Hodgkin lymphoma, MAL, is also expressed in a subset of thymic medullary B cells (Copie-Bergman et al., 2002). Thus, it is possible that PMBL and some cases of Hodgkin lymphoma inherit a gene expression program that is characteristic of these thymic B cells. That being said, the origin and function of thymic B cells is obscure at present. Thymic B cells have a high load of somatic mutation in their immunoglobulin genes without evidence of ongoing somatic hypermutation, as do PMBLs (Csernus et al., 2004; Flores et al., 2001; Kuppers et al., 1997; Leithauser et al., 2001; Pileri et al., 2003; Tonnelle et al., 1997). These data suggest that thymic B cells are post-germinal center in origin, but it is unclear whether these B cells diversify in the thymus or diversify elsewhere and home to this organ. The gene expression similarities between PMBL and Hodgkin lymphoma are also due to the fact that they share several oncogenic mechanisms. The NF-kB signaling pathway is constitutively active in both the lymphoma types, leading to the shared expression of many NF-kB target genes (see later discussion) (Rosenwald et al., 2003a; Savage et al., 2003). In addition, both PMBL and Hodgkin lymphoma have recurrent gains and amplifications of a chromosomal region in cytoband 9p24. These abnormalities are present in 40–50% of PMBL tumors, and 10% have a high-level amplification (Rosenwald et al., 2003a). This region is also amplified in Hodgkin lymphoma cell lines and in primary Hodgkin Reed-Sternberg cells (Bentz et al., 2001; Copie-Bergman

174

L O UIS M. STA UDT A ND SA NDE EP DAVE

et al., 2002; Joos et al., 1996, 2000; Rosenwald et al., 2003a). The amplicon contains the genes encoding the tyrosine kinase JAK2, the B7 family members PDL1 and PDL2, and the putative chromatin regulator SMARCA2 (Rosenwald et al., 2003a). Of these, JAK2 has received the most attention due to its involvement in stimulating cytokine-signaling pathways. Several PMBL signature genes are known to be induced by IL-4, including IL4I/ Fig1 (Chu and Paul, 1997), and MAL (Kovanen et al., 2003). STAT6, which mediates signaling by IL-4 and IL-13, is phosphorylated and present in the nucleus of PMBL cell lines and most primary PMBL tumors (Guiter et al., 2004). Indeed, JAK2 is phosphorylated and potentially active in PMBL cell lines and primary tumors (Guiter et al., 2004). The mechanisms underlying the activity of JAK2 remain to be elucidated, as neither IL-4 nor IL-13 are expressed in PMBL. Genomic copy number changes in cancer may be selected because they simultaneously alter the expression or activity of several genes in the affected region. It is therefore possible that the 9p24 amplicon is important in PMBL and Hodgkin lymphoma because it causes overexpression of PDL1 and/or PDL2, in addition to JAK2. PDL2 was found to be the gene that distinguished PMBL from other DLBCLs most significantly (Rosenwald et al., 2003a). PDL2 was found to be highly expressed in PMBL tumors even in the absence of a 9p24 gain or amplification and was further upregulated in those tumors in which the gene was amplified. PDL1 was likewise overexpressed in PMBL tumors but to a lesser degree than PDL2. Both genes are also expressed in Hodgkin lymphoma cells (Rosenwald et al., 2003a). PDL1 and PDL2 belong to the B7 family of surface proteins that affect T-cell responses (Curiel et al., 2003; Dong et al., 1999, 2002; Freeman et al., 2000; Latchman et al., 2001). Both proteins bind PD-1, a cell surface receptor on T cells that bears sequence homology with CD28 and CTLA4 (Dong et al., 1999; Freeman et al., 2000; Latchman et al., 2001). Engagement of PD-1 by PDL1 or PDL2 delivers a negative signal that inhibits signaling through the T-cell receptor (Freeman et al., 2000; Latchman et al., 2001). It has been suggested that tumors that express PDL1 or PDL2 may inhibit tumor-specific T-cell responses (Dong and Chen, 2003; Dong et al., 2002). In addition, however, PDL2 can costimulate T cells under certain conditions, which is mediated by another receptor that has not yet been identified (Liu et al., 2003). Given the probable origin of PMBL from a thymic B cell, the cell surface expression of PDL2 or PDL1 may allow these lymphoma cells to coexist with the T cells in the thymus. On one hand, PDL1 and PDL2 may inhibit T-cell responses to the lymphoma. On the other hand, the ability of PDL2 to costimulate thymic T cells may lead to the production of cytokines that promote proliferation and/or survival of the PMBL cells.

b io l og y of h um an ly mp ho mas

175

2.4. Oncogenic Pathways in DLBCL Subgroups One of the most compelling arguments supporting the view that the DLBCL subgroups represent distinct diseases is that they utilize distinct oncogenic mechanisms (Fig. 1C). The t(14;18) translocation deregulates the BCL-2 gene by placing it near the enhancer elements of the immunoglobulin heavy chain locus. This oncogenic event was found to be common in GCB DLBCL, occurring in 45% (29/65) of cases analyzed, and also was detected in 18% (2/11) of PMBL cases (Huang et al., 2002; Rosenwald et al., 2002). By contrast, out of 42 cases of ABC DLBCL analyzed, none had a BCL-2 translocation. Nonetheless, the majority of ABC DLBCLs express BCL-2 mRNA at high levels, presumably due to transcriptional deregulation of BCL-2 (Rosenwald et al., 2002; Wright et al., 2003). Furthermore, the anti-apoptotic BCL-2 family member A1 is expressed in ABC DLBCLs due to their constitutive NF-kB activity (see later discussion). In the face of the high expression of BCL2 and/or A1 in ABC DLBCL, there would be no selective pressure for a t(14;18) translocation. On the other hand, normal germinal center centroblasts express little if any BCL-2, which favors apoptosis as the default pathway in these cells (Martinez-Valdez et al., 1996; Tuscano et al., 1996). Therefore, the t(14;18) translocation would provide a strong selective advantage in a germinal center B-cell–derived lymphomas such as GCB DLBCL. Another recurrent oncogenic abnormality in DLBCL is amplification of the c-rel locus on chromosome arm 2p. This oncogenic event occurs in 16% of GCB DLBCLs and in 25% of PMBLs, but it has never been detected in ABC DLBCLs (Rosenwald et al., 2002; Zettl et al., 2004). c-rel encodes a member of the anti-apoptotic NF-kB family of transcription factors. However, it is currently unclear what selective advantage is conferred by c-rel amplification in GCB DLBCLs since cases with this abnormality do not express NF-kB target genes at higher levels than those with a wild type c-rel copy number (unpublished). This conundrum might be explained by the fact that NF-kB transcription factors require the activity of IkB kinase to become localized to the nucleus. During a germinal center reaction, stimulation through the antigen receptor or through CD40 can activate IkB kinase, and a germinal center B cell with a c-rel amplification might receive a stronger anti-apoptotic signal and be positively selected. In this scenario, c-rel amplifications may confer a selective advantage early in the genesis of a GCB DLBCL, but may not be functionally important in the tumor at diagnosis. Comparative genomic hybridization analysis has revealed multiple differences in chromosomal copy number alterations between the DLBCL subgroups (unpublished). In ABC DLBCLs, trisomy 3 or gain of the chromosome 3q arm was detected in 18% (11/62) and 24% (15/62) of cases, respectively, but these

176

L O UIS M. STA UDT A ND SA NDE EP DAVE

abnormalities were never detected in GCB DLBCLs (0/63). High-level amplification of chromosome band 18q21, which contains the BCL-2 gene, was detected in one sixth of ABC DLBCLs but in only 3% of GCB DLBCLs. This abnormality may occur more commonly in ABC DLBCL because BCL-2 is transcriptionally active in the majority of these tumors. Interestingly, among the few GCB DLBCLs with this abnormality, 75% (3/4) had a t(14;18) translocation and thus expressed BCL-2. Finally, as discussed in detail previously, 43% of PMBLs have a gain or amplification of cytoband 9p24 whereas this abnormality has not been detected in GCB DLBCLs (0/63) and only rarely in ABC DLBCLs (2/62). The uneven distribution of these chromosomal abnormalities among the DLBCL subgroups suggests that the subgroups utilize distinct oncogenic pathways. One of the most important differences among the DLBCL subgroups is the constitutive activity of the NF-kB pathway in ABC DLBCL and PMBL but not GCB DLBCL. By gene expression profiling, ABC DLBCLs were found to have high expression of known NF-kB target genes when compared with GCB DLBCLs (Davis et al., 2001). Cell line models of ABC DLBCL have constitutive nuclear NF-kB due to constitutive activity of IkB kinase, and these were not features of GCB DLBCL cell lines. Inhibition of the NF-kB pathway using a dominant active form of IkBa or a dominant negative version of the IkB kinase b subunit was toxic to ABC DLBCL cell lines but not to GCB DLBCL cell lines. More recently, PMBL cells were found to express NF-kB target genes and have nuclear NF-kB, demonstrating the activity of the NF-kB pathway in this DLBCL subgroup as well (Rosenwald et al., 2003a; Savage et al., 2003). In this respect, PMBLs again resemble Hodgkin lymphoma, which is also characterized by constitutive NF-kB activity (Bargou et al., 1996, 1997; Cabannes et al., 1999; Emmerich et al., 1999; Krappmann et al., 1999; Wood et al., 1998). These findings suggest that the NF-kB pathway is a potential therapeutic target for ABC DLBCL and PMBL, but not GCB DLBCL. In support of this idea, small molecule inhibitors of IkB kinase were found to be selectively toxic for ABC DLBCL and PMBL cell lines, but had no effect on GCB DLBCL cell lines (Fig. 1D) (Lam et al., 2005). These findings support the further development of IkB kinase inhibitors for the treatment of lymphomas with NF-kB activity. In addition, an understanding of the mechanism(s) underlying the constitutive IkB kinase activity in ABC DLBCL and PMBL may lead to the identification of further molecular targets. 2.5. Clinical Differences Between DLBCL Subgroups Given that the DLBCL subgroups appear to arise from different stages of B-cell differentiation, utilize distinct oncogenic pathways, and differ in the expression of thousands of genes, it is perhaps not surprising that they differ

b io l og y of h um an ly mp ho mas

177

in their care rate following anthracycline-based multiagent chemotherapy (Alizadeh et al., 2000; Rosenwald et al., 2002; Wright et al., 2003). Patients with ABC DLBCL, GCB DLBCL, and PMBL have 5 year survival rates of 30%, 59%, and 64%, respectively (Fig. 1B). Relatively few relapses occur beyond 5 years, so these survival rates roughly reflect the probability that patients in each DLBCL subgroup will be cured by chemotherapy. To determine if these survival differences are reproducible in different DLBCL cohorts, it is necessary to establish a reliable method for the molecular diagnosis of the DLBCL subgroups. To this end, a method based on Bayesian statistics was developed that is able to assign a probability that a given DLBCL tumor sample belongs to the GCB DLBCL, ABC DLBCL, or PMBL subgroups (Rosenwald et al., 2003a; Wright et al., 2003). An important feature of this method is that in allows a patient sample to be declared ‘‘unclassified’’ if its gene expression profile does not closely match that of any of the subgroups. Based on this method, GCB DLBCL, ABC DLBCL, and PMBL account for roughly 40%, 34%, and 8% of DLBCLs. Thus, 18% of DLBCL samples remain unclassified and may represent additional DLBCL subgroups. Alternatively, some samples may fail to yield a diagnosis for technical reasons, such as inadequate content of malignant cells within the biopsy sample, which may happen in cases with an abundant infiltration of nonmalignant immune cells. An attractive feature of this molecular diagnosis method is that the statistical algorithm is independent of the type of DNA microarray used to generate the gene expression data (Wright et al., 2003). When this Bayesian predictor was applied to data from 58 DLBCL samples profiled using Affymetrix oligonucleotide microarrays, GCB DLBCL and ABC DLBCL subgroups were identified, and their 5-year survival rates were 62% and 26%, respectively (Wright et al., 2003). More recently, many groups have used immunohistochemical stains for a few proteins to approximate the distinction between GCB DLBCL and ABC DLBCL and have consistently found that GCB DLBCL is the more favorable prognostic group (Chang et al., 2004; de Leval et al., 2003; Hans et al., 2004; Tzankov et al., 2003). 3. Gene Expression-Based Survival Predictors The distinction between the DLBCL subgroups detailed previously was discovered using an ‘‘unsupervised’’ approach that relies on methods that detect prominent patterns in the gene expression data. A complementary analytical approach is termed ‘‘supervised’’ because it uses statistical methods to find associations between gene expression data and external clinical, pathological,

178

L O UIS M. STA UDT A ND SA NDE EP DAVE

or molecular data (Staudt, 2003). Supervised methods typically begin by determining the statistical correlation between the expression pattern of each gene on a microarray and an external parameter such as the length of survival following diagnosis. Supervised methods face a mammoth multiple comparisons problem: thousands of statistical correlations can be made using the expression profiles of each gene on the microarray, and a large number of these correlations will occur by chance alone. Because of this, the indiscriminant choice of the genes with the best correlations can lead to the development of statistical models that are overfit to the particular data set on which they were based (Ransohoff, 2004). Dividing samples into a ‘‘training’’ set that is used to generate a statistical model and a ‘‘test’’ set that is used to validate the model can circumvent this problem. Nevertheless, it is likely that most models generated in the training set will fail to validate in the test set unless the model is formed from genes whose expression patterns reflect meaningful biological variation that is related to the external parameter. A useful method to derive biological meaning from gene expression data is to classify genes into gene expression ‘‘signatures’’ (Shaffer et al., 2001). This method relies on the fact that genes that encode proteins with similar biological function are often transcriptionally coregulated. A gene expression signature may reflect a particular cellular lineage or stage of differentiation, the activity of an intracellular signaling pathway, or an aspect of cellular physiology such as the proliferation rate (Shaffer et al., 2001). A successful approach has been to use supervised methods to identify genes with expression patterns that are correlated with an external parameter, and then to classify these genes into gene expression signatures (Rosenwald et al., 2002). Next, a statistical model of the external parameter is created using the gene expression signatures instead of the individual genes. By creating statistical models from a limited number of gene expression signatures, the multiple comparisons problem is mitigated. The following sections demonstrate that this analytical approach has been used successfully to create survival predictors in DLBCL, mantle cell lymphoma, and follicular lymphoma. 3.1. A Gene Expression-Based Survival Predictor for Diffuse Large B-Cell Lymphoma The subdivision of diffuse large B-cell lymphoma into subgroups explains some, but not all, of the variation in survival of these patients. Although patients with GCB DLBCL have a relatively favorable prognosis, roughly 30% die within the first 2 years of diagnosis. Patients with ABC DLBCL have a relatively poor prognosis, yet roughly 20% are long-term survivors. Some of this clinical heterogeneity is explained by the fact that certain clinical

b io l og y of h um an ly mp ho mas

179

variables have prognostic significance in DLBCL, and their influence may not be reflected in tumor gene expression. Clinical parameters with prognostic significance in DLBCL include age, tumor stage, serum lactate dehydrogenase level (a measure of tumor bulk), performance status, and the number of extranodal sites (Shipp, 1993). These five clinical parameters form the International Prognostic Index (IPI), which is a strong predictor of survival in DLBCL (Shipp, 1993). The DLBCL subgroup distinction is statistically independent of the IPI in predicting survival, demonstrating that the subgroup distinction is not acting as a surrogate for clinical prognostic variables (Alizadeh et al., 2000; Rosenwald et al., 2002). Further scrutiny of DLBCL gene expression profiling data using a supervised method revealed that the DLBCL subgroup distinction captures only part of the biological variation among DLBCL tumors that influences survival (Rosenwald et al., 2002). This study began by identifying ‘‘survival predictor’’ genes with expression patterns in DLBCL tumor biopsies that were statistically associated with the length of survival of the patients (Rosenwald et al., 2002). Separately, gene expression signatures were defined as sets of genes that were coordinately expressed across the DLBCL tumor samples. Notably, more than half of the survival predictor genes could be classified into one of four gene expression signatures, termed germinal center B cell, lymph node, MHC class II, and proliferation (Fig. 3). A multivariate model of survival was formed from these four signatures and one additional predictive gene, BMP6, which was not part of a gene expression signature. This model was used to assign a ‘‘survival predictor score’’ to each patient based on tumor gene expression. Patients were ranked according to these survival predictor scores and divided into four quartile groups that had 5‐year survival rates of 73%, 71%, 34%, and 15% (Rosenwald et al., 2002). This model had a stronger statistical association with survival than the DLBCL subgroup distinction, demonstrating that it accounted for a greater degree of the clinical heterogeneity among these patients. This model may prove useful clinically: it places one half of the patients in a favorable prognostic group for whom anthracycline-based chemotherapy has a reasonable chance of producing a cure, and one quarter of the patients in a poor prognosis group for whom alternative therapies should be considered. 3.2. Germinal Center B-Cell Signature The components of this multivariate model reveal biological features of DLBCL tumors that appear to influence the probability of being cured by chemotherapy. The germinal center B-cell signature includes those genes that are characteristically expressed at the germinal center B-cell stage of

180

b io l og y of h um an ly mp ho mas

181

differentiation. This signature mirrors the DLBCL subgroup distinction in that both GCB DLBCLs and PMBLs express germinal center B-cell signature genes, but ABC DLBCLs do not. The three germinal center B-cell signature genes that were found to be most strongly associated with survival were BCL6, SERPINA9, and GCET2 (Rosenwald et al., 2002). Both SERPINA9 and GCET2 were identified as germinal center B-cell signature genes because the Lymphochip DNA microarrays used for this study included a large number of DNA clones selected from a germinal center B-cell cDNA library (Alizadeh et al., 1999). SERPINA9 (GCET1) encodes a serine protease inhibitor whereas GCET2 (HGAL) is of unknown function (Lossos et al., 2003; Pan et al., 2003). Interestingly, GCET2 is the human homologue of the mouse M17 gene, which is restricted in expression to germinal center B cells (Christoph et al., 1994). It is important to emphasize, however, that these three germinal center B-cell signature genes may not themselves influence survival in DLBCL, they merely represent the fact that those tumors of germinal center B-cell origin have a more favorable prognosis. A second study also used supervised methods to discover survival predictor genes in DLBCL (Shipp et al., 2002). Two of the survival predictor genes highlighted in the study were protein kinase C b (PRKCB1) and phosphodiesterase 4B (PDE4B), both of which are more highly expressed in ABC DLBCLs than in GCB DLBCLs (Davis and Staudt, 2002; Rosenwald et al., 2002). Likewise, another study generated a model of survival in DLBCL using six genes, two of which are germinal center B-cell signature genes (BCL6, LMO2) and two others that are more highly expressed in ABC DLBCLs (CCND2, BCL2) (Lossos et al., 2004). Thus, not unexpectedly, each supervised model of DLBCL survival that has been created incorporates the DLBCL subgroup distinction since this is a predominant biological distinction among DLBCLs that is associated with survival.

Figure 3 A gene expression‐based multivariate model of survival following chemotherapy for DLBCL. The left panel shows the expression of the four gene expression signatures used to create the survival model in 201 DLBCL biopsy samples. Expression of the germinal center, lymph node, and MHC class II signatures is associated with favorable prognosis, while expression of the proliferation signature is associated with poor prognosis (Rosenwald et al., 2002). Representative genes from each signature that were used to create the survival model are shown. For each signature, patients were divided into four equal quartiles based on the expression of the signature in their biopsy samples. The four Kaplan-Meier plots in the center depict the survival of patients in each signature quartile. These four signatures were combined into a multivariate model of survival, and patients were divided into four quartile groups based on this model (Rosenwald et al., 2002). The Kaplan-Meier plot at the right depicts the overall survival of each quartile group of the multivariate model.

182

L O UIS M. STA UDT A ND SA NDE EP DAVE

3.3. Lymph Node Signature The ‘‘lymph node’’ signature includes many genes whose expression patterns were found to be associated with favorable survival in DLBCL (Rosenwald et al., 2002). Despite its name, the genes in this signature do not merely reflect normal lymph node cells, but rather reflect a host response to malignant DLBCL cells in the lymph node. In keeping with this notion, the lymph node signature genes are not highly expressed in most lymph node biopsies from mantle cell lymphoma, small lymphocytic lymphoma, or follicular lymphoma. The survival predictor genes within the lymph node signature include genes that are expressed in macrophages (e.g., a actinin 1/ACTN1, urokinase plasminogen activator/PLAU) or fibroblasts (e.g., connective tissue growth factor/ CTGF), and genes that encode extracellular matrix components (Fibronectin/ FN1, collagen III a1/COL3A1) (Rosenwald et al., 2002). Involved lymph nodes from some DLBCL patients are heavily infiltrated with macrophages, and some have a prominent sclerotic reaction; expression of the lymph node signature genes appears to reflect these host responses. Indeed, gene expression profiling analysis of separated malignant and nonmalignant cells from DLBCL biopsy specimens confirms that the large majority of lymph node signature genes are expressed in the nonmalignant cells (unpublished). The DLBCLs subgroups have a somewhat different expression of lymph node signature genes, with PMBLs having universally high expression, GCB DLBCLs having intermediate expression, and most ABC DLBCLs having a relatively low expression. Nonetheless, among ABC DLBCLs, lymph node signature expression is associated with a more favorable prognosis, even though this DLBCL subgroup as a whole has a relatively poor prognosis. The mechanisms accounting for the association of lymph node signature gene expression and favorable outcome in DLBCL have not been elucidated. One possibility is that the infiltrating cells that generate the lymph node signature are mounting an immune response to the tumor. The immune response might be ineffective on its own, but it might help contribute to curative responses following chemotherapy. A second possibility is that some DLBCLs rely on signals derived from host cells for survival and/or proliferation. In this ‘‘extracellular signal addiction’’ hypothesis, the dependence of the malignant cells on microenvironmental signals in the lymph node may prevent their spread to other anatomical sites in which these signals are absent. In this regard, it is interesting that high expression of the lymph node signature is associated with low tumor stage (unpublished). Tumor stage is a measure of how many lymph node groups and extranodal sites are involved in a patient, and thus, it could reflect how well a malignant DLBCL cell can survive and proliferate in new anatomical locations. In DLBCLs with high lymph node signature expression, the malignant cells may receive signals by direct cell-cell

b io l og y of h um an ly mp ho mas

183

contact with tumor-infiltrating host cells. Alternatively, since many lymph node signature genes encode extracellular matrix components, it is conceivable that cytokine or chemokine signaling could be augmented by their deposition on extracellular matrix in DLBCLs with high lymph node signature expression. Comparative genomic hybridization analysis has revealed that some chromosomal copy number changes in DLBCLs are associated with high or low lymph node signature expression (unpublished). This observation suggests that particular oncogenic events create a DLBCL that is dependent upon lymph node microenvironmental signals, while other oncogenic events create a DLBCL that is independent of such signals. Whereas most oncogenes render a cell independent of extracellular signals, it has been postulated that certain oncogenes may create an interdependence between the malignant cell and its microenvironment (Hanahan and Weinberg, 2000). One example is the oncogene c-maf, which is frequently overexpressed in multiple myeloma (Hurt et al., 2004). c-maf encodes a B-ZIP transcription factor, and gene expression profiling was used to discover the genes that it transactivates (Hurt et al., 2004). One c-maf target gene is integrin b7, which encodes an adhesion molecule that, together with integrin aE, binds to E-cadherin. E-cadherin is expressed on the surface of bone marrow stromal cells, and the up-regulation of integrin b7 by c-maf causes myeloma cells to adhere more strongly to these stromal cells. As a result, the bone marrow stromal cells secrete more vascular endothelial growth factor (VEGF), which is a proliferation factor for myeloma cells as well as an angiogenic factor. This example suggests that an understanding of mechanisms that may cause certain DLBCLs to be ‘‘addicted’’ to the lymph node microenvironment could provide new targets for therapeutic intervention. 3.4. MHC Class II Signature Expression of the MHC class II signature predicts favorable survival in DLBCL (Rosenwald et al., 2002). This signature is comprised of the genes encoding the alpha and beta chains of MHC class II molecules as well as the gene encoding invariant chain, which plays a role in the MHC class II antigen presentation pathway. The variation in MHC class II signature expression among DLBCLs is due to differences in expression within the malignant cells, and not to differences in MHC class II-expressing non-malignant cells (Rimsza et al., 2004). In fact, some DLBCLs are virtually devoid of MHC class II molecules on their surface. Since the MHC class II molecules and invariant chain are encoded on different chromosomes, a single structural alteration in the genome cannot account for differences in MHC class II signature expression. Therefore, these differences are likely due to variation in a regulatory factor that coordinates the expression of all of the MHC class II signature

184

L O UIS M. STA UDT A ND SA NDE EP DAVE

genes. Notably, the prognostic significance of the MHC class II signature is independent of the DLBCLs subgroup distinction. The mechanisms accounting for the favorable prognosis associated with MHC class II signature expression are not clear. One possibility is that loss of MHC class II molecules on the surface of DLBCLs prevents an immune response from developing against the malignant cells. In this regard, it is interesting that DLBCLs lacking MHC class II expression have significantly fewer CD8þ T cells infiltrating the tumor (List et al., 1993; Rimsza et al., 2004). MHC class II is associated with antigen presentation to CD4þ T cells, which are known to be required for the generation of optimal CD8þ T cell responses. However, further work is needed to evaluate whether such immunological mechanisms account for the prognostic significance of MHC class II signature expression. 3.5. Proliferation Signature Many of the genes with expression patterns that are correlated with poor outcome in DLBCL belong to the proliferation signature (Rosenwald et al., 2002). The proliferation signature includes genes that are expressed highly in proliferating cells and at low levels in quiescent cells (Shaffer et al., 2001). Typically, one eighth of all the genes represented on a DNA microarray belong to the proliferation signature. These genes encode a functionally diverse set of proteins that play roles in cell cycle progression, DNA replication and repair, protein translation, cell growth, and cellular metabolism. To some degree, the proliferation signature genes that encode proteins involved in the same aspect of cellular proliferation (e.g., mitosis regulators, glycolysis enzymes, ribosomal proteins) are more tightly coregulated than are proliferation signature genes as a whole (Shaffer et al., 2001). Given the breadth of the proliferation signature, it is important to understand which subset of these genes is associated with survival in DLBCL. Interestingly, proliferation signature genes that encode cell cycle progression proteins are not associated with survival in DLBCL. Instead, the oncogene c-myc is one of the genes in the proliferation signature that is most strongly associated with the outcome (Rosenwald et al., 2002). c-myc is a transcription factor that controls many aspects of cellular metabolism, but it is particularly associated with cell growth (i.e., an increase in cell size) (Levens, 2002). c-myc is well known to play a role in the pathogenesis of non-Hodgkin lymphomas in that it is deregulated by translocation to the immunoglobulin locus in all cases of Burkitt lymphoma and in a small subset of DLBCLs. Notably, normal germinal center B cells have very low expression of c-myc and some of its known target genes such as those that encode the ribosomal proteins (Klein et al., 2003; Shaffer et al., 2001). Teleologically, germinal center centroblasts may have low c-myc so as to avoid expending energy on cell growth, thereby favoring maximal proliferation. This

b io l og y of h um an ly mp ho mas

185

bias in favor of proliferation allows centroblasts to undergo the multiple rounds of somatic hypermutation that are required for optimal positive selection of B cells with high affinity for antigen. Among the DLBCL subgroups, ABC DLBCL has the highest expression of cmyc, which is consistent with its origin from a post-germinal center cell that has engaged signaling pathways that transcriptionally activate c-myc. Stimulation of normal B cells through a variety of cell surface receptors increases c-myc expression and, in particular, the NF-kB signaling pathway can increase c-myc transcription (Schauer et al., 1996). Therefore, the constitutive activity of the NF-kB pathway in ABC DLBCL may contribute to its high c-myc expression. The transcriptional regulation of c-myc is complex, however, and this is unlikely to be the entire explanation for elevated c-myc expression in ABC DLBCLs. Interestingly, another proliferation signature gene associated with poor outcome in DLBCL is nucleostemin (Rosenwald et al., 2002), which encodes a nucleolar protein that is a direct transcriptional target of c-myc (O’Connell et al., 2003; Schlosser et al., 2003; Tsai and McKay, 2002; Zeller et al., 2003). One function of nucleostemin may be to bind p53 and sequester it in the nucleolus (Tsai and McKay, 2002). However, nucleostemin is likely to have other functions since RNA interference-mediated knockdown of nucleostemin decreases the number of cells in the S-phase of the cell cycle (Tsai and McKay, 2002). c-myc transactivates a large number of genes that encode nucleolar proteins and also enhances processing of ribosomal RNA in the nucleolus (Schlosser et al., 2003). A third proliferation signature gene in the DLBCL survival predictor is nucleophosmin-3/NPM3, which is structurally related to the nucleolar protein nucleophosmin-1/NPM1 (Rosenwald et al., 2002). Together, these observations suggest that the particular subset of proliferation signature genes that is associated with poor outcome in DLBCL is that which contributes to nucleolar function, many of which are c-myc targets. c-myc thus appears to play an important role in lymphoma biology that extends beyond those malignancies in which c-myc is translocated. 3.6. A Gene Expression-Based Survival Predictor for Mantle Cell Lymphoma Mantle cell lymphoma accounts for roughly 6% of non-Hodgkin lymphomas, but it contributes disproportionately to the deaths from lymphoma because there is no curative treatment. This lymphoma type is derived in the vast majority of cases from a pre-germinal center B cell since its rearranged immunoglobulin genes are usually unmutated. As a consequence, the biology of mantle cell lymphoma is substantially different from that of other nonHodgkin lymphomas that originate from B cells that have traversed the germinal center. The characteristic t(11;14) translocation of mantle cell

186

L O UIS M. STA UDT A ND SA NDE EP DAVE

Figure 4 (A) Differential expression of proliferation signature genes in biopsy samples from mantle cell lymphoma. The expression patterns of 20 genes from the proliferation signature are shown across 92 mantle cell lymphoma biopsy samples. The expression of each of these genes was found to be associated with short survival (Rosenwald et al., 2003b). The expression levels of these 20 genes were averaged to create the proliferation signature average, which was divided into four quartile groups as shown. (B) Kaplan-Meier plot of overall survival of patients with mantle cell

b io l og y of h um an ly mp ho mas

187

lymphoma deregulates the cyclin D1 gene by placing it in proximity to the immunoglobulin heavy chain locus. D-type cyclins form heterodimers with the cyclin-dependent kinases cdk4 and cdk6, thereby forming active kinase complexes that are key regulators of the G1/S phase transition of the cell cycle (Sherr and McCormick, 2002). Of the three D-type cyclins, cyclin D1 is not normally expressed by B lymphocytes, whereas cyclin D2 is expressed by mitogenically activated B cells, and cyclin D3 is expressed by germinal center B cells. The t(11;14) translocation points to the central role of cell cycle dysregulation in the pathophysiology of mantle cell lymphoma. Mantle cell lymphoma has a characteristic gene expression signature that can be used to distinguish it from other non-Hodgkin lymphomas (Rosenwald et al., 2003b). The vast majority (>95%) of cases that are histologically compatible with a diagnosis of mantle cell lymphoma have cyclin D1 overexpression (Rosenwald et al., 2003b). However, gene expression profiling revealed a rare subtype of mantle cell lymphoma in which cyclin D1 is not expressed, but which is nevertheless histologically and molecularly indistinguishable from cyclin D1-positive mantle cell lymphoma (Rosenwald et al., 2003b). Not surprisingly, cyclin D2 or cyclin D3 is expressed in some of these cases by mechanisms that are as yet unknown. The survival of patients with mantle cell lymphoma ranges from under one year to more than 5 years. Using the gene expression signature of mantle cell lymphoma, it was possible to identify some patients with an extremely indolent form of this lymphoma who survived more than 10 years following diagnosis (Rosenwald et al., 2003b). Although mantle cell lymphoma patients receive a variety of chemotherapy regimens, none has been shown to alter the length of survival. A supervised analysis of gene expression in biopsy specimens from patients with cyclin D1-positive mantle cell lymphoma was used to uncover the molecular basis for the varying lengths of survival of these patients (Rosenwald et al., 2003b). All of the genes whose expression was associated with short survival belonged to the proliferation gene expression signature (Fig. 4A). In contrast to DLBCL, the proliferation signature genes that were associated with survival lymphoma, divided into four quartile groups according to the expression of the proliferation signature in their tumors. (C) The proliferation signature integrates distinct oncogenic events in mantle cell lymphoma. The expression of the cyclin D1 mRNA was determined by a quantitative RT‐PCR assay for the coding region. Deletion of the INK4a/ARF locus was determined by a quantitative PCR using genomic DNA. Yellow indicates heterozygous or homozygous deletion; black indicates wild-type copy number. Tumors with higher expression of the proliferation signature tend to have higher cyclin D1 mRNA expression and/or deletion of the INK4a/ARF locus. (D) Model depicting how increased cyclin D1 expression and deletion of the INK4a/ARF locus may contribute to a higher proliferation rate in mantle cell lymphoma. See text for details.

188

L O UIS M. STA UDT A ND SA NDE EP DAVE

in mantle cell lymphoma included genes involved in cell cycle progression and DNA synthesis, but did not include c-myc. This suggests that proliferation signature genes that quantitatively reflect the tumor proliferation rate predict the length of survival in mantle cell lymphoma. The average expression of 20 proliferation signature genes was used to calculate a survival predictor score for each mantle cell lymphoma patient (Rosenwald et al., 2003b). The patients were ranked according to these scores and divided into four equal quartile groups with median survival times of 0.8 years, 2.3 years, 3.3 years, and 6.7 years (Fig. 4B). Other studies have used immunohistochemical or cytological techniques to estimate the proliferation rate in mantle cell lymphoma. At best, however, the prognostic groups that were identified differed by 2.7 years in median survival (Argatoff et al., 1997; Bosch et al., 1998; Raty et al., 2002; Velders et al., 1996). The proliferation signature average provides a more quantitative measure of tumor proliferation rate than these other methods, which most likely explains why it is able to identify risk groups of patients that differ by 5.9 years in median survival. From a clinical standpoint, this molecular predictor of survival could provide valuable prognostic information to these patients. For those patients with indolent forms of this lymphoma, a watchful waiting approach is appropriate. Patients with aggressive forms of the disease should be considered for newer therapeutic approaches, some of which show early promise (see later discussion). From a biological standpoint, the most remarkable feature of the proliferation signature average is that it quantitatively integrates the effects of multiple oncogenic events that affect cell cycle progression. Unexpectedly, a quantitative RT-PCR assay for the cyclin D1 coding region revealed higher expression in many of the mantle cell lymphomas with the highest proliferation signature average (Fig. 4C) (Rosenwald et al., 2003b). Consequently, higher expression of cyclin D1 coding region mRNA was associated with shorter survival. Several molecular mechanisms account for the varying expression of cyclin D1 coding region transcripts. Various forms of cyclin D1 mRNA have been observed in mantle cell lymphoma: a full-length 4.7 kb isoform that contains a 3 kb 30 untranslated region and shorter isoforms that essentially consist of the coding region. The cyclin D1 30 untranslated region contains an mRNA destabilizing element and therefore, with an equivalent amount of transcription, the long mRNA isoform will accumulate to lower levels than the short mRNA isoforms (Lebwohl et al., 1994; Lin et al., 2000; Rimokh et al., 1994). Some mantle cell lymphomas were found to lack expression of the long mRNA isoform and exclusively expressed shorter, more stable, mRNA isoforms, and these were the cases with higher levels of cyclin D1 coding region transcripts (Rosenwald et al., 2003b). In some mantle cell lymphoma cases, the genomic region encoding the cyclin D1 30 untranslated region is deleted, resulting in

b io l og y of h um an ly mp ho mas

189

expression of a short cyclin D1 mRNA (de Boer et al., 1995; Rimokh et al., 1994; Seto et al., 1992; Withers et al., 1991). Another molecular mechanism leading to exclusive expression of a short cyclin D1 mRNA is the acquisition of a somatic mutation immediately downstream of the stop codon that generates a new polyadenylation signal, thereby truncating the mRNA (unpublished). Deletion of the INK4a/ARF locus is a frequent oncogenic event in mantle cell lymphoma, occuring in roughly one fifth of cases (Dreyling et al., 1997; Pinyol et al., 1997, 1998, 2000; Rosenwald et al., 2003b). INK4a/ARF deletions are more frequent in mantle cell lymphomas with high expression of the proliferation signature and are consequently associated with short survival (Fig. 4C) (Rosenwald et al., 2003b). The INK4a/ARF locus encodes two key tumor suppressor proteins, p16INK4a and p14ARF (Sherr and McCormick, 2002). p16INK4a prevents the assembly of cdk4 and cdk6 with D-types cyclins and thus is an important negative regulator of the G1/S phase transition of the cell cycle. p14ARF blocks the ability of MDM2 to target p53 for degradation, and thus potentiates p53-dependent apoptosis and cell cycle arrest. In addition, loss of p14ARF retards proliferation of mouse embryonic fibroblasts and pre-B cells (Kamijo et al., 1998; Randle et al., 2001). Unlike some cancers, mantle cell lymphomas do not sustain inactivating mutations in the p16INK4a coding region (Pinyol et al., 1997, 2000), suggesting that the selective advantage of INK4a/ARF deletions in mantle cell lymphoma is due to loss of both p16INK4a and p14ARF. Interestingly, INK4a/ARF deletions and elevated cyclin D1 expression were statistically independent in their associations with high proliferation signature expression and short survival (Rosenwald et al., 2003b). In fact, many mantle cell lymphomas have deletions of both the cyclin D1 30 untranslated region and the INK4a/ARF locus. This observation suggests a model in which these two oncogenic events act synergistically to accelerate cell cycle progression. One way this could occur is by increasing the frequency (or probability) that a lymphoma cell moves from G1 to S phase. A current view of this cell cycle transition is that cyclin D/cdk4(6) complexes bind the cyclin-dependent kinase inhibitors p21 and p27kip1, but are not inhibited by them. In so doing, they titrate these inhibitors away from cyclin E/cdk2 complexes, whose activity is critical for entry into S phase (Fig. 4D). In keeping with this model, most p27kip1 in mantle cell lymphomas is physically associated with cyclin D1/cdk4 (6) (Quintanilla-Martinez et al., 2003). This model could have therapeutic implications. p27kip1 is ubiquitinylated by Skp2, which targets it for proteosomal degradation. Intriguingly, the proteasome inhibitor Velcade/PS-341 has shown activity against mantle cell lymphoma in early clinical trials. If p27kip1 levels are quantitatively titrated by cyclin D1/cdk4(6) complexes, proteosomal inhibition might allow more

190

L O UIS M. STA UDT A ND SA NDE EP DAVE

p27kip1 to accumulate and tip this regulatory balance in favor of a block in G1 phase of the cell cycle. A theoretically ideal inhibitor of the G1/S phase transition, yet to be developed, would be a small molecule mimetic of p16INK4a that would inhibit assembly of cdk4 and cdk6 with cyclin D1. As mentioned above, loss of p14ARF in some mantle cell lymphomas may also contribute to cell growth and a higher proliferation rate. Many of the mantle cell lymphomas with INK4a/ARF deletions are histologically ‘‘blastic,’’ meaning that they have a large cell size compared with other mantle cell lymphomas (Pinyol et al., 1997; Rosenwald et al., 2003b). This phenotype could be caused by increased c-myc activity, due to the absence of p14ARF (Datta et al., 2004; Qi et al., 2004). In this regard, it is notable that the BMI-1 gene is amplified in some mantle cell lymphoma cases that lack INK4a/ARF deletions (Bea et al., 2001; Rosenwald et al., 2003b). BMI-1 is a transcriptional repressor that blocks the expression of p16INK4a and p14ARF and cooperates with c-myc in malignant transformation (Sherr and McCormick, 2002). Thus, enhanced c-myc function may be another way in which INK4a/ARF deletions synergize with cyclin D1 upregulation to enhance the proliferation rate of mantle cell lymphomas. This analysis leads to the view that tumor proliferation rate is a rheostat that can vary continuously over a broad range. The setpoint of this rheostat results from the cellular integration of multiple oncogenic changes that affect the rate or probability that the cancer cell transits the G1/S phase boundary of the cell cycle. The fact that the proliferation rate in mantle cell lymphoma (as measured by the proliferation signature expression) can be modeled using cyclin D1 expression and INK4a/ARF deletion illustrates that complex cancer phenotypes may become mathematically tractable as more precise measurements become available. Mathematical models can have heuristic value in that they can highlight areas where further biological research is needed. For example, the multivariate model of survival based on cyclin D1 expression and INK4a/ARF deletion was not as statistically significant as the model based on proliferation signature expression (Rosenwald et al., 2003b). This observation suggests that additional oncogenic mechanisms remain to be discovered that affect the proliferation rate and, consequently, the length of survival in mantle cell lymphoma. An important implication of this rheostat model of proliferation is that therapies that can dial down the proliferation setpoint in mantle cell lymphoma might be of great clinical benefit, even if they are not curative. In other words, it is possible to imagine a therapy that could decrease the proliferation rate so significantly that it might be able to move a patient from the least favorable prognostic group to the most favorable prognostic group, thereby prolonging the patient’s life by more than 5 years. Although such therapies

b io l og y of h um an ly mp ho mas

191

might affect normal cells as well, studies in mice have shown that the D-type cyclins have cell type-restricted functions. For example, mice deficient in cyclin D1 have discrete defects in embryonic retinal development and in pregnancy-associated mammary development but are normal in other respects (Sicinski et al., 1995). Indeed, in mice lacking all D-type cyclins, many cell types proliferate normally, although hematopoietic stem cell proliferation is compromised (Kozar et al., 2004). It therefore seems conceivable that a therapeutic window may exist to allow prolonged treatment of mantle cell lymphoma patients with agents targeted at cyclin D/cdk4(6) complexes. 3.7. A Gene Expression-Based Survival Predictor for Follicular Lymphoma Follicular lymphoma, the second most common form of non-Hodgkin lymphoma, has a highly variable clinical course. Although the median survival is approximately 10 years following diagnosis, some patients have an aggressive illness that is fatal in under 1 year, while others may live more than 20 years. Patients with follicular lymphoma are treated with a variety of chemotherapeutic and immunotherapeutic regimens (Bendandi et al., 1999; Colombat et al., 2001; Czuczman et al., 1999; Kwak et al., 1992; Maloney et al., 1997; Timmerman et al., 2002; Witzig et al., 2002), but it has not been demonstrated that these approaches change the length of survival of these patients (Horning, 2000). Follicular lymphomas are derived from germinal center B cells and maintain the gene expression program of this stage of differentiation (Dave et al., 2004; Flenghi et al., 1995; Shaffer et al., 2002b). In addition, most follicular lymphomas grow as pseudo-germinal centers that include T cells and follicular dendritic cells intermingled with the malignant cells. Unlike normal germinal center B cells, roughly 90% of follicular lymphomas express BCL-2 as a result of the characteristic t(14;18) translocation. In some cases, follicular lymphoma ‘‘transforms’’ into an aggressive lymphoma resembling DLBCL, and this transformation can be associated with a variety of oncogenic changes (Lossos and Levy, 2003). However, there is no evidence that these stochastic events affect the overall survival rate. Gene expression profiling has revealed that the length of survival following diagnosis of follicular lymphoma can be predicted by biological differences among the tumors at the time of diagnosis (Dave et al., 2004). A new form of supervised analysis, termed survival signature analysis, was used to develop a gene expression-based survival predictor for follicular lymphoma (Dave et al., 2004). Like other supervised methods, survival signature analysis begins with the subdivision of the cases into a training set and a test set, followed by the

192

L O UIS M. STA UDT A ND SA NDE EP DAVE

Figure 5 (A) Two sets of coordinately expressed genes, termed the immune response‐1 and immune response‐2 signatures, are associated with survival in follicular lymphoma. The expression pattern of each gene in these two signatures is shown for 191 follicular lymphoma biopsy samples. Expression of the immune response‐1 signature is associated with long survival following diagnosis, and expression of the immune response‐2 signature is associated with short survival (Dave et al., 2004). These signatures are combined into a multivariate model of survival that generates a survival predictor score for each patient. Patients are ranked according to this survival predictor and divided into four equal quartiles as shown. (B) Kaplan‐Meier plot of overall survival of patients in the four quartiles of the survival predictor. (C) Expression of the genes that constitute the immune response‐1 and immune response‐2 signatures in normal immune cells. Tonsillar germinal center B cells, peripheral blood B cells, peripheral blood T cells, and peripheral blood monocytes from healthy donors were profiled for gene expression. Relative expression of the immune response‐1 and immune response‐2 signature genes in the malignant (CD19‐positive) and the

b io l og y of h um an ly mp ho mas

193

identification of genes with expression patterns associated with long survival (good prognosis genes) or short survival (bad prognosis genes) within the training set. Hierarchical clustering is then used to organize the good prognosis genes and the bad prognosis genes according to their expression patterns within the training set of cases. Clusters of genes with highly correlated expression patterns are grouped together into survival-associated signatures. Within each survival-associated signature, the expression levels of the component genes are averaged, creating a signature average for each patient. Multivariate statistical models are created using signature averages from the training set of cases, and these models are then tested for their reproducibility in the test set of cases. This algorithm is based on the assumption that within a particular survival-associated signature, the expression profile of each component gene reflects the same biological process that influences survival. By grouping coordinately expressed genes together and creating a limited set of survival-associated signatures, the multiple comparisons problem associated with the supervised analysis of DNA microarray data is avoided (Ransohoff, 2004). Using this method, an optimal survival model in follicular lymphoma was created using two survival-associated signatures, one from the good prognosis gene set and one from the bad prognosis gene set (Fig. 5A). These two signatures were termed immune response-1 and immune response-2 because their component genes included genes known to be expressed in normal immune cells. Although the immune response-1 and immune response-2 signatures each predicted survival as single variables, they were strongly synergistic in a multivariate model. This suggests that the relative level of these two signatures is more important in predicting survival than the absolute level of either one alone. The model generated a ‘‘survival predictor score’’ for each patient, with a high score associated with long survival and a low score associated with short survival. The patients were ranked according to their survival predictor scores and divided into four equal quartile groups that had strikingly different median survival times of 3.9 years, 10.8 years, 11.1 years, and 13.6 years, respectively (Fig. 5B). This finding demonstrates that although the follicular lymphoma genome may continue to be altered following diagnosis, such stochastic changes do not have a large effect on the length of survival. Rather, the biological heterogeneity already present in

non‐malignant (CD19‐negative) cells isolated from follicular lymphoma biopsy samples is shown. (D) A schematic of how the immune response hypothesis and immune cell dependence hypothesis might explain the clinical behavior of tumors with high expression of the immune response‐1 signature or the immune response‐2 signature (see text for details).

194

L O UIS M. STA UDT A ND SA NDE EP DAVE

follicular lymphoma at the time of diagnosis dictates, in large measure, the clinical aggressiveness of the disease. The immune response-1 and immune response-2 signatures were found to be preferentially expressed in the CD19-negative nonmalignant cells isolated from follicular lymphoma biopsies by flow sorting (Fig. 5C) (Dave et al., 2004). Furthermore, the genes that make up these two signatures are not highly expressed in normal germinal center B cells, the cell of origin of follicular lymphoma, but rather in normal blood T cells or monocytes. These results point to an interplay between the malignant cells and the host immune system that influences the clinical behavior of follicular lymphoma. The immune response-1 signature includes a number of genes that encode T cell-restricted proteins, such as CD7, CD8b1, ITK, and LEF1 (Fig. 5C) (Dave et al., 2004). However, this signature is not merely a measure of the number of T cells in the follicular lymphoma because the expression patterns of a number of genes encoding T cell markers (e.g., CD2, CD4, LAT, TRIM, SH2D1A) were not found to be associated with survival in follicular lymphoma. This suggests that the expression of the T-cell genes in the immune response-1 signature reflects the presence of a particular T-cell subset(s) within the tumor. In this regard, it is notable that one of the immune response-1 signature genes is CD8b1, which could indicate the presence of cytotoxic T cells. However, this signature also includes several genes that are highly expressed in macrophages, such as ACTN1 (Allen and Aderem, 1996) and TNFSF13B (BLYS/BAFF) (Craxton et al., 2003). Therefore, the immune response-1 signature appears to reflect a complex mixture of immune cells, including T cells, which is associated with long survival in follicular lymphoma. The immune response-2 signature, by contrast, does not include genes encoding T-cell markers, but rather includes genes expressed in macrophages and/or dendritic cells (Fig. 5C) (Dave et al., 2004). For instance, FCGR1A (CD64), TLR5, and C3AR encode receptors for immunoglobulin Fc regions, flagellin and complement component C3A, respectively, that are typically expressed in monocytic cells (Ames et al., 1996; Hayashi et al., 2001; Muzio et al., 2000; Roglic et al., 1996). Other immune response-2 signature genes are expressed in mature dendritic cells, including SEPT10, which encodes a septin, and LGMN, which encodes a lysosomal protease (Li et al., 2003; Sui et al., 2003). Also included are the genes encoding the complement components C1qA, C1qB, and C4A, which are synthesized by phagocytes in the myeloid lineage (McPhaden and Whaley, 1993; Tsukamoto et al., 1992). The immune response-2 signature therefore reflects an immune infiltrate dominated by macrophages and/or dendritic cells that is associated with short survival in follicular lymphomas.

b io l og y of h um an ly mp ho mas

195

It is important to emphasize the strong statistical synergism of the immune response-1 and the immune response-2 signatures in predicting the length of survival (Dave et al., 2004). Biologically, this means that many follicular lymphoma tumors express both signatures, but their relative ratio is what is most predictive of the length of survival. This observation highlights the ability of gene expression profiling to reveal quantitative differences in immune responses that are clinically meaningful. Why do patients with follicular lymphoma differ with respect to the type of immune infiltrate in their tumors? One possibility is that these differences reflect quantitative traits of the immune system that influence the way in which it responds to the malignant follicular lymphoma cells. Genetic differences in key regulators of the immune response are well known to influence susceptibility to autoimmune and inflammatory disorders and may therefore play a role in anti-tumor responses as well. A second possibility is that the molecular features of the malignant cells in follicular lymphoma dictate the character of the immune infiltrate. To address this possibility comprehensively, the gene expression profiles and genetic abnormalities of sorted malignant cells from a large number of cases will need compared with the gene expression profiles of the corresponding nonmalignant cells. Why is the immune response-1 signature associated with good prognosis and the immune response-2 signature associated with bad prognosis? Two hypotheses, which are not mutually exclusive, can be entertained: an ‘‘immune response’’ hypothesis and an ‘‘immune cell dependence’’ hypothesis (Fig. 5D). In the immune response hypothesis, the immune response-1 signature could reflect an active anti-tumor immune response. There is abundant clinical evidence suggesting that anti-tumor immune responses can be mounted in some patients with follicular lymphoma. Follicular lymphoma is the only lymphoma in which spontaneous regressions are observed, albeit rarely (Horning and Rosenberg, 1984). When this occurs in other cancers, such as melanoma and renal cell carcinoma, it is taken as an indication of an effective anti-tumor immune response. In addition, anti-idiotype vaccines can elicit long-term remissions in some patients with follicular lymphoma, demonstrating that this is one of the few cancers, so far, in which an immune response is clinically effective (Bendandi et al., 1999; Kwak et al., 1992; Timmerman et al., 2002). The immune response-1 signature may therefore reflect an anti-tumor immune response that is able to prolong the survival of these patients, but not eradicate the tumor. The inclusion of T-cell genes in this signature, especially the cytotoxic T cell gene CD8b1, is consistent with this possibility. The immune response-2 signature lacks T-cell–restricted genes, and instead includes genes expressed in innate immune cells. It is conceivable that the

196

L O UIS M. STA UDT A ND SA NDE EP DAVE

follicular lymphomas with high expression of the immune response-2 signature have evolved mechanisms to evade an adaptive immune response, and the short survival of these patients reflects this immune evasion. If this ‘‘immune response’’ hypothesis is correct, the success of immunebased therapies in follicular lymphoma may depend upon the molecular profile of the tumor-infiltrating immune cells. Therapies that rely on T-cell responses, such as anti-idiotype vaccination, may be more successful in tumors that express the immune response-1 signature highly since this signature reflects a type of T-cell infiltrate that is associated with long survival. Furthermore, it is conceivable that non-specific augmentation of T-cell responses, as can be achieved with anti-CTLA-4 therapy (Egen et al., 2002), could be of benefit to patients whose tumors express the immune response-1 signature highly. The ‘‘immune cell dependence’’ hypothesis suggests that the tumor-infiltrating immune cells in some follicular lymphomas provide trophic signals that promote survival and/or proliferation of the malignant cells (Fig. 5D). The immune response-1 signature could reflect a pseudo-germinal center reaction in which the malignant cells receive signals from T cells and/or follicular dendritic cells. The malignant cells may be dependent upon these microenvironmental signals, making it difficult for them to leave the lymph node. This microenvironmental ‘‘addiction’’ could account for the relatively favorable prognosis associated with the immune response-1 signature. Follicular lymphomas with high expression of the immune response-2 signature may have lost their dependence on microenvironmental signals and may therefore be able to survive in other anatomical sites, possibly accounting for the relatively short survival of these patients. Precedent for the immune cell dependence hypothesis is provided by certain mouse B-cell lymphomas that require T cells for their growth (Ponzio and Thorbecke, 2000). These mouse lymphomas express an MMTV-encoded superantigen that stimulates CD4-positive T cells, which in turn secrete cytokines such as IL-4 that may provide survival signals to the lymphomas. Some intriguing data suggest that such mechanisms should be considered in human lymphomas as well. In follicular lymphoma, the pattern of immunoglobulin mutations is consistent with a role for antigen selection at some point in the genesis of the tumor (Bahler and Levy, 1992). In addition, the immunoglobulin variable regions in follicular lymphoma acquire N-linked glycosylation sites during the hypermutation process in 79% of cases, which is far in excess of the 9% frequency observed in normal B cells (Zhu et al., 2002). This surprising finding raises the possibility that carbohydrates attached to the immunoglobulin receptor of a follicular lymphoma could interact with lectin-like proteins present within the germinal center microenvironment. Further investigations will be needed to understand whether such interactions are of functional significance at the time of clinical presentation or play a role in the early

b io l og y of h um an ly mp ho mas

197

development of these cancers. If malignant cells and immune cells communicate in follicular lymphoma, a molecular understanding of this communication could provide new targets for therapy. 4. Concluding Remarks The comprehensive and exploratory nature of gene expression profiling has provided fresh insights into the pathobiology of human lymphomas, many of which are applicable to a wider range of cancers. Principles of cancer biology revealed by gene expression profiling include the following: 1. The clinical and biological behavior of cancers can be understood and modeled with mathematical precision. Complex clinical phenotypes such as the length of survival and the response to therapy can now be understood as the consequence of quantitative differences in tumor biology. The proliferation rate in cancer can be modeled as a continuous variable that integrates multiple oncogenic events that influence cell cycle progression. It is likely that other cancer phenotypes such as susceptibility to apoptosis or response to DNA damage can also be modeled, leading ultimately to a quantitative, systems biology view of cancer. 2. Existing diagnostic categories of cancer consist of multiple molecularly distinct diseases that differ in their cell of origin, oncogenic mechanisms, and clinical outcome. The example of diffuse large B cell lymphoma illustrates this principle well, but gene expression profiling has also uncovered distinct disease entities in other cancers also (Sorlie et al., 2003). 3. The clinical heterogeneity of cancer can be ascribed to heterogeneity in the tumor at the time of diagnosis. This observation holds for each lymphoma type studied thus far, as well as for other cancers (Bernards and Weinberg, 2002). Although the stepwise acquisition of oncogenic changes certainly contributes to the cancer phenotype, stochastic changes in the tumor genome after diagnosis do not appear to strongly influence subsequent clinical behavior. 4. Multiple independently varying biological features of tumors contribute to their clinical behavior. Gene expression signatures can be used to measure different biological attributes in tumor samples. Optimal models of survival usually incorporate several gene expression signatures, reflecting tumor cell intrinsic properties as well as the influence of the tumor microenvironment. 5. Interactions between the tumor and its microenvironment are biologically and clinically crucial. For DLBCL and follicular lymphoma, survival was found to be strongly associated with the nature of the tumor-infiltrating immune cells. An understanding of the interaction between the malignant cell and its microenvironment should provide new therapeutic options.

198

L O UIS M. STA UDT A ND SA NDE EP DAVE

6. Molecular diagnosis leads to new molecular targets. Of the three gene expression subgroups of DLBCL, two respond to inhibitors of the NF-kB pathway, whereas the third is impervious to these agents. The importance of molecular diagnosis will continue to increase as drugs become available to target specific signaling pathways. Several research challenges must be surmounted in order to build upon the recent progress in understanding human lymphomas. We must develop a comprehensive view of the abnormal signaling networks inside lymphoma cells, and functional genomics techniques such as RNA interference will be critical in this effort. Coupling RNA interference with gene expression profiling will lead to an understanding of the wiring diagrams of lymphoma cells, which will ultimately enable rational manipulation of signaling pathways. By necessity, much of this work must be carried out in lymphoma cell lines, and we must take care to use cell lines that are faithful mimics of particular lymphoma types. Concerted efforts should be made to generate additional cell lines that are representative of each lymphoma type. Finally, given the importance of the tumor-infiltrating immune cells in lymphomas, methods need to be developed to maintain patient tumor samples in tissue culture or in immunodeficient mice in a fashion that preserves the interactions between the malignant cells and their microenvironment. In all of these efforts, gene expression profiling will continue to provide a comprehensive and biologically informative framework. References Addis, B. J., and Isaacson, P. G. (1986). Large cell lymphoma of the mediastinum: A B-cell tumour of probable thymic origin. Histopathology 10, 379–390. Albagli, O., Dhordain, P., Bernardin, F., Quief, S., Kerkaert, J. P., and Leprince, D. (1996). Multiple domains participate in distance-independent LAZ3/BCL6-mediated transcriptional repression. Biochem. Biophys. Res. Commun. 220, 911–915. Alizadeh, A., Eisen, M., Davis, R. E., Ma, C., Sabet, H., Tran, T., Powell, J., Yang, L., Marti, G., Moore, T., Hudson, J., Chan, W. C., Greiner, T., Weisenburger, D., Armitage, J. O., Lossos, I., Levy, R., Botstein, D., Brown, P. O., and Staudt, L. M. (1999). The lymphochip: A specialized cDNA microarray for the genomic-scale analysis of gene expression in normal and malignant lymphocytes. Cold Spring Harbor. Symp. Quant. Biol. 64, 71–78. Alizadeh, A. A., Eisen, M. B., Davis, R. E., Ma, C., Lossos, I. S., Rosenwald, A., Boldrick, J. C., Sabet, H., Tran, T., Yu, X., Powell, J. I., Yang, L., Marti, G. E., Moore, T., Hudson, J., Lu, L., Lewis, D. B., Tibshirani, R., Sherlock, G., Chan, W. C., Greiner, T. C., Weisenburger, D. D., Armitage, J. O., Warnke, R., Levy, R., Wilson, W., Grever, M. R., Byrd, J. C., Botstein, D., Brown, P. O., and Staudt, L. M. (2000). Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503–511. Allen, L. A., and Aderem, A. (1996). Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages. J. Exp. Med. 184, 627–637.

b io l og y of h um an ly mp ho mas

199

Allman, D., Jain, A., Dent, A., Maile, R. R., Selvaggi, T., Kehry, M. R., and Staudt, L. M. (1996). BCL-6 expression during B-cell activation. Blood 87, 5257–5268. Ames, R. S., Li, Y., Sarau, H. M., Nuthulaganti, P., Foley, J. J., Ellis, C., Zeng, Z., Su, K., Jurewicz, A. J., Hertzberg, R. P., Bergsma, D. J., and Kumar, C. (1996). Molecular cloning and characterization of the human anaphylatoxin C3a receptor. J. Biol. Chem. 271, 20231–20234. Angelin-Duclos, C., Cattoretti, G., Lin, K. I., and Calame, K. (2000). Commitment of B lymphocytes to a plasma cell fate is associated with Blimp-1 expression in vivo. J. Immunol. 165, 5462–5471. A predictive model for aggresive non-Hodgkin’s lymphoma. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. (1993). N. Engl. J. Med. 329, 987–994. Argatoff, L. H., Connors, J. M., Klasa, R. J., Horsman, D. E., and Gascoyne, R. D. (1997). Mantle cell lymphoma: A clinicopathologic study of 80 cases. Blood 89, 2067–2078. Bahler, D. W., and Levy, R. (1992). Clonal evolution of a follicular lymphoma: Evidence for antigen selection. Proc. Natl. Acad. Sci. USA 89, 6770–6774. Bargou, R. C., Emmerich, F., Krappmann, D., Bommert, K., Mapara, M. Y., Arnold, W., Royer, H. D., Grinstein, E., Greiner, A., Scheidereit, C., and Dorken, B. (1997). Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin’s disease tumor cells. J. Clin. Invest. 100, 2961–2969. Bargou, R. C., Leng, C., Krappmann, D., Emmerich, F., Mapara, M. Y., Bommert, K., Royer, H. D., Scheidereit, C., and Dorken, B. (1996). High-level nuclear NF-kappa B and Oct-2 is a common feature of cultured Hodgkin/Reed-Sternberg cells. Blood 87, 4340–4347. Bea, S., Tort, F., Pinyol, M., Puig, X., Hernandez, L., Hernandez, S., Fernandez, P. L., van Lohuizen, M., Colomer, D., and Campo, E. (2001). BMI-1 gene amplification and overexpression in hematological malignancies occur mainly in mantle cell lymphomas. Cancer. Res. 61, 2409–2412. Bendandi, M., Gocke, C. D., Kobrin, C. B., Benko, F. A., Sternas, L. A., Pennington, R., Watson, T. M., Reynolds, C. W., Gause, B. L., Duffey, P. L., Jaffe, E. S., Creekmore, S. P., Longo, D. L., and Kwak, L. W. (1999). Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma. Nat. Med. 5, 1171–1179. Bentz, M., Barth, T. F., Bruderlein, S., Bock, D., Schwerer, M. J., Baudis, M., Joos, S., Viardot, A., Feller, A. C., Muller-Hermelink, H. K., Lichter, P., Dohner, H., and Moller, P. (2001). Gain of chromosome arm 9p is characteristic of primary mediastinal B-cell lymphoma (MBL): Comprehensive molecular cytogenetic analysis and presentation of a novel MBL cell line. Genes Chromosomes Cancer 30, 393–401. Bereshchenko, O. R., Gu, W., and Dalla-Favera, R. (2002). Acetylation inactivates the transcriptional repressor BCL6. Nat. Genet. 32, 606–613. Bernards, R., and Weinberg, R. A. (2002). A progression puzzle. Nature 418, 823. Bosch, F., Lopez-Guillermo, A., Campo, E., Ribera, J. M., Conde, E., Piris, M. A., Vallespi, T., Woessner, S., and Montserrat, E. (1998). Mantle cell lymphoma: Presenting features, response to therapy, and prognostic factors. Cancer 82, 567–575. Cabannes, E., Khan, G., Aillet, F., Jarrett, R. F., and Hay, R. T. (1999). Mutations in the IkBa gene in Hodgkin’s disease suggest a tumour suppressor role for IkappaBalpha. Oncogene 18, 3063–3070. Cattoretti, G., Chang, C.-C., Cechova, K., Zhang, J., Ye, B. H., Falini, B., Louie, D. C., Offit, K., Chaganti, R. S. K., and Dalla-Favera, R. (1995). BCL-6 protein is expressed in germinal-center B cells. Blood 86, 45–53. Chang, C. C., McClintock, S., Cleveland, R. P., Trzpuc, T., Vesole, D. H., Logan, B., KajdacsyBalla, A., and Perkins, S. L. (2004). Immunohistochemical expression patterns of germinal center and activation B-cell markers correlate with prognosis in diffuse large B-cell lymphoma. Am. J. Surg. Pathol. 28, 464–470.

200

L O UIS M. STA UDT A ND SA NDE EP DAVE

Chang, C. C., Ye, B. H., Chaganti, R. S., and Dalla-Favera, R. (1996). Proc. Natl. Acad. Sci. USA 93, 6947–6952. Christoph, T., Rickert, R., and Rajewsky, K. (1994). M17: A novel gene expressed in germinal centers. Int. Immunol. 6, 1203–1211. Chu, C. C., and Paul, W. E. (1997). Fig1, an interleukin 4-induced mouse B-cell gene isolated by cDNA representational difference analysis. Proc. Natl. Acad. Sci. USA 94, 2507–2512. Colombat, P., Salles, G., Brousse, N., Eftekhari, P., Soubeyran, P., Delwail, V., Deconinck, E., Haioun, C., Foussard, C., Sebban, C., Stamatoullas, A., Milpied, N., Boue, F., Taillan, B., Lederlin, P., Najman, A., Thieblemont, C., Montestruc, F., Mathieu-Boue, A., Benzohra, A., and Solal-Celigny, P. (2001). Rituximab (anti-CD20 monoclonal antibody) as single first-line therapy for patients with follicular lymphoma with a low tumor burden: Clinical and molecular evaluation. Blood 97, 101–106. Copie-Bergman, C., Plonquet, A., Alonso, M. A., Boulland, M. L., Marquet, J., Divine, M., Moller, P., Leroy, K., and Gaulard, P. (2002). MAL expression in lymphoid cells: Further evidence for MAL as a distinct molecular marker of primary mediastinal large B-cell lymphomas. Mod. Pathol. 15, 1172–1180. Craxton, A., Magaletti, D., Ryan, E. J., and Clark, E. A. (2003). Macrophage- and dendritic cell– dependent regulation of human B-cell proliferation requires the TNF family ligand BAFF. Blood 101, 4464–4471. Csernus, B., Timar, B., Z, F. U., Bognar, A., Szepesi, A., Laszlo, T., Jakso, P., Warnke, R., Kopper, L., and Matolcsy, A. (2004). Mutational analysis of IgVH and BCL-6 genes suggests thymic B-cells origin of mediastinal (thymic) B-cell lymphoma. Leuk. Lymphoma. 45, 2105–2110. Curiel, T. J., Wei, S., Dong, H., Alvarez, X., Cheng, P., Mottram, P., Krzysiek, R., Knutson, K. L., Daniel, B., Zimmermann, M. C., David, O., Burow, M., Gordon, A., Dhurandhar, N., Myers, L., Berggren, R., Hemminki, A., Alvarez, R. D., Emilie, D., Curiel, D. T., Chen, L., and Zou, W. (2003). Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity. Nat. Med. 9, 562–567. Czuczman, M. S., Grillo-Lopez, A. J., White, C. A., Saleh, M., Gordon, L., LoBuglio, A. F., Jonas, C., Klippenstein, D., Dallaire, B., and Varns, C. (1999). Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy. J. Clin. Oncol. 17, 268–276. Datta, A., Nag, A., Pan, W., Hay, N., Gartel, A. L., Colamonici, O., Mori, Y., and Raychaudhuri, P. (2004). Myc-ARF (alternate reading frame) interaction inhibits the functions of Myc. J. Biol. Chem. 279, 36698–36707. Dave, S. S., Wright, G., Tan, B., Rosenwald, A., Gascoyne, R. D., Chan, W. C., Fisher, R. I., Braziel, R. M., Rimsza, L. M., Grogan, T. M., Miller, T. P., LeBlanc, M., Greiner, T. C., Weisenburger, D. D., Lynch, J. C., Vose, J. M., Armitage, J. O., Smeland, E. B., Kvaloy, S., Holte, H., Delabie, J., Connors, J. M., Lansdorp, P., Ouyang, Q., Lister, T. A., Davies, A. J., Norton, A. J., Muller-Hermelink, H. K., Ott, G., Campo, E., Montserrat, E., Wilson, W. H., Jaffe, E. S., Simon, R., Yang, L., Powell, J., Zhao, H., Goldschmidt, N., Chiorazzi, M., and Staudt, L. M. (2004). Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N. Engl. J. Med. 351, 2159–2169. Davis, R. E., Brown, K. D., Siebenlist, U., and Staudt, L. M. (2001). Constitutive nuclear factor kappaB activity is required for survival of activated B-cell–like diffuse large B-cell lymphoma cells. J. Exp. Med. 194, 1861–1874. Davis, R. E., and Staudt, L. M. (2002). Molecular diagnosis of lymphoid malignancies by gene expression profiling. Curr. Opin. Hematol. 9, 333–338.

b io l og y of h um an ly mp ho mas

201

de Boer, C. J., van Krieken, J. H., Kluin-Nelemans, H. C., Kluin, P. M., and Schuuring, E. (1995). Cyclin D1 messenger RNA overexpression as a marker for mantle cell lymphoma. Oncogene 10, 1833–1840. de Leval, L., Braaten, K. M., Ancukiewicz, M., Kiggundu, E., Delaney, T., Mankin, H. J., and Harris, N. L. (2003). Diffuse large B-cell lymphoma of bone: An analysis of differentiationassociated antigens with clinical correlation. Am. J. Surg. Pathol. 27, 1269–1277. Dent, A. L., Shaffer, A. L., Yu, X., Allman, D., and Staudt, L. M. (1997). Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science 276, 589–592. Dhordain, P., Albagli, O., Lin, R. J., Ansieau, S., Quief, S., Leutz, A., Kerckaert, J. P., Evans, R. M., and Leprince, D. (1997). Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein. Proc. Natl. Acad. Sci. USA 94, 10762–10767. Dong, H., and Chen, L. (2003). B7-H1 pathway and its role in the evasion of tumor immunity. J. Mol. Med. 81, 281–287. Dong, H., Strome, S. E., Salomao, D. R., Tamura, H., Hirano, F., Flies, D. B., Roche, P. C., Lu, J., Zhu, G., Tamada, K., Lennon, V. A., Celis, E., and Chen, L. (2002). Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. Nat. Med. 8, 793–800. Dong, H., Zhu, G., Tamada, K., and Chen, L. (1999). B7-H1, a third member of the B7 family, costimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 5, 1365–1369. Dreyling, M. H., Bullinger, L., Ott, G., Stilgenbauer, S., Muller-Hermelink, H. K., Bentz, M., Hiddemann, W., and Dohner, H. (1997). Alterations of the cyclin D1/p16-pRB pathway in mantle cell lymphoma. Cancer Res. 57, 4608–4614. Egen, J. G., Kuhns, M. S., and Allison, J. P. (2002). CTLA-4: New insights into its biological function and use in tumor immunotherapy. Nat. Immunol. 3, 611–618. Emmerich, F., Meiser, M., Hummel, M., Demel, G., Foss, H. D., Jundt, F., Mathas, S., Krappmann, D., Scheidereit, C., Stein, H., and Dorken, B. (1999). Overexpression of I kappa B alpha without inhibition of NF-kappaB activity and mutations in the I kappa B alpha gene in Reed-Sternberg cells. Blood 94, 3129–3134. Falini, B., Fizzotti, M., Pucciarini, A., Bigerna, B., Marafioti, T., Gambacorta, M., Pacini, R., Alunni, C., Natali-Tanci, L., Ugolini, B., Sebastiani, C., Cattoretti, G., Pileri, S., Dalla-Favera, R., and Stein, H. (2000). A monoclonal antibody (MUM1p) detects expression of the MUM1/ IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells. Blood 95, 2084–2092. Flenghi, L., Ye, B. H., Fizzotti, M., Bigerna, B., Cattoretti, G., Venturi, S., Pacini, R., Pileri, S., Lo Coco, F., and Pescarmona, E., et al (1995). A specific monoclonal antibody (PG-B6) detects expression of the BCL-6 protein in germinal center B cells. Am. J. Pathol. 147, 405–411. Flores, K. G., Li, J., and Hale, L. P. (2001). B cells in epithelial and perivascular compartments of human adult thymus. Hum. Pathol. 32, 926–934. Freeman, G. J., Long, A. J., Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., Fitz, L. J., Malenkovich, N., Okazaki, T., Byrne, M. C., Horton, H. F., Fouser, L., Carter, L., Ling, V., Bowman, M. R., Carreno, B. M., Collins, M., Wood, C. R., and Honjo, T. (2000). Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192, 1027–1034. Fujita, N., Jaye, D. L., Kajita, M., Geigerman, C., Moreno, C. S., and Wade, P. A. (2003). MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 113, 207–219. Gonzalez, C. L., Medeiros, L. J., and Jaffe, E. S. (1991). Composite lymphoma. A clinicopathologic analysis of nine patients with Hodgkin’s disease and B-cell non-Hodgkin’s lymphoma. Am. J. Clin. Pathol. 96, 81–89.

202

L O UIS M. STA UDT A ND SA NDE EP DAVE

Guiter, C., Dusanter-Fourt, I., Copie-Bergman, C., Boulland, M. L., Le Gouvello, S., Gaulard, P., Leroy, K., and Castellano, F. (2004). Constitutive STAT6 activation in primary mediastinal large B-cell lymphoma. Blood 104, 543–549. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57–70. Hans, C. P., Weisenburger, D. D., Greiner, T. C., Gascoyne, R. D., Delabie, J., Ott, G., MullerHermelink, H. K., Campo, E., Braziel, R. M., Jaffe, E. S., Pan, Z., Farinha, P., Smith, L. M., Falini, B., Banham, A. H., Rosenwald, A., Staudt, L. M., Connors, J. M., Armitage, J. O., and Chan, W. C. (2004). Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 103, 275–282. Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett, D. R., Eng, J. K., Akira, S., Underhill, D. M., and Aderem, A. (2001). The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103. Horning, S. J. (2000). Follicular lymphoma: Have we made any progress? Ann. Oncol. 11, 23–27. Horning, S. J., and Rosenberg, S. A. (1984). The natural history of initially untreated low-grade non-Hodgkin’s lymphomas. N. Engl. J. Med. 311, 1471–1475. Huang, J. Z., Sanger, W. G., Greiner, T. C., Staudt, L. M., Weisenburger, D. D., Pickering, D. L., Lynch, J. C., Armitage, J. O., Warnke, R. A., Alizadeh, A. A., Lossos, I. S., Levy, R., and Chan, W. C. (2002). The t(14; 18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile. Blood 99, 2285–2290. Hurt, E. M., Wiestner, A., Rosenwald, A., Shaffer, A. L., Campo, E., Grogan, T., Bergsagel, P. L., Kuehl, W. M., and Staudt, L. M. (2004). Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma. Cancer Cell 5, 191–199. Huynh, K. D., Fischle, W., Verdin, E., and Bardwell, V. J. (2000). BCoR, a novel corepressor involved in BCL-6 repression. Genes. Dev. 14, 1810–1823. Jaffe, E. S., and Muller-Hermelink, K. (1999). In ‘‘Hodgkin’s Disease’’ (P. M. Mauch, J. O. Armitage, V. Diehl, R. T. Hoppe, and L. M. Weiss, Eds.), p. 181. Lippincott Williams & Wilkens, Philadelphia. Joos, S., Kupper, M., Ohl, S., von Bonin, F., Mechtersheimer, G., Bentz, M., Marynen, P., Moller, P., Pfreundschuh, M., Trumper, L., and Lichter, P. (2000). Genomic imbalances including amplification of the tyrosine kinase gene JAK2 in CD30þ Hodgkin cells. Cancer Res. 60, 549–552. Joos, S., Otano-Joos, M. I., Ziegler, S., Bruderlein, S., du Manoir, S., Bentz, M., Moller, P., and Lichter, P. (1996). Primary mediastinal (thymic) B-cell lymphoma is characterized by gains of chromosomal material including 9p and amplification of the REL gene. Blood 87, 1571–1578. Kamijo, T., Weber, J. D., Zambetti, G., Zindy, F., Roussel, M. F., and Sherr, C. J. (1998). Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc. Natl. Acad. Sci. USA 95, 8292–8297. Klein, U., Tu, Y., Stolovitzky, G. A., Keller, J. L., Haddad, J., Jr., Miljkovic, V., Cattoretti, G., Califano, A., and Dalla-Favera, R. (2003). Transcriptional analysis of the B-cell germinal center reaction. Proc. Natl. Acad. Sci. USA 100, 2639–2644. Kovanen, P. E., Rosenwald, A., Fu, J., Hurt, E. M., Lam, L. T., Giltnane, J. M., Wright, G., Staudt, L. M., and Leonard, W. J. (2003). Analysis of gamma c-family cytokine target genes. Identification of dual-specificity phosphatase 5 (DUSP5) as a regulator of mitogen-activated protein kinase activity in interleukin-2 signaling. J. Biol. Chem. 278, 5205–5213. Kozar, K., Ciemerych, M. A., Rebel, V. I., Shigematsu, H., Zagozdzon, A., Sicinska, E., Geng, Y., Yu, Q., Bhattacharya, S., Bronson, R. T., Akashi, K., and Sicinski, P. (2004). Mouse development and cell proliferation in the absence of D-cyclins. Cell 118, 477–491.

b io l og y of h um an ly mp ho mas

203

Krappmann, D., Emmerich, F., Kordes, U., Scharschmidt, E., Dorken, B., and Scheidereit, C. (1999). Molecular mechanisms of constitutive NF-kappaB/Rel activation in Hodgkin/Reed-Sternberg cells. Oncogene 18, 943–953. Kuppers, R., Rajewsky, K., and Hansmann, M. L. (1997). Diffuse large cell lymphomas are derived from mature B cells carrying V region genes with a high load of somatic mutation and evidence of selection for antibody expression. Eur. J. Immunol. 27, 1398–1405. Kwak, L. W., Campbell, M. J., Czerwinski, D. K., Hart, S., Miller, R. A., and Levy, R. (1992). Induction of immune responses in patients with B-cell lymphoma against the surfaceimmunoglobulin idiotype expressed by their tumors. N. Engl. J. Med. 327, 1209–1215. Lam, L. T., Davis, R. E., Wright, G., Rosenwald, A., Hurt, E. M., X., Y., Pierce, J., Hepperle, M., Xu, Y., Adams, J., Dang, L., and Staudt, L. M. (2005). Clin. Cancer Res. 11, 28–40. Latchman, Y., Wood, C. R., Chernova, T., Chaudhary, D., Borde, M., Chernova, I., Iwai, Y., Long, A. J., Brown, J. A., Nunes, R., Greenfield, E. A., Bourque, K., Boussiotis, V. A., Carter, L. L., Carreno, B. M., Malenkovich, N., Nishimura, H., Okazaki, T., Honjo, T., Sharpe, A. H., and Freeman, G. J. (2001). PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat. Immunol. 2, 261–268. Lebwohl, D. E., Muise-Helmericks, R., Sepp-Lorenzino, L., Serve, S., Timaul, M., Bol, R., Borgen, P., and Rosen, N. (1994). A truncated cyclin D1 gene encodes a stable mRNA in a human breast cancer cell line. Oncogene 9, 1925–1929. Leithauser, F., Bauerle, M., Huynh, M. Q., and Moller, P. (2001). Isotype-switched immunoglobulin genes with a high load of somatic hypermutation and lack of ongoing mutational activity are prevalent in mediastinal B-cell lymphoma. Blood 98, 2762–2770. Levens, D. (2002). Disentangling the MYC web. Proc. Natl. Acad. Sci. USA 99, 5757–5759. Li, D. N., Matthews, S. P., Antoniou, A. N., Mazzeo, D., and Watts, C. (2003). Multistep autoactivation of asparaginyl endopeptidase in vitro and in vivo. J. Biol. Chem. 278, 38980–38990. Lin, K. I., Angelin-Duclos, C., Kuo, T. C., and Calame, K. (2002). Blimp-1-dependent repression of Pax-5 is required for differentiation of B cells to immunoglobulin M-secreting plasma cells. Mol. Cell. Biol. 22, 4771–4780. Lin, S., Wang, W., Wilson, G. M., Yang, X., Brewer, G., Holbrook, N. J., and Gorospe, M. (2000). Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover. Mol. Cell. Biol. 20, 7903–7913. Lin, Y., Wong, K., and Calame, K. (1997). Repression of c-myc transcription by Blimp-1, an inducer of terminal B-cell differentiation. Science 276, 596–599. List, A. F., Spier, C. M., Miller, T. P., and Grogan, T. M. (1993). Deficient tumor-infiltrating T-lymphocyte response in malignant lymphoma: Relationship to HLA expression and host immunocompetence. Leukemia 7, 398–403. Liu, X., Gao, J. X., Wen, J., Yin, L., Li, O., Zuo, T., Gajewski, T. F., Fu, Y. X., Zheng, P., and Liu, Y. (2003). B7DC/PDL2 promotes tumor immunity by a PD-1-independent mechanism. J. Exp. Med. 197, 1721–1730. Lossos, I. S., Alizadeh, A. A., Eisen, M. B., Chan, W. C., Brown, P. O., Botstein, D., Staudt, L. M., and Levy, R. (2000). Ongoing immunoglobulin somatic mutation in germinal center B-cell–like but not in activated B-cell–like diffuse large cell lymphomas. Proc. Natl. Acad. Sci. USA 97, 10209–10213. Lossos, I. S., Alizadeh, A. A., Rajapaksa, R., Tibshirani, R., and Levy, R. (2003). HGAL is a novel interleukin-4-inducible gene that strongly predicts survival in diffuse large B-cell lymphoma. Blood 101, 433–440. Lossos, I. S., Czerwinski, D. K., Alizadeh, A. A., Wechser, M. A., Tibshirani, R., Botstein, D., and Levy, R. (2004). Prediction of survival in diffuse large-B-cell lymphoma based on the expression of six genes. N. Engl. J. Med. 350, 1828–1837.

204

L O UIS M. STA UDT A ND SA NDE EP DAVE

Lossos, I. S., and Levy, R. (2003). Higher grade transformation of follicular lymphoma: Phenotypic tumor progression associated with diverse genetic lesions. Semin. Cancer Biol. 13, 191–202. Maloney, D. G., Grillo-Lopez, A. J., White, C. A., Bodkin, D., Schilder, R. J., Neidhart, J. A., Janakiraman, N., Foon, K. A., Liles, T. M., Dallaire, B. K., Wey, K., Royston, I., Davis, T., and Levy, R. (1997). IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood 90, 2188–2195. Martinez-Valdez, H., Guret, C., de Bouteiller, O., Fugier, I., Banchereau, J., and Liu, Y. J. (1996). Human germinal center B cells express the apoptosis-inducing genes Fas, c-myc, P53, and Bax but not the survival gene bcl-2. J. Exp. Med. 183, 971–977. McPhaden, A. R., and Whaley, K. (1993). Complement biosynthesis by mononuclear phagocytes. Immunol. Res. 12, 213–232. Melnick, A., Carlile, G., Ahmad, K. F., Kiang, C. L., Corcoran, C., Bardwell, V., Prive, G. G., and Licht, J. D. (2002). Critical residues within the BTB domain of PLZF and Bcl-6 modulate interaction with corepressors. Mol. Cell. Biol. 22, 1804–1818. Monod, J., and Jacob, F. (1961). Teleonomic mechanisms in cellular metabolism, growth, and differentiation. Cold Spring Harb. Symp. Quant. Biol. 26, 389–401. Muscat, G. E., Burke, L. J., and Downes, M. (1998). The corepressor N-CoR and its variants RIP13a and RIP13Delta1 directly interact with the basal transcription factors TFIIB, TAFII32 and TAFII70. Nucleic Acids Res. 26, 2899–2907. Muzio, M., Bosisio, D., Polentarutti, N., D’Amico, G., Stoppacciaro, A., Mancinelli, R., van’t Veer, C., Penton-Rol, G., Ruco, L. P., Allavena, P., and Mantovani, A. (2000). Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: Selective expression of TLR3 in dendritic cells. J. Immunol. 164, 5998–6004. Niu, H., Ye, B. H., and Dalla-Favera, R. (1998). Antigen receptor signaling induces MAP kinasemediated phosphorylation and degradation of the BCL-6 transcription factor. Genes Dev. 12, 1953–1961. O’Connell, B. C., Cheung, A. F., Simkevich, C. P., Tam, W., Ren, X., Mateyak, M. K., and Sedivy, J. M. (2003). A large scale genetic analysis of c-Myc-regulated gene expression patterns. J. Biol. Chem. 278, 12563–12573. Onizuka, T., Moriyama, M., Yamochi, T., Kuroda, T., Kazama, A., Kanazawa, N., Sato, K., Kato, T., Ota, H., and Mori, S. (1995). BCL-6 gene product, a 92- to 98-kD nuclear phosphoprotein, is highly expressed in germinal center B cells and their neoplastic counterparts. Blood 86, 28–37. Pan, Z., Shen, Y., Du, C., Zhou, G., Rosenwald, A., Staudt, L. M., Greiner, T. C., McKeithan, T. W., and Chan, W. C. (2003). Two newly characterized germinal center B-cell-associated genes, GCET1 and GCET2, have differential expression in normal and neoplastic B cells. Am. J. Pathol. 163, 135–144. Pasqualucci, L., Bereschenko, O., Niu, H., Klein, U., Basso, K., Guglielmino, R., Cattoretti, G., and Dalla-Favera, R. (2003). Molecular pathogenesis of non-Hodgkin’s lymphoma: The role of Bcl-6. Leuk. Lymphoma. 44, S5–S12. Peh, S. C., Kim, L. H., and Poppema, S. (2001). TARC, a CC chemokine, is frequently expressed in classic Hodgkin’s lymphoma but not in NLP Hodgkin’s lymphoma, T-cell-rich B-cell lymphoma, and most cases of anaplastic large cell lymphoma. Am. J. Surg. Pathol. 25, 925–929. Pileri, S. A., Gaidano, G., Zinzani, P. L., Falini, B., Gaulard, P., Zucca, E., Pieri, F., Berra, E., Sabattini, E., Ascani, S., Piccioli, M., Johnson, P. W., Giardini, R., Pescarmona, E., Novero, D., Piccaluga, P. P., Marafioti, T., Alonso, M. A., and Cavalli, F. (2003). Primary mediastinal B-cell lymphoma: High frequency of BCL-6 mutations and consistent expression of the transcription factors OCT-2, BOB.1, and PU.1 in the absence of immunoglobulins. Am. J. Pathol. 162, 243–253.

b io l og y of h um an ly mp ho mas

205

Pinyol, M., Cobo, F., Bea, S., Jares, P., Nayach, I., Fernandez, P. L., Montserrat, E., Cardesa, A., and Campo, E. (1998). p16(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin’s lymphomas. Blood 91, 2977–2988. Pinyol, M., Hernandez, L., Cazorla, M., Balbin, M., Jares, P., Fernandez, P. L., Montserrat, E., Cardesa, A., Lopez-Otin, C., and Campo, E. (1997). Deletions and loss of expression of p16INK4a and p21Waf1 genes are associated with aggressive variants of mantle cell lymphomas. Blood 89, 272–280. Pinyol, M., Hernandez, L., Martinez, A., Cobo, F., Hernandez, S., Bea, S., Lopez-Guillermo, A., Nayach, I., Palacin, A., Nadal, A., Fernandez, P. L., Montserrat, E., Cardesa, A., and Campo, E. (2000). INK4a/ARF locus alterations in human non-Hodgkin’s lymphomas mainly occur in tumors with wild-type p53 gene. Am. J. Pathol. 156, 1987–1996. Piskurich, J. F., Lin, K. I., Lin, Y., Wang, Y., Ting, J. P. Y., and Calame, K. (2000). BLIMP-I mediates extinction of major histocompatibility class II transactivator expression in plasma cells. Nature Immunology 1, 526–532. Ponzio, N. M., and Thorbecke, G. J. (2000). Requirement for reverse immune surveillance for the growth of germinal center-derived murine lymphomas. Semin. Cancer Biol. 10, 331–340. Qi, Y., Gregory, M. A., Li, Z., Brousal, J. P., West, K., and Hann, S. R. (2004). p19ARF directly and differentially controls the functions of c-Myc independently of p53. Nature 431, 712–717. Quintanilla-Martinez, L., Davies-Hill, T., Fend, F., Calzada-Wack, J., Sorbara, L., Campo, E., Jaffe, E. S., and Raffeld, M. (2003). Sequestration of p27Kip1 protein by cyclin D1 in typical and blastic variants of mantle cell lymphoma (MCL): Implications for pathogenesis. Blood 101, 3181–3187. Randle, D. H., Zindy, F., Sherr, C. J., and Roussel, M. F. (2001). Differential effects of p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages. Proc. Natl. Acad. Sci. USA 98, 9654–9659. Ransohoff, D. F. (2004). Rules of evidence for cancer molecular-marker discovery and validation. Nat. Rev. Cancer 4, 309–314. Raty, R., Franssila, K., Joensuu, H., Teerenhovi, L., and Elonen, E. (2002). Ki-67 expression level, histological subtype, and the International Prognostic Index as outcome predictors in mantle cell lymphoma. Eur. J. Haematol. 69, 11–20. Reimold, A. M., Iwakoshi, N. N., Manis, J., Vallabhajosyula, P., Szomolanyi-Tsuda, E., Gravallese, E. M., Friend, D., Grusby, M. J., Alt, F., and Glimcher, L. H. (2001). Plasma cell differentiation requires the transcription factor XBP-. Nature 412, 300–307. Reimold, A. M., Ponath, P. D., Li, Y. S., Hardy, R. R., David, C. S., Strominger, J. L., and Glimcher, L. H. (1996). Transcription factor B-cell lineage-specific activator protein regulates the gene for human X-box binding protein 1. J. Exp. Med. 183, 393–401. Rimokh, R.Berger, F.Bastard, C.Klein, B.French, M.Archimbaud, E.Rouault, J. P.Santa Lucia, B. Duret, L.Vuillaume, M., et al (1994). Rearrangement of CCND1 (BCL1/PRAD1) 30 untranslated region in mantle-cell lymphomas and t(11q13)-associated leukemias. Blood 83, 3689–3696. Rimsza, L. M., Roberts, R. A., Miller, T. P., Unger, J. M., LeBlanc, M., Braziel, R. M., Weisenberger, D. D., Chan, W. C., Muller-Hermelink, H. K., Jaffe, E. S., Gascoyne, R. D., Campo, E., Fuchs, D. A., Spier, C. M., Fisher, R. I., Delabie, J., Rosenwald, A., Staudt, L. M., and Grogan, T. M. (2004). Loss of MHC class II gene and protein expression in diffuse large Bcell lymphoma is related to decreased tumor immunosurveillance and poor patient survival regardless of other prognostic factors: A follow-up study from the Leukemia and Lymphoma Molecular Profiling Project. Blood 103, 4251–4258.

206

L O UIS M. STA UDT A ND SA NDE EP DAVE

Roglic, A., Prossnitz, E. R., Cavanagh, S. L., Pan, Z., Zou, A., and Ye, R. D. (1996). cDNA cloning of a novel G protein-coupled receptor with a large extracellular loop structure. Biochim. Biophys. Acta 1305, 39–43. Rosenwald, A., Wright, G., Chan, W. C., Connors, J. M., Campo, E., Fisher, R. I., Gascoyne, R. D., Muller-Hermelink, H. K., Smeland, E. B., Giltnane, J. M., Hurt, E. M., Zhao, H., Averett, L., Yang, L., Wilson, W. H., Jaffe, E. S., Simon, R., Klausner, R. D., Powell, J., Duffey, P. L., Longo, D. L., Greiner, T. C., Weisenburger, D. D., Sanger, W. G., Dave, B. J., Lynch, J. C., Vose, J., Armitage, J. O., Montserrat, E., Lopez-Guillermo, A., Grogan, T. M., Miller, T. P., LeBlanc, M., Ott, G., Kvaloy, S., Delabie, J., Holte, H., Krajci, P., Stokke, T., and Staudt, L. M. (2002). The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N. Engl. J. Med. 346, 1937–1947. Rosenwald, A., Wright, G., Leroy, K., Yu, X., Gaulard, P., Gascoyne, R. D., Chan, W. C., Zhao, T., Haioun, C., Greiner, T. C., Weisenburger, D. D., Lynch, J. C., Vose, J., Armitage, J. O., Smeland, E. B., Kvaloy, S., Holte, H., Delabie, J., Campo, E., Montserrat, E., Lopez-Guillermo, A., Ott, G., Muller-Hermelink, H. K., Connors, J. M., Braziel, R., Grogan, T. M., Fisher, R. I., Miller, T. P., LeBlanc, M., Chiorazzi, M., Zhao, H., Yang, L., Powell, J., Wilson, W. H., Jaffe, E. S., Simon, R., Klausner, R. D., and Staudt, L. M. (2003a). Molecular diagnosis of primary mediastinal B-cell lymphoma identifies a clinically favorable subgroup of diffuse large B-cell lymphoma related to Hodgkin lymphoma. J. Exp. Med. 198, 851–862. Rosenwald, A., Wright, G., Wiestner, A., Chan, W. C., Connors, J. M., Campo, E., Gascoyne, R. D., Grogan, T. M., Muller-Hermelink, H. K., Smeland, E. B., Chiorazzi, M., Giltnane, J. M., Hurt, E. M., Zhao, H., Averett, L., Henrickson, S., Yang, L., Powell, J., Wilson, W. H., Jaffe, E. S., Simon, R., Klausner, R. D., Montserrat, E., Bosch, F., Greiner, T. C., Weisenburger, D. D., Sanger, W. G., Dave, B. J., Lynch, J. C., Vose, J., Armitage, J. O., Fisher, R. I., Miller, T. P., LeBlanc, M., Ott, G., Kvaloy, S., Holte, H., Delabie, J., and Staudt, L. M. (2003b). The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer Cell 3, 185–197. Savage, K. J., Monti, S., Kutok, J. L., Cattoretti, G., Neuberg, D., De Leval, L., Kurtin, P., Dal Cin, P., Ladd, C., Feuerhake, F., Aguiar, R. C., Li, S., Salles, G., Berger, F., Jing, W., Pinkus, G. S., Habermann, T., Dalla-Favera, R., Harris, N. L., Aster, J. C., Golub, T. R., and Shipp, M. A. (2003). The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin lymphoma. Blood 102, 3871–3879. Schauer, S. L., Wang, Z., Sonenshein, G. E., and Rothstein, T. L. (1996). Maintenance of nuclear factor-kappa B/Rel and c-myc expression during CD40 ligand rescue of WEHI 231 early B cells from receptor-mediated apoptosis through modulation of I kappa B proteins. J. Immunol. 157, 81–86. Schlosser, I., Holzel, M., Murnseer, M., Burtscher, H., Weidle, U. H., and Eick, D. (2003). A role for c-Myc in the regulation of ribosomal RNA processing. Nucleic Acids Res. 31, 6148–6156. Schwab, U., Stein, H., Gerdes, J., Lemke, H., Kirchner, H., Schaadt, M., and Diehl, V. (1982). Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin’s disease and a subset of normal lymphoid cells. Nature 299, 65–67. Seto, M.Yamamoto, K.Iida, S.Akao, Y.Utsumi, K. R.Kubonishi, I.Miyoshi, I.Ohtsuki, T.Yawata, Y. Namba, M., et al (1992). Gene rearrangement and overexpression of PRAD1 in lymphoid malignancy with t(11; 14)(q13; q32) translocation. Oncogene 7, 1401–1406. Seyfert, V. L., Allman, D., He, Y., and Staudt, L. M. (1996). Transcriptional repression by the protooncogene BCL-6. Oncogene 12, 2331–2342.

b io l og y of h um an ly mp ho mas

207

Shaffer, A. L., Lin, K. I., Kuo, T. C., Yu, X., Hurt, E. M., Rosenwald, A., Giltnane, J. M., Yang, L., Zhao, H., Calame, K., and Staudt, L. M. (2002a). Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B-cell gene expression program. Immunity 17, 51–62. Shaffer, A. L., Rosenwald, A., Hurt, E. M., Giltnane, J. M., Lam, L. T., Pickeral, O. K., and Staudt, L. M. (2001). Signatures of the immune response. Immunity 15, 375–385. Shaffer, A. L., Rosenwald, A., and Staudt, L. M. (2002b). Lymphoid malignancies: The dark side of B-cell differentiation. Nat. Rev. Immunol. 2, 920–932. Shaffer, A. L., Shapiro-Shelef, M., Iwakoshi, N. N., Lee, A.-H., Qian, S.-B., Zhao, H., Yu, X., Yang, L., Tan, B. K., Rosenwald, A., Hurt, E. M., Petroulakis, E., Sonenberg, N., Yewdell, J. W., Calame, K., Glimcher, L. H., and Staudt, L. M. (2004). XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation. Immunity 21, 81–93. Shaffer, A. L., Yu, X., He, Y., Boldrick, J., Chan, E. P., and Staudt, L. M. (2000). BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity 13, 199–212. Shapiro-Shelef, M., Lin, K. I., McHeyzer-Williams, L. J., Liao, J., McHeyzer-Williams, M. G., and Calame, K. (2003). Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 19, 607–620. Sherr, C. J., and McCormick, F. (2002). The RB and p53 pathways in cancer. Cancer Cell 2, 103–112. Shipp, M. A., Ross, K. N., Tamayo, P., Weng, A. P., Kutok, J. L., Aguiar, R. C. T., Gaasenbeek, M., Angelo, M., Reich, M., Pinkus, G. S., Ray, T. S., Koval, M. A., Last, K. W., Norton, A., Lister, T. A., Mesirov, J., Neuberg, D. S., Lander, E. S., Aster, J. C., and Golub, T. R. (2002). Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nature Med. 8, 68–74. Sicinski, P., Donaher, J. L., Parker, S. B., Li, T., Fazeli, A., Gardner, H., Haslam, S. Z., Bronson, R. T., Elledge, S. J., and Weinberg, R. A. (1995). Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82, 621–630. Skinnider, B. F., Elia, A. J., Gascoyne, R. D., Trumper, L. H., von Bonin, F., Kapp, U., Patterson, B., Snow, B. E., and Mak, T. W. (2001). Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood 97, 250–255. Sorlie, T., Tibshirani, R., Parker, J., Hastie, T., Marron, J. S., Nobel, A., Deng, S., Johnsen, H., Pesich, R., Geisler, S., Demeter, J., Perou, C. M., Lonning, P. E., Brown, P. O., Borresen-Dale, A. L., and Botstein, D. (2003). Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc. Natl. Acad. Sci. USA 100, 8418–8423. Staudt, L. M. (2003). Molecular diagnosis of the hematologic cancers. N. Engl. J. Med. 348, 1777–1785. Sui, L., Zhang, W., Liu, Q., Chen, T., Li, N., Wan, T., Yu, M., and Cao, X. (2003). Cloning and functional characterization of human septin 10, a novel member of septin family cloned from dendritic cells. Biochem. Biophys. Res. Commun. 304, 393–398. Timmerman, J. M., Czerwinski, D. K., Davis, T. A., Hsu, F. J., Benike, C., Hao, Z. M., Taidi, B., Rajapaksa, R., Caspar, C. B., Okada, C. Y., van Beckhoven, A., Liles, T. M., Engleman, E. G., and Levy, R. (2002). Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: Clinical and immune responses in 35 patients. Blood 99, 1517–1526. Tonnelle, C., D’Ercole, C., Depraetere, V., Metras, D., Boubli, L., and Fougereau, M. (1997). Human thymic B cells largely overexpress the VH4 Ig gene family. A possible role in the control of tolerance in situ? Int. Immunol. 9, 407–414. Tsai, R. Y., and McKay, R. D. (2002). A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev. 16, 2991–3003.

208

L O UIS M. STA UDT A ND SA NDE EP DAVE

Tsukamoto, H., Nagasawa, K., Ueda, Y., Mayumi, T., Furugo, I., Tsuru, T., and Niho, Y. (1992). Effects of cell differentiation on the synthesis of the third and fourth component of complement (C3, C4) by the human monocytic cell line U937. Immunology 77, 621–623. Tunyaplin, C., Shaffer, A. L., Angelin-Duclos, C. D., Yu, X., Staudt, L. M., and Calame, K. L. (2004). Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation. J. Immunol. 173, 1158–1165. Tuscano, J. M., Druey, K. M., Riva, A., Pena, J., Thompson, C. B., and Kehrl, J. H. (1996). Bcl-x rather than Bcl-2 mediates CD40-dependent centrocyte survival in the germinal center. Blood 88, 1359–1364. Tzankov, A., Pehrs, A. C., Zimpfer, A., Ascani, S., Lugli, A., Pileri, S., and Dirnhofer, S. (2003). Prognostic significance of CD44 expression in diffuse large B-cell lymphoma of activated and germinal centre B-cell–like types: A tissue microarray analysis of 90 cases. J. Clin. Pathol. 56, 747–752. Velders, G. A., Kluin-Nelemans, J. C., De Boer, C. J., Hermans, J., Noordijk, E. M., Schuuring, E., Kramer, M. H., Van Deijk, W. A., Rahder, J. B., Kluin, P. M., and Van Krieken, J. H. (1996). Mantle-cell lymphoma: A population-based clinical study. J. Clin. Oncol. 14, 1269–1274. Withers, D. A., Harvey, R. C., Faust, J. B., Melnyk, O., Carey, K., and Meeker, T. C. (1991). Characterization of a candidate bcl-1 gene. Mol. Cell. Biol. 11, 4846–4853. Witzig, T. E., Gordon, L. I., Cabanillas, F., Czuczman, M. S., Emmanouilides, C., Joyce, R., Pohlman, B. L., Bartlett, N. L., Wiseman, G. A., Padre, N., Grillo-Lopez, A. J., Multani, P., and White, C. A. (2002). Randomized controlled trial of yttrium-90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin’s lymphoma. J. Clin. Oncol. 20, 2453–2463. Wong, C. W., and Privalsky, M. L. (1998). Transcriptional repression by the SMRT-mSin3 corepressor: Multiple interactions, multiple mechanisms, and a potential role for TFIIB. Mol. Cell. Biol. 18, 5500–5510. Wood, K. M., Roff, M., and Hay, R. T. (1998). Defective IkappaBalpha in Hodgkin cell lines with constitutively active NF-kappaB. Oncogene 16, 2131–2139. Wright, G., Tan, B., Rosenwald, A., Hurt, E. H., Wiestner, A., and Staudt, L. M. (2003). A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B-cell lymphoma. Proc. Natl. Acad. Sci. USA 100, 9991–9996. Ye, B. H., Cattoretti, G., Shen, Q., Zhang, J., Hawe, N., de Waard, R., Leung, C., Nouri-Shirazi, M., Orazi, A., Chaganti, R. S., Rothman, P., Stall, A. M., Pandolfi, P. P., and Dalla-Favera, R. (1997). The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation. Nat. Genet. 16, 161–170. Zarate-Osorno, A., Medeiros, L. J., Longo, D. L., and Jaffe, E. S. (1992). Non-Hodgkin’s lymphomas arising in patients successfully treated for Hodgkin’s disease. A clinical, histologic, and immunophenotypic study of 14 cases. Am. J. Surg. Pathol. 16, 885–895. Zeller, K. I., Jegga, A. G., Aronow, B. J., O’Donnell, K. A., and Dang, C. V. (2003). An integrated database of genes responsive to the Myc oncogenic transcription factor: Identification of direct genomic targets. Genome. Biol. 4, R69. Zettl, A., Bea, S., Wright, G., Salaverria, I., Jehn, P., Ott, G., Chan, W.-C., Jaffe, E. S., Weisenburger, D. D., Greiner, T. C., Armitage, J. O., Gascoyne, R. D., Connors, J. M., Grogan, T. M., Miller, T. P., Fisher, R. I., Delabie, J., Kvaloy, s., Mueller-Hermelink, H. K., Moreno, V., Montserrat, E., Wilson, W. H., Staudt, L. M., Rosenwald, A., and Campo, E. (2004). submitted. Zhu, D., McCarthy, H., Ottensmeier, C. H., Johnson, P., Hamblin, T. J., and Stevenson, F. K. (2002). Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma. Blood 99, 2562–2568.

New Insights into Alternative Mechanisms of Immune Receptor Diversification Gary W. Litman,*,{,{ John P. Cannon,* and Jonathan P. Rast§ *Department of Pediatrics, University of South Florida College of Medicine, USF/ACH Children’s Research Institute, St. Petersburg, Florida 33701 { All Children’s Hospital, Department of Molecular Genetics, St. Petersburg, Florida 33701 { H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612 § Sunnybrook and Women’s College, Health Sciences Centre, Toronto, Ontario, Canada M4N 3M5

1. 2. 3. 4.

Abstract............................................................................................................. Introduction ....................................................................................................... Variation in Innate Receptors ................................................................................ Alternative Mechanisms that Diversify Immune Receptors.......................................... Conclusions........................................................................................................ References .........................................................................................................

209 210 211 216 228 231

Abstract The clonal commitment, selection, and expansion of B and T lymphocytes expressing diversified receptors provide the underlying basis for the jawed vertebrates adaptive immune response. At the core of this process is the rearrangement and somatic modification of segmental genetic elements that encode the constituent components of immunoglobulins and T-cell antigen receptors. No evidence has been found for a similar mechanism outside of jawed vertebrates; however, invertebrates and jawless vertebrates are subjected to continuous exposure to pathogenic bacteria, viruses, and parasites. The invertebrates and jawless vertebrates as well as jawed vertebrates all encode a variety of mediators of innate immunity. Several reports of extensive germline diversification of conventional innate receptors, as well as molecules that resemble innate receptors but undergo germline and somatic modification, have been made recently. The range of such molecules, which include the fibrinogen-related proteins (FREPs) in a mollusc, variable region-containing chitin-binding proteins (VCBPs) in a cephalochordate, variable lymphocyte receptors (VLRs) in jawless vertebrates, and novel immune-type receptors (NITRs) in bony fish, encompasses both the immunoglobulin gene superfamily (IgSF) and leucine-rich repeat (LRR) proteins. Although these molecules vary markedly in form and likely in function, growing evidence suggests that they participate in various types of host defense and thereby represent significant alternatives to current paradigms of innate and adaptive immune receptors. Unusual genetic mechanisms for diversifying recognition proteins may be a widespread characteristic of animal immunity.

209 advances in immunology, vol. 87 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)87006-3

210

g a ry w. li tm an

E T AL .

1. Introduction The current paradigm of antibody and T-cell antigen receptor (TCR) gene rearrangement provides a rational explanation for somatic commitment of individual lymphocytes to single immune specificities. At the core of this process is an elaborate, multicomponent system of gene reorganization and reassembly that occurs in individual T and B lymphocyte precursors and introduces regionalized structural variation in the antigen-combining sites of B-cell receptors (BCRs) and TCRs expressed on the surfaces of mature lymphocytes. During the course of somatic reorganization, an extraordinary degree of structural diversity is created through both combinatorial (Tonegawa, 1983) and junctional diversification mechanisms (Alt and Baltimore, 1982). Many of the molecules that mediate this process are common to DNA damage-responsive repair processes, whereas other molecules such as the RAG proteins and TdT effect functions that are unique to differentiating lymphocytes (Jung and Alt, 2004). Further refinement of antigen-binding specificity and secondary biological function in BCRs is achieved through somatic hypermutation and isotype/class switching. An RNA processing event during the terminal differentiation of the B lymphocyte to a plasma cell converts the membrane-bound immunoglobulin (Ig) that serves as an antigen binding receptor to a soluble, circulating antibody; daughter cells expressing the parent receptor serve memory functions, and the somatically rearranged Ig genes remain as templates for additional somatic change. The origins of the mechanisms that diversify the BCRs and TCRs can be traced at least to the last common ancestor of the modern jawed vertebrates. Despite the conservation of the overall somatic rearrangement process, specific mechanisms used to generate BCR diversity vary throughout the vertebrates (e.g., gene conversion is used to diversify Ig genes in avians [Reynaud et al., 1994] and a number of mammals, but not in humans and mice) (Knight and Crane, 1994; Reynaud et al., 1994). In cartilaginous fish (chondrichthyans), which possess large numbers of independent Ig gene clusters, what otherwise appear to be typical chondrichthyan Ig loci can be fully or partially joined in the germline (i.e., the receptor specificity is committed or partially committed in an innate fashion) (Kokubu et al., 1988; Litman et al., 1999). Still other species of fish encode Ig loci that are in both mammalianlike and Ig cluster organizations. The lack of consistency in the mechanisms that diversify BCRs throughout vertebrate phylogeny is remarkable, whereas generation of TCR variability seems far more consistent between phylogenetically divergent species insofar as it has been characterized (Litman et al., 1999).

novel immune mechanisms

211

2. Variation in Innate Receptors 2.1. Traditional Considerations Regarding Relative Complexity in Adaptive Versus Innate Receptors Adaptive immunity, as considered conventionally, is an ancient process that involves the generation of a defined receptor specificity on the surface of a single lymphocyte through a stochastic mechanism that recombines individual segmental elements. The most significant variability in BCR structure is introduced through nontemplated somatic change at junctional boundaries. Clonal expansion of B cells is associated with the accumulation and selection of further somatic change in Ig genes. The current operational paradigm of innate immune receptor function centers on a relatively limited number of recognition proteins that are expressed by a large number of cells and recognize determinants or certain metabolic products that are indicative of an entire ‘‘class’’ of potential pathogen (Medzhitov, 2001; Vasta et al., 2004). Genes encoding traditional innate receptors do not undergo genetic rearrangement, somatic modification, or clonal selection and do not partner functionally with MHC I and II, as is the case for vertebrate TCRs. Rather, innate immunity represents the concerted activities of many recognition, signaling, and effector systems (Beutler, 2004b). The evolutionary origins of some innate immune mechanisms are far more ancient than those of the rearranging adaptive receptors (Hoffmann and Reichhart, 2002; Nicholas and Hodgkin, 2004), whereas other innate immune functions, including those mediated by IgSF members (e.g., killer cell Ig-like receptors [KIRs]), represent relatively recent adaptations (Khakoo et al., 2000; Trowsdale, 2001). Innate immune molecules in certain organisms, such as Drosophila, effect recognition of a number of different targets, including gram-positive bacteria, gram-negative bacteria, fungi, and Drosophila C virus. The antiviral response is not associated with the antimicrobial peptides seen in the systemic response to bacteria and fungi (J.-L. Imler and J. Hoffmann, personal communication). Taken as a whole, our current concepts of receptor-based immune protection against pathogens have innate immunity with limited germline diversity at one extreme and adaptive immunity with extensive variation in receptor diversity expressed by individual somatic cells at the other extreme. When approaching the broad topic of the evolution of immunity, one immediate question relates to the limitations of innate immune responses; specifically, how do species with exclusively innate immune systems combat highly variable and quickly evolving pathogens? The answer to this overriding question is only now becoming clear and in part lies in the complexity and longevity of the animal as well as the evolutionary stability of hostpathogen interactions and other aspects of the animal’s natural history that may

212

g a ry w. li tm an

E T AL .

be difficult to generalize across phyla. Somewhat contrary to the view of static innate receptors, a number of studies have identified polymorphisms in several innate immune genes. Some of these germline-inherited differences have been shown to afford functional advantage, suggesting that innate mediators likely are under strong selective pressure to diversify. Alternative immune and immune-type recognition systems have now been characterized in a sufficient number of species to permit an overview that would not have been possible until recently. A phylogenetic tree is illustrated (Fig. 1) in order to facilitate an understanding of the phylogenetic radiations of the protostomes and deuterostomes and the relationships of the principal model organisms discussed in this review to one another. 2.2. Diversification of Innate Immune Receptors During Evolution Studies of innate immunity in individual, representative invertebrate and vertebrate species have demonstrated a tremendous level of complexity in the array of molecules that mediate recognition of pathogens, as well as in those that effect their elimination. In contrast to the complexity introduced into adaptive immune receptors through gene rearrangement and other somatic processes within the lifetime of an organism, evolutionary pressures shape innate immunity over a succession of generations. One of the central themes of the evolution of innate receptors is the selection for recognition of molecular signatures, often referred to as pathogen-associated molecular patterns (PAMPs), which generally are essential to the survival or virulence of the pathogen and thus have been conserved through evolution. A particularly dramatic example of evolutionary diversification of a relatively small family of immune receptors is provided by the Toll-like receptors (TLRs), which take their name from the prototypic developmental/immune gene Toll in Drosophila melanogaster and subsequently have been identified in several other animal radiations, including that of the mammals (Beutler, 2004b; Medzhitov, 2001). TLRs are involved in immune responses in both Drosophila and mammals and signal through a relatively conserved cytosolic Toll/interleukin-1 receptor (TIR) homologous domain, which is involved in a cascade with downstream molecules, such as MyD88 and NF-kB orthologs, which have been conserved across the two phylogenetic groups (Beutler, 2004a). Despite the conservation of intracellular Toll-signaling mechanisms between insects and mammals, the specific roles of the TLR leucinerich receptor (LRR) ectodomains are somewhat different. Specifically, in Drosophila, Toll mediates immune responses after ligation of an endogenous factor (cleaved Spaetzle), whereas in mammals, the TLRs directly recognize a wide array of PAMP molecules expressed by potential pathogens (Hoffmann and Reichhart, 2002; Medzhitov, 2001).

novel immune mechanisms

213

Figure 1 A simplified phylogeny showing the interrelationships of various species and higher taxonomic groups discussed in this review, as well as C. elegans, in which evidence has been presented to suggest an innate immune response, (reviewed in Nicholas and Hodgkin, 2004; Young and Dillin, 2004). Bilaterian animals can be divided into two major groups: the protostomes and deuterostomes. The deuterostomes include the Chordates (the phylum that contains the vertebrates) and Echinoderms (along with the hemichordates and Xenoturbella, not shown). The protostomes can be divided into the Ecdysozoa, including the arthropods and nematodes, and the Lophotrochozoa, including mollusks and annelids. The bilaterians comprise
30 phyla; however, most of our knowledge of animal immunity comes from representatives in only two phyla (the chordates and arthropods). This assemblage is highly diverse in terms of species richness and variety of life histories. The mechanisms that are employed in animal immunity are expected to be similarly diverse. Numbers at the base of the tree show rough estimates of the number of phyla in the protostomes and deuterostomes (from Maddison, D. R., and Schulz, K.‐S. [ed.] [2004]. The Tree of Life Web Project. http://tolweb.org). Commonly used laboratory designations have been used in place of genus/species.

The known molecular ligands of mammalian TLRs comprise an extremely wide array of structures, including lipopolysaccharide (LPS), peptidoglycan, lipopeptide, bacterial flagellin, RNA (both single- and double-stranded), unmethylated CpG DNA, and other molecules (Beutler, 2004b; Heil et al., 2004; Medzhitov, 2001). In general, a given TLR interacts with one of the very

214

g a ry w. li tm an

E T AL .

specific biochemical classes indicated above (e.g., TLR4 interacts with LPS, whereas TLR5 interacts with flagellin, although combinatorial effects, such as heterodimerization, may increase this range of recognition) (Wyllie et al., 2000). Given the small number of TLR proteins in most animals, it is likely that each recognizes a variety of conserved antigens. As might be expected for such different biochemical functions, the ectodomain of each particular TLR has diverged extensively over time from those of other TLRs within the same species, whereas orthologous TLR ectodomains are relatively conserved. Indeed, the extracellular domain of any given human TLR shares more amino acid identity with its mouse ortholog than with any other human TLR, in marked contrast to other immune receptors, such as Ig V region families, which are highly divergent even between mouse and human. Furthermore, although polymorphism of TLR genes (e.g., TLR4) within mammalian species has been described, the resulting diversity of TLR4 proteins is not even remotely comparable to that observed for the rearranging antigen-binding receptors in that organism (Smirnova et al., 2000, 2001; White et al., 2003). Selection for evolutionary diversification of LRR domain-containing innate immune receptors, such as TLRs, is not restricted to the animal kingdom. Plant R-genes, which encode proteins with diverse LRR domains that recognize either factors expressed by various pathogens or endogenous factors indicative of infection, exhibit extensive diversity (McDowell and Woffenden, 2003). Furthermore, there is strong evidence for positive selection in the LRR domains of certain R-genes, especially at sites encoding the solventexposed residues of the LRR domains that may interact with variable ligands (Bergelson et al., 2001), consistent with observations that have been made in LRR-containing immune receptors found in animals. As will become apparent later, the LRR, as a unit of genetic variation, has a particularly long history and seems adaptable to many different roles relating to host defense. Coevolution of hosts and pathogens is well recognized (Carton et al., 2005; Donelson, 2003), and many gene families encoding innate immune molecules have become diversified from selective pressure over time. Such families include the peptidoglycan recognition proteins (Dziarski, 2003), antimicrobial peptides such as defensins (Huttner et al., 1994; Lehrer and Ganz, 2002; Maxwell et al., 2003), and cryptdin-related sequence proteins (Hornef et al., 2004), as well as a particularly wide array of lectins (Kilpatrick, 2002; Lee and So¨ derha¨ll, 2001; Suzuki et al., 2003; Vasta et al., 2004). As a rule, representative members of these families can be found in a large number of different phyla and have diversified to meet the specific needs of each particular species to combat its own pathogens. Together with the LRR domain-containing receptors, such as TLRs or R-gene products, the sum of all of these molecules

novel immune mechanisms

215

constitutes a highly complex and diversified set of immune mediators that are quite variable across different phylogenetic groups. Although variation within any particular family in a given species does not approach that produced by adaptive immune systems, a recent study in Drosophila suggests that significant variability persists in innate immune sensors, effectors and signal transduction molecules in wild populations and correlates with variable resistance to pathogens (Lazzaro et al., 2004). 2.3. Large Diversified Multigene Families Encoding Innate Immune Receptors Two main points emerge from the previous descriptions: (1) a variety of different molecular mechanisms confer immunity in species that lack an adaptive immune system, and (2) a typical solution to the problem appears to have been to evolve and diversify single genes or relatively small gene families. In contrast to these observations, unprecedented expansion of several multigene families encoding immune-type receptors has been noted in the sea urchin, an echinoderm that is classed along with the chordates within the deuterostomes and has long served as an important developmental model (Fig. 1). The purple sea urchin, Strongylocentrotus purpuratus, expresses extremely large numbers of different scavenger receptor genes that are upregulated upon foreign stimulation (Pancer, 2000; Pancer et al., 1999). Furthermore, a greatly expanded family of TLR genes is encoded in the genome of this species, consistent with a highly developed repertoire of innate immune receptors (Rast and Pancer, unpublished). Large-scale expansion and diversification of various gene families represents one of many strategies to solve the problem of creating immunological diversity. Additional studies, described later, suggest that the sea urchin somatically diversifies another family of genes that likely are integral to host defense. Notably, none of the immunetype molecules described above in the sea urchin are of the Ig type; however, scans of the partially resolved sea urchin genome have identified a large number of Ig-related gene segments that presently are under investigation (Rast, unpublished observations). Although relatively few of the IgSF candidate genes are of the V type, it is important to note that KIR-type molecules expressed by natural killer (NK) cells exhibit complex specificities through variation in C2-type domains, demonstrating that immune specificity is not solely dependent on differences in variable (V) region structure. Patterned variation in Ig-related molecules in sponge has been interpreted to be associated with immune function (Pancer et al., 1998). Diversified Ig-type molecules also have been identified in both a protostome invertebrate and a cephalochordate but have not yet been identified in jawless vertebrates; rather

216

g a ry w. li tm an

E T AL .

it appears that a highly derived form of LRR-containing receptor contributes to immune recognition in the two extant orders of this latter major phylogenetic radiation (described later). 3. Alternative Mechanisms that Diversify Immune Receptors 3.1. Variable Lymphocyte Receptors (VLRs) as Adaptive Immune Receptors in Jawless Vertebrates As indicated previously, despite the extraordinary variation in the generation of B-cell receptor complexity, the basic components of the genetic mechanisms that diversify both BCR and TCR in mammals are present in the modern representatives of the jawed vertebrates that are most phylogenetically distant from mammals. A clear demarcation currently exists between jawed and jawless vertebrates with regard to the mediators of classical adaptive immunity (Cannon et al., 2004b; Litman et al., 1999). A variety of molecular genetic approaches, including EST screens (Mayer et al., 2002; Uinuk-Ool et al., 2002), used in both lamprey and hagfish (Suzuki et al., 2004), which represent the two extant orders of jawless vertebrates, have failed to identify gene homologs sharing significant sequence identity with Igs and TCRs. However, utilizing a selective cloning approach (Cannon et al., 2002) we recently have identified a gene in the sea lamprey (Petromyzon marinus) that is predicted to encode a product that shares several characteristics with VpreB, an integral component of the pre-BCR (Melchers et al., 2000; Ohnishi and Melchers, 2003). The relative expression of the VpreB-like gene is increased significantly in circulating lymphocytes and myeloid cells obtained from leukocytes recovered from animals that have been challenged with a cocktail of immunogens (Cannon et al., 2004a). Understanding the nature of the association of the VpreB-like gene product with other lymphocyte proteins is of considerable potential significance. Along these lines, it remains possible that distantly homologous forms of Ig, TCR and MHC I/II, as well as accessory molecules that function in concert with Ig and TCR, will be identified in the genomes of jawless vertebrates. However, the gene products in lamprey that have been interpreted as being related to TCR and CD4 (Pancer et al., 2004b) underscore the difficulties in assigning such relationships. In a broader evolutionary context, the progenitor of rearranging Ig/TCR is thought to have arisen by way of a genetic transposition event involving RAG1/ 2 in a common ancestor of the modern jawed vertebrates, after this lineage separated from the evolutionary line leading to the living jawless vertebrates (Agrawal et al., 1998; Hiom et al., 1998). It should be noted that the speculation regarding when this event occurred (i.e., before or after the emergence of

novel immune mechanisms

217

the vertebrates) is based on very incomplete data. Genome sequences emerging from other deuterostome species change the current view of the timing and mechanism of this critical event. Specifically, the recent finding of RAG-like genes in the sea urchin may alter this view with regard to when the components of adaptive immunity appear in the deuterostomes (Rast, manuscript in preparation). There is no reason to suspect that jawless vertebrates, which were diverse and numerous at the time of the emergence of the jawed vertebrates, were in any way exempt from pressure to respond to pathogens. Furthermore, earlier studies demonstrate that the sea lamprey mounts an induced response to immunization with bacterial or foreign erythrocytes, exhibits allograft rejection, and demonstrates delayed hypersensitivity (Finstad and Good, 1964; Good et al., 1972; Litman et al., 1999). Lampreys also possess a cell type that bears a histological resemblance to a lymphocyte and is found in the circulation as well as in certain tissues (Finstad and Good, 1964; Uinuk-Ool et al., 2002). In one tissue (protovertebral arch), this cell type undergoes proliferation during routine immunization (Good et al., 1972) (Litman, unpublished observation). Finally, the lamprey encodes genes that represent convincing orthologs of hematopoietic transcription factors (Anderson et al., 2001; Shintani et al., 2000) that are involved in lymphocyte development in jawed vertebrates. More recently, a concentrated effort was initiated to further characterize lamprey lymphocytes and to define receptor diversification at the molecular genetic level (Pancer et al., 2004a). This work is based on the observation that the number of large lymphocytes in the peripheral leukocyte population of larval lamprey increased
13-fold upon injection (stimulation) with an antigen/mitogen cocktail. The large lymphocytes are two times greater in diameter relative to small lymphocytes, possess azurophilic cytoplasm, and exhibit prominent nucleoli. A subtractive cDNA cloning approach was adapted to identify possible response-related gene products in these cells. Twenty percent of clones recovered using this approach are predicted to encode proteins with diverse LRR motifs. After further inspection, 239 uniquely diverse LRR-encoding ESTs were identified. The molecular structure of the predicted products of these ESTs consists of six essential features: (1) a 30–38 residue N-terminal region, (2) nine 16–24 residue LRRs, (3) a 48–58 residue C-terminal LRR, (4) a threonine/proline stalk, (5) a glycosylphosphatidyl-inositol anchor, and (6) a hydrophobic tail (Fig. 2A). The number of core LRR motifs is variable, but the N- and C-terminal domains are remarkably invariant. The molecules that are predicted to be encoded at this gene locus are designated variable lymphocyte receptors (VLRs). Whereas the extensive database of VLR ESTs indicates an exceptional degree of overall genetic variability, PCR amplicons of VLRs from individual cells were shown

218

g a ry w. li tm an

E T AL .

Figure 2 Representative structures of mature proteins from genes that likely effect alternative mechanisms of immune recognition. (A) Variable lymphocyte receptor (VLR) consisting of leucine‐ rich repeats (LRRs), including N‐terminal (LRRNT) and C‐terminal (LRRCT) regions, a connecting peptide (CP), a stalk, a glycosyl‐phosphatidyl‐inositol (GPI) anchor, and a hydrophobic tail. (B) Fibrinogen‐related protein (FREP) consisting of two Ig‐type domains (that cannot be classified as V, I, C1, or C2 types), a small connecting region (SCR), an interceding region (ICR) and a fibrinogen domain (FBG). (C) Variable (V) region‐containing chitin‐binding protein (VCBP) consisting of two V domains and a chitin‐binding domain (CBD). (D) Two different forms of novel immune‐type receptors (NITRs), consisting of Ig/TCR‐type V and I domains, and a C terminus in which the activating form (left) contains a transmembrane region bearing a positively charged residue as well as a tyrosine (Y) in the cytoplasmic tail, the inhibitory form (right) contains a neutral transmembrane region and a cytoplasmic domain encoding a prototypic ITIM and variant (itim).

to represent a single molecular species in each instance described; such monoallelic expression is an integral characteristic of lymphocyte-mediated immunity and a central component of a clonal commitment/expansion form of adaptive immunity. Conventional Southern blotting using a probe specific to the C-terminal region of the VLR suggested that the receptor was encoded at a single locus; this configuration was confirmed through additional physical mapping and sequence analysis of VLR-encoding PACs (P1 artificial chromosomes). Southern blot analysis of pulsed field gel electrophoresis-separated, restriction enzyme-digested lamprey erythrocyte DNA was shown to be consistent with the VLR locus spanning at least 100–150 kb. PAC library screening, using probes complementing the 50 and 30 ends of a VLR, yielded five clones of which three were informative across the entire predicted VLR genomic locus.

novel immune mechanisms

219

The basic genomic structure of the VLR consists of a split exon encoding a 50 untranslated region; a second exon encoding the remainder of the 50 untranslated region and the N-terminal half of the N-terminal LRR; a third exon encoding the 50 half of the C-terminal LRR; and a fourth exon encoding the 30 half of the C-terminal LRR, the remainder of the VLR protein, and the 30 untranslated region. Notably, the core of the genomic locus lacked the 30 portion of the N-terminal LRR, the first LRR (LRR1), or any of the 24 residue LRRs that were predicted from the cDNA analyses. Six cassettes of singlet-or doublet-variable LRRs as well as the N-terminal LRR and LRR1 were identified
14 kb upstream of the locus, contained within the PAC in which the entire C-terminal portion of the VLR is encoded. The LRR cassettes are in both forward and reverse orientation. PAC library screening identified clones encoding multiple LRRs, as well as the 50 untranslated region and 50 N-terminal LRR segment. Sequence analysis indicates the presence of 30 diverse LRR segments, which are arranged in 17 cassettes of 1-3 motifs. The LRR cassettes are
15 kb upstream of the core genomic VLR sequences. VLR diversity is generated through the insertion of upstream and downstream LRR-containing blocks. Specifically, it was possible to identify unique amplicons from lymphoid genomic DNA that would encode VLRs. When the same types of amplifications were attempted from erythrocytes or muscle DNA, ‘‘genomic VLR’’ amplicons (
14 kb) were identified, whereas lymphocytes produced both
14 kb and
1 kb (rearranged) amplicons. It has been suggested that the mechanism(s) underlying the germline variation may be related to gene conversion but also could be mediated through a unique genetic process that is restricted to individual cells. Unlike Ig and TCRs, VLRs do not utilize a recombination signal sequence-mediated recombination mechanism to effect somatic reorganization. Recently, VLR-related genes have been identified in hagfish (Pancer and Amemiya, personal communication), presenting the possibility that these molecules may contribute to immune recognition throughout the jawless vertebrates; however, these latter studies may prove somewhat more challenging than those in lamprey given that inducible immunity in hagfish is not well documented. Several additional points are relevant in considering these data. First, no relationship has been reported yet between the VLRs and the induced antibacterial and antierythrocyte adaptive responses described earlier in lamprey. The molecular size of the agglutinins directed at both of these categories of antigens is
315 kD, suggesting that if VLRs account for these activities, they would need to function as complex multimers. Such size homogeneity would not be a predicted feature of the VLRs. It remains to be seen if the induced, specific responses to bacteria and erythrocytes are VLR-mediated.

220

g a ry w. li tm an

E T AL .

A number of questions of broad significance are raised by the studies in lamprey. First, do cells expressing VLR molecules undergo true antigenresponsive lymphocyte-type clonal proliferation or is the response to immunization more akin to an acute phase response? What, if any, aspect of VLR selection is analogous to that seen in conventional adaptive immune responses? The restricted expression of a single receptor on each cell suggests that clonal selection may be a significant factor in the immune response in jawless vertebrates. Is there a memory component to the VLR response? Do VLRs sustain additional change such as somatic hypermutation? What is the range of actual specificities of VLRs for different challenging immunogens? This last question is particularly important as multiple ligands and molecules, which tend to elicit broad ‘‘activation’’-type responses, were used to induce the VLR-based responses described thus far. A particularly interesting aspect of the VLRs is the relationship that they suggest between innate receptors of the LRR type (e.g., TLRs) and what appears to be the potential for a LRR-based ‘‘adaptive’’ immune function. Molecular variation in LRRs has been implicated previously in immune protection (Bergelson et al., 2001). Furthermore, mechanisms that can modify genetic information have an ancient history in a wide range of bacterial and viral pathogens, as well as in parasites as fundamental host response-evasion mechanisms. The VLRs depart from our current concepts of LRR-based immunity through what likely represents a unique mechanism for somatic diversification. Furthermore, this variation is introduced in the context of a single cell that may be capable of selective proliferation, representing a potential jawless vertebrate counterpart of the clonal expansion of lymphocytes seen in jawed vertebrates. Collectively, these characteristics may well create an alternative form of an adaptive immune system. Ongoing investigations in both protochordates and jawed vertebrates are aimed at detecting VLRs and other forms of somatically varied LRRs. At this point in our understanding of alternative mechanisms to generate immune receptor diversity, we consider the variability in LRRs as an integral feature of a large set of diverse immune receptors that have undergone a complex evolution that likely exhibits divergence, convergence, and perhaps parallel effects. The jawless vertebrate VLRs, which could assume at least some of the roles of the rearranging Ig/TCRs, represent the most genetically derived form of LRR-type immune proteins yet seen. Although these genes are presently not known to function in jawed vertebrates or invertebrates, it is notable that the various strategies employed to date to identify conventional immune-type genes in these species likely would not have identified such molecules. Further elucidation of the role of diversified LRR-containing receptors in the context of both innate and adaptive immune responses is of compelling interest. Despite their obvious

novel immune mechanisms

221

biological significance, the VLRs are not the only form of somatically variable immune effector molecules found outside of the jawed vertebrates. 3.2. Variation in IgSF-Type Immune Receptors in an Invertebrate Invertebrates, which comprise a particularly heterogeneous group of phyla with extensive diversity in body forms, lifespans, and life histories, are susceptible to a wide range of viral, bacterial, fungal, and parasitic infections; furthermore, in many cases, the pathogens that affect these organisms undergo genetic changes that would potentially evade traditional innate defenses, reviewed in (Loker et al., 2004). Studies of invertebrate immunity at the gene level have focused on relatively few models; the most significant work relating to conserved innate responses has been carried out in two dipterans, Drosophila and Anopheles, owing to the extraordinary value of the former as a genetic model (Hoffmann and Reichhart, 2002) and the significance of the latter as a vector in the transmission of human disease (Dimopoulos et al., 2002). Even the most polymorphic innate receptors that have been described exhibit relatively little variation compared with the receptors that mediate adaptive immunity. Considering that the invertebrates are enormously diverse, constituting nearly 30 phyla, as opposed to the vertebrates, which constitute only a portion of the phylum Chordata, it is reasonable to assume that investigations of invertebrate immunity would result in the discovery of many unique immune mechanisms, which indeed seems to be the case. Remarkable insights into the generation of complex ‘‘immune-type’’ diversity in a protostome invertebrate have been provided by studies of the immune interaction of Biomphalaria glabrata, a freshwater snail, and parasitic flatworms (trematodes). B. glabrata possesses a family of hemolymph proteins termed fibrinogen-related proteins (FREPs), which consist of one or two N-terminal Ig superfamily (IgSF) domains and a C-terminal fibrinogen-like domain (Adema et al., 1997; Zhang and Loker, 2004; Zhang et al., 2001) (Fig. 2B). Notably, fibrinogen-like domains have been implicated previously in recognition of nonself in other invertebrates (Loker et al., 2004). FREPs are produced in defense cells of snails following infection with trematode parasites such as Schistosoma mansoni and Echinostoma paraensei (Loker et al., 2004). FREPs are associated with a lectin-like activity that precipitates soluble parasitic antigens (Adema et al., 1997). FREP genes can be organized into subfamilies, and within a subfamily, genomic sequences vary in both the IgSF-related and fibrinogen-like domains (Leonard et al., 2001; Zhang and Loker, 2004). This variation was studied more intensively in a region encoding 110 amino acids in the N-terminal IgSF1 domain of FREP subfamily 3 (IgSF1/FREP3) (Zhang et al., 2004). An

222

g a ry w. li tm an

E T AL .

indication of variation in this genetic region is revealed in comparisons between sequences amplified from two different individual snails. Forty-five different sequences were found in one animal and 37 different sequences in another; only one sequence was common to both animals. Ultimately, it was possible to identify 314 different IgSF1/FREP3 sequences from 22 animals, and the substitution patterns observed were interpreted to be nonrandom. It was concluded by using comparable amplification methods that the differences were four-fold higher in IgSF1 coding sequences than in several different control amplicons from unrelated genes. Southern blot analysis of genomic DNA from individual snails placed the number of genes encoding IgSF1/ FREP3 sequences at two to five per individual, which can be equated with the presence of 2–10 alleles in any given organism. Through a mathematical treatment of the large sequence data set that was acquired in these investigations, it was concluded that nine germline sequences could account for all of the known variant sequences through the introduction of nucleotide point mutation and recombination diversification (concatenation of segments from pairs of what the investigators called ‘‘source’’ sequences). This conclusion implies that the high level of observed diversity was generated by somatic alteration of FREP genes, which was confirmed in cDNA analyses that demonstrate elevated diversity of FREP3 cDNAs from three snails relative to control genes from the same animals. The offspring of the hermaphroditic B. glabrata were found to exhibit mutational and recombinatorial diversification, but source sequences (i.e., those that give rise to the genetic variation), were similar or identical to parental genes (Zhang et al., 2004). The sequence diversification in IgSF1/ FREP3 occurs in several distinct tissues; however, it is not clear if such tissues are populated by the same cell type(s). A selective capacity to effect somatic changes in different parts of the body could be highly advantageous. Additional variation in FREPs may be introduced through the formation of heterotypic multimers. It will be of great interest to determine how the various FREPs interact with the purported target pathogen, characterize further the individual cells from which the FREPs derive, identify the precise mechanisms underlying the observed genetic change, and determine whether there is any immunological memory component in FREP-based immunity as reported in another invertebrate model (Kurtz and Franz, 2003). Collectively, the studies of FREPs raise fundamental questions about the occurrence and nature of genetic changes in other ‘‘innate’’ molecules found in invertebrates, jawless vertebrates, and jawed vertebrates. Studies in several other model systems suggest that the capacity to diversify immune-type genes may be widespread throughout the invertebrates. The penaeidins, a family of antimicrobial peptides found in shrimp, are encoded

novel immune mechanisms

223

by a relatively small number of genetic loci. Diversity in penaeidins is not readily reconciled with simple allelic polymorphisms but may reflect the activities of alternative transcriptional mechanisms (Cuthbertson et al., 2002). Recently, a novel family of genes that are upregulated upon challenge from LPS in the sea urchin was identified using a subtracted probe to screen an arrayed cDNA library that was constructed from LPS-activated coelomocytes (C. Smith et al., unpublished observations). Nearly three-fourths of the ESTs could be matched to an unknown sequence, designated 185/333, consistent with the interpretation that they represent components of a major inflammatory response mounted by coelomocytes. The deduced 185/333 peptides possess a leader sequence, a glycine-rich region containing an arginine-glycine-aspartic acid (RGD) motif, which has been implicated in binding to integrins and may be involved in inducing phagocytosis, and a histidine-rich region. Expression patterns of 185/333 mRNAs in coelomocytes from individual animals indicate that message sizes are different before challenge from LPS compared to after challenge and that nucleotide and deduced amino acid diversity increases postinjection. Alignments of full-length cDNAs have revealed a surprising level of apparent alternative splicing in addition to variations at specific nucleotide positions that translate to amino acid sequence diversity. Furthermore, certain domain usage patterns were identified only before or only after challenge. Searches of the partially assembled sea urchin genome indicate that the leader sequence and the remainder of the coding region in each transcript are encoded by two distinct exons, ruling out the possibility that some of the diversity is generated as a product of alternative splicing of numerous exons, although cryptic splicing may be involved. Taken together with the observations regarding scavenger receptors and TLR-like genes in the sea urchin described above, these data indicate that this species utilizes a variety of mechanisms to generate and maintain significant diversity in large sets of proteins that are potentially involved in inflammatory and/or immune functions. 3.3. Variable Region-Containing Chitin-Binding Proteins (VCBPs) in Protochordates A second example of diverse IgSF-like receptors is seen with the protochordate VCBPs. Unlike the FREPs, the members of this multigene family encode authentic V regions (Fig. 2C). VCBP genes were characterized first in amphioxus (Branchiostoma floridae) in the course of our efforts to identify orthologs of V region-containing immune-type receptors in a cephalochordate (Cannon et al., 2002). Our rationale for focusing on this subphylum was that such species might possess aspects of a more basal (less derived) immune receptor system that would not necessarily be present in the highly derived jawless

224

g a ry w. li tm an

E T AL .

vertebrates. Homologous forms of VCBPs were identified subsequently from the assembled genome of Ciona (Azumi et al., 2003; Dehal et al., 2002); however, the number and complexity of VCBPs appear to be reduced significantly relative to what we described initially in amphioxus. Three features of the VCBPs, which consist of two N-terminal tandem V regions and a chitinbinding domain, suggest that they are immune receptor candidates: (1) the V regions of VCBPs possess the same core residues as the V regions found in Ig, TCR, and other IgSF members (Chothia et al., 1998), (2) like Ig and TCR, the VCBPs can be organized into at least five families that differ from one another by at least 30% at the predicted peptide sequence level. Such an effect resembles variation seen in the large V region families of Ig, TCR, and novel immune-type receptors (NITRs; see later discussion) and suggests that an immune-related diversification has taken place, and (3) with some VCBP families, regionalized sequence variation that is predicted to influence receptor function is present, although such focal hypervariation is not positionally synonymous with complementarity determining region (CDR)-type variation seen in Ig and TCR. The initial interpretations of sequence variation in VCBPs were based on cDNAs that were derived from tissue isolated from pooled animals. Although relatively few cDNA sequences were available in the original studies, their complexity was inconsistent with the limited number of genes observed in genomic Southern blots of single source DNA isolates. A parallel series of observations noted that VCBP expression is restricted entirely to specific cells of the gut in this filter-feeding organism. The known presence of chitin in bacteria, fungi, and other pathogens and the extreme variability in the load of marine pathogens that likely confront amphioxus (Fuhrman, 1999) collectively reinforce speculation that the VCBPs are involved in some form of specific host defense. The findings that both VCBPs and FREPs consist of N-terminal Ig-related domains and a C-terminal protein that is reactive with potential pathogens suggests a basic and potentially significant relationship between these otherwise unrelated molecules. The source of sequence diversity observed in the cDNA analyses was examined further by amplification of V exons from genomic DNA isolated from individual specimens as well as from overlapping BAC (bacterial artificial chromosome) clones derived from a single animal. On average, we were able to amplify six copies of an extended region of a particular V1 exon of one VCBP gene family (VCBP2) from both genomic DNA and the overlapping BAC clones that cover the VCBP encoding region of the genome (Cannon et al., 2004c) (and unpublished). Analyses of the genomic complexity of the different VCBPs amplified from individual animals indicate that certain sequences are shared in common by nearly all animals but that the majority of polymorphic sequences derived from a single animal DNA source are unique to that

novel immune mechanisms

225

individual (Cannon et al., 2004c). Notably, the differences involve both single positional replacements and overall length differences; the latter bears a superficial resemblance to N-region variation seen at Ig-joining junctions. Such complex polymorphisms are consistent with the variation that was observed initially in the pooled cDNAs that were employed as template sources and suggest an immune function for VCBPs in which these genes are undergoing strong diversifying selection and adaptive evolution. We have speculated that the VCBP gene locus may not exhibit stable Mendelian transmission and presently are investigating this possibility by comparing VCBP complexity in parents and offspring. A significant portion of the VCBP locus has been sequenced and annotated. We are able to relate, with a considerable degree of confidence, certain genomic sequences to specific transcripts. At least some VCBP genes are variable at the mRNA (cDNA) level, and some of this variation may arise through recombination and differential splicing within the locus (Dishaw, unpublished observation). The generation of extraordinarily large numbers of isoform variants of single IgSF genes has been described previously in other systems (Schmucker et al., 2000; Wojtowicz et al., 2004). At this point in our studies, it is reasonable to conclude that a number of different genetic processes/effects may influence VCBP structure. More conclusive statements will be possible once the entire VCBP locus is resolved. The high level of diversity seen in VCBPs led us to speculate that, in addition to the variation seen in the amphioxus VCBPs, generalized diversification of immune-type molecules (not only of the Ig-V type) in both invertebrates and jawless vertebrates may be mediated by both genomic and somatic processes (Cannon et al., 2004b). The studies described in this review are consistent with this proposition (Pancer et al., 2004a; Zhang et al., 2004). Notwithstanding other conclusions that have been drawn regarding alternative mechanisms of receptor diversification, what sets the VCBPs apart from VLRs and FREPs is that variability is introduced on the background of a prototypic V region. Although somatic reorganization as seen in Ig and TCR does not occur in VCBPs, these disparate forms of V region-containing immune receptors could share other features that introduce somatic change in conventional adaptive immune receptors. In addition to genetic studies currently underway, our interests also have focused on the structural biology of VCBPs. We have described the characteristics of VCBP crystals (Hernandez Prada et al., 2004) and more recently, the crystal structure of the N-terminal V domain of one representative member of the VCBP family has been solved at a level of structural refinement of 1.15 A˚ . As predicted from primary sequence data, this VCBP domain is unequivocally of the V-type (Hernandez Prada, Haire, Cannon, Litman, and Ostrov, unpublished observation). The level of

226

g a ry w. li tm an

E T AL .

structural inferences that can be drawn from the solved structure far exceeds that possible for any Ig or TCR V described to date. A number of screening strategies utilizing solubilized VCBP receptors currently are being applied in efforts to define their ligand specificities. 3.4. NITRs – An Unusual Leukocyte Regulatory Receptor that Combines Features of Adaptive and Innate Receptors In addition to their capacity to undergo somatic change, both Ig and TCR genes exhibit considerable germline complexity through structurally diversified V region families. In a sense, these gene segments represent the innate component of the conventional adaptive receptors. A number of other immune-type molecules also possess V regions, including NKp30 (Pende et al., 1999), NKp44 (Cantoni et al., 1999), and OX-2 (Wright et al., 2000). However, extensive V region diversity in mammals has not been attributed to gene families other than those encoding Igs and TCRs. In addition, resolution and annotation of several whole mammalian genomes has not revealed any other candidate V families. We hypothesized that other species outside of mammals potentially could utilize V regions for immune recognition. In order to test this possibility, we utilized the pufferfish as an experimental model. Its small genome size affords two strategic advantages in searching for novel genes: (1) the number of artifacts generated using PCR-based approaches with short primers complementing V regions is very low owing to short introns and diminished intergenic regions (the principal sources of spurious amplicons in short primer-based PCR analysis) (Rast et al., 1997), and (2) when the studies described later were conducted (prior to the resolution of the pufferfish genome), the genomic structures of candidate genes could be determined efficiently. We initially described a single unique gene (Rast et al., 1995) and ultimately described a family of NITRs possessing highly diversified V regions (Strong et al., 1999). Like TCRs, the NITR proteins are generally of the transmembrane type and typically consist of an N-terminal V-type ectodomain and a C-terminal intermediate (I)-type ectodomain, although some NITRs possess only a single, V-type ectodomain. NITRs either possess a positively charged residue in their transmembrane region (putative activating forms), a cytoplasmic tail with one or two immunoreceptor tyrosinebased inhibitory motifs (ITIMs; putative inhibitory forms), or less frequently, a cytoplasmic tail without a signaling motif (Fig. 2D). A single example of an NITR possessing a cytoplasmic tail that may contain both an ITIM and an immunoreceptor tyrosine-based activation motif (ITAM) also has been identified. Some NITRs possess unequivocal Ig/TCR-like J regions, some possess J-like motifs, and others lack J-related sequences. Certain NITRs

novel immune mechanisms

227

lack a transmembrane region and resemble decoy molecules (Yoder et al., 2004) that have been identified in other types of leukocyte regulatory receptors (Dietrich et al., 2000). High-confidence molecular modeling of NITRs has shown them to closely resemble Ig/TCR V domains. In the specific case of NITR1g, the greatest similarity is with a VH gene; however, the stem of the second hypervariable region (described later) of this NITR deviates from the VH region at Ser49 of the NITR1g V domain in central portions of the C0 strand, causing the loop to shift 15 A˚ compared with CDR2 of the most similar VH region for which a crystal structure has been solved. This structural shift creates a ‘‘pocket-like’’ effect that is predicted to influence ligand recognition. If V family classification (see later discussion) is taken into consideration, more than 40 different variants of NITRs have been described in zebrafish (Yoder et al., 2004) of which only a single unequivocal activating form and splice variant are present. NITRs are expressed on several different cell lineages in bony fish, including NK-type cells and cytotoxic lymphocytes (Shen et al., 2004) expressing TCR genes (Hawke et al., 2001) (Miller, personal communication). In addition to this remarkable structural diversity, the NITR V regions can be categorized into families using equivalent criteria to those that are applied in distinguishing families of Igs and TCRs. In zebrafish, 14 (12 [Yoder et al., 2004] and 2 [Cannon, unpublished observations]) different V families have been identified, 13 are present in pufferfish, and, thus far, four different V families constituting 12 different structural forms have been characterized in catfish (Hawke et al., 2001). It is likely that as many V families as have been defined in pufferfish and zebrafish will be identified in catfish once the genomic sequence of the catfish NITR locus has been established. Interestingly, the gene families found in one species, with the exception of only a few individual sequences, are not found in the other species. Rather, it appears as if each class of fish has rapidly evolved and diversified NITR gene families. Furthermore, in each species one family is comprised of a relatively large number of individual members, but the other families only infrequently consist of more than a few unique members. Our efforts to identify NITR orthologs outside of teleost fish have been unsuccessful thus far, although these experiments have resulted in the identification of a separate, large family of IgSF receptors in the clearnose skate (Raja eglanteria), a representative cartilaginous fish (Cannon, unpublished observation). It is possible to examine patterns of sequence variation within members of the large, single families of NITRs (Yoder et al., 2001). Differences are regionally concentrated, with the N-terminal hypervariable region corresponding to CDR1 as defined in Ig and TCR V regions; however, the other regions of hypervariation are shifted, a finding that is not unexpected

228

g a ry w. li tm an

E T AL .

given the differences in the predicted structure of the NITR ligand-binding site. No evidence has been found for the introduction of somatic mutation into NITR genes, but their complex isotype polymorphism is well documented. Even within individual lines of zebrafish, considerable allelic and haplotypic variation are present within the NITR gene complex (Yoder et al., 2004). Such interindividual variation relates to the manner in which the principal lines of this species are maintained and likely is driven by heterozygote advantage. The NITRs resemble KIRs (Vilches and Parham, 2002) in many regards, including differential expression by individual cells as well as expression in both NK and certain classes of T cells. NITRs could well represent the bony fish functional corollary of primate KIR-type receptors (Litman et al., 2001). Although NITRs do not supplant the role of the rearranging receptors, they stand out as a compelling example of an ‘‘adaptive’’-type structural form being utilized, presumably in an innate context. 4. Conclusions The discoveries of new mechanisms of immune diversification are changing the commonly held view that adaptive immunity is the exclusive domain of jawed vertebrates. In combination with findings of extensively diversified innate immune systems in widely divergent animal phyla, these data are consistent with the premise that immunity is under similar intense selective pressures in both invertebrates and vertebrates. Presently there is no indication that either the jawless vertebrate VLR or the molluscan FREP system uses mechanisms of immune receptor diversification that are related to the recombination signal sequence-mediated VDJ recombination found in all jawed vertebrates. Even so, and although our understanding of these and other receptor diversification systems is far from thorough, there is no denying the conclusion that the outcomes of germline and somatic change in immune receptor genes of invertebrates (including protochordates) and jawless vertebrates share many features in common with the processes that create receptor diversity in jawed vertebrates. While it certainly is too early to draw firm conclusions about the forces that drive the evolution of adaptive immunity, these new findings, as well as the high likelihood that further unusual mechanisms of immune receptor diversification will be identified in other species, invite speculation regarding possible scenarios under which these alternative molecular systems emerged. The most plausible pathway for the evolutionary emergence of adaptive systems is through modification of preexisting innate receptors. In this way, the complex genetic and cellular machinery that regulates the use of receptor specificity is already in place and must only be retargeted. This process, in

novel immune mechanisms

229

turn, raises the possibility that some of the regulatory mechanisms that control clonal selection, proliferation, and memory in the VDJ recombination-based systems of jawed vertebrates may already have been adapted to control complex innate immune responses. There is increasing evidence for a significant level of regulatory conservation in the function and development of immune cells among protostome invertebrates, deuterostome invertebrates, and vertebrates (Barreau-Roumiguiere et al., 2003; Cho et al., 2002; Hoffmann, 2003; Lebestky et al., 2000; Millet and Ewbank, 2004; Pancer et al., 1999; Rast et al., 2000; Waltzer et al., 2002). While these data are far from conclusive in describing the regulatory state of primitive bilaterian immune cells (e.g., there is some discrepancy regarding orthology among protostome and deuterostome ‘‘blood cell’’ regulators), it begins to suggest a conserved genetic program upon which novel immune receptor diversity was structured during evolution. While the studies described above permit us to classify several distinct phases in the acquisition of immune recognition complexity and to recognize far earlier roles for somatic variation than considered previously, they also underscore an increasingly blurred distinction between adaptive and innate immunity (Fig. 3). Adaptive receptors have a large, potentially innate component to their specificity (i.e., they primarily are encoded directly in the genome) and certain specificities, simply as the result of somatic reorganization, are statistically certain to emerge even in the absence of somatic selection. Innate specificity in adaptive systems can range in importance from critical, as may be the case for the molluscan FREP genes, where somatic mutation slightly alters an existing innate specificity, to nearly insignificant in the case of an entirely novel antibody specificity. At the extremes, there are some clear differences between the innate and adaptive systems that may have some bearing on the evolutionary transitions from one to the other. For both receptor types, specificity is the result of selection; however, while innate immune receptors are selected at the level of the organism over the course of evolution, somatic diversity is selected at the level of the cells that express the diversified receptor within the individual and, in the case of Ig and TCR, at a resolution of a single lymphocyte and its subsequent progeny. Diversification of innate systems may reach a threshold beyond which genespecific regulatory mechanisms that are able to control smaller receptor sets become unwieldy or evolve at a rate that is insufficient to keep pace with the addition of new receptor specificity. At this point, cellular mechanisms for selective receptor amplification may come to dominate. Thus, for multigene systems such as those seen in the sea urchin, in which scavenger receptor or TLR genes number in the hundreds, cellular control akin to that which modulates T- and B-cell specificities may become essential. A prediction of

230

g a ry w. li tm an

E T AL .

Figure 3 Different mechanisms that account for generation and maintenance of structural diversity in immune receptors. Different classes of immune diversity are outlined using examples from two protein structure families. Progression between particular gene types is not implied, although progression from simple innate receptors to highly diverse multigene families to somatic diversification mechanisms is a plausible scenario. In the top row, members of the IgSF effect both innate specificities (NKp30, NKp44, and NITRs) as well as somatically variable (FREPs and Ig light chain) and clonally selectable (Ig light chain) adaptive immunity. A number of different Ig‐like domains, specifically; V (NKp30, NKp44, NITR, Ig‐light chain), I (NITR), C1 (Ig light chain), C2 (which forms the ligand-binding site of KIRs, not shown), and unclassified Ig domains (as seen in FREPs), effect ligand recognition. In the bottom row, receptors containing LRRs mediate both innate (TLR and TLR‐like) and adaptive (VLR) immunity. The cytoplasmic tail of the TLRs contains a TIR domain. General parallels between IgSF‐based and LRR‐based variation are highlighted by border shading; the basic processes underlying the genetic variation (or lack thereof) are specified. Clonal selection may play a role in the immune regulatory function of the multigene expansion and classes of somatic variation immune diversity, although this possibility has not yet been investigated. Shading differences in IgSF‐ and LRR‐type receptors are used to underscore family and protein domain variants. Circles within the Ig domains of FREPs and Ig light chains (top) represent replacement substitutions; contiguous circles (in the case of light chain genes) are used to signify nontemplated junctional variation. Variation in FREPs has been resolved primarily in the N‐terminal Ig‐like domain; other substitutions illustrated here are inferred. Shading variations in the LRR portion of VLRs (see Fig. 2) reflect the differential utilization of different LRR cassettes.

novel immune mechanisms

231

this hypothesis, which will be directly testable, is that the expression of these highly diverse innate receptors exhibits some degree of clonal restriction. It was impossible to infer a general understanding of the evolutionary forces that led to the emergence of adaptive immunity from the single example of jawed vertebrate VDJ systems. The newly recognized systems of somatic diversification described here provide independent cases that will help to unravel how and why such complex immune systems tend to emerge. More importantly, these findings suggest that somatic diversification may be widespread in animal immunity. Unfortunately, these systems are initially difficult to identify and characterize, especially against the background of diversified multigene families and in the context of complex genomes that often are only partially characterized. What has been uncovered to date is remarkable, given that the foundations of the work rest on observations in only a few of the
30 animal phyla and, with the exception of the vertebrates, only a handful of species have been studied in any depth. Current and planned resolution of genome sequences and a renewed interest in the immune mechanisms of invertebrates will greatly accelerate these findings, which in turn are expected to alter radically the perceived landscape of animal immunity. Acknowledgments We thank Barbara Pryor for editorial assistance and Mike Sexton for graphic design. Drs. L. Courtney Smith, Chris T. Amemiya, Jules Hoffmann, Jean-Luc Imler, Larry Dishaw, and Zeev Pancer provided preliminary information that is cited in the manuscript. We thank Coen Adema, Chris Amemiya, and Sam Loker for comments regarding the manuscript. This work was supported by grants to GWL from the National Institutes of Health. JPC was supported by a fellowship from the H. Lee Moffitt Cancer Center and Research Institute.

References Adema, C. M., Hertel, L. A., Miller, R. D., and Loker, E. S. (1997). A family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection. Proc. Natl. Acad. Sci. USA 94, 8691–8696. Agrawal, A., Eastman, Q. M., and Schatz, D. G. (1998). Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system [see comments]. Nature 394, 744–751. Alt, F. W., and Baltimore, D. (1982). Joining of immunoglobulin heavy chain gene segments: Implications from a chromosome with evidence of three D-JH fusions. Proc. Natl. Acad. Sci. USA 79, 4118–4122. Anderson, M. K., Sun, X., Miracle, A. L., Litman, G. W., and Rothenberg, E. V. (2001). Evolution of hematopoiesis: Three members of the PU.1 transcription factor family in a cartilaginous fish, Raja eglanteria. Proc. Natl. Acad. Sci. USA 98, 553–558. Azumi, K., DeSantis, R., Tomaso, A., Rigoutsos, I., Yoshizaki, F., Pinto, M. R., Marino, R., Shida, K., Ikeda, M., Ikeda, M., Arai, M., Inoue, Y., Shimizu, T., Satoh, N., Rokhsar, D. S., Du Pasquier, L.,

232

g a ry w. li tm an

E T AL .

Kasahara, M., Satake, M., and Nonaka, M. (2003). Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: ‘‘Waiting for Godot.’’ Immunogenetics 55, 570–581. Barreau-Roumiguiere, C., Montagnani, C., and Escoubas, J. M. (2003). Characterization of a Tal/ SCL-like transcription factor in the pacific oyster Crassostrea gigas. Dev. Comp. Immunol. 27, 793–800. Bergelson, J., Kreitman, M., Stahl, E. A., and Tian, D. (2001). Evolutionary dynamics of plant R-genes. Science 292, 2281–2285. Beutler, B. (2004a). Inferences, questions and possibilities in Toll-like receptor signalling. Nature 430, 257–263. Beutler, B. (2004b). Innate immunity: An overview. Mol. Immunol. 40, 845–859. Cannon, J. P., Haire, R. N., and Litman, G. W. (2002). Identification of diversified immunoglobulin-like variable region-containing genes in a protochordate. Nature Immunol. 3, 1200–1207. Cannon, J. P., Haire, R. N., Pancer, Z., Mueller, M. G., Skapura, D., Cooper, M. D., and Litman, G. W. (2004a). Variable domains and a VpreB-like molecule are present in a jawless vertebrate. Immunogenetics 56, 924–929. Cannon, J. P., Haire, R. N., Rast, J. P., and Litman, G. W. (2004b). The phylogenetic origins of the antigen binding receptors and somatic diversification mechanisms. Immunol. Rev. 200, 12–22. Cannon, J. P., Haire, R. N., Schnitker, N., Mueller, M. G., and Litman, G. W. (2004c). Individual protochordates possess unique immune-type receptor repertoires. Curr. Biol. 14, R465–R466. Cantoni, C., Bottino, C., Vitale, A., Pessino, A., Augugliaro, R., Malaspina, A., Parolini, S., Moretta, L., Moretta, A., and Biassoni, R. (1999). NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily. J. Exp. Med. 189, 787. Carton, Y., Nappi, A. J., and Poirie, M. (2005). Genetics of anti-parasite resistance in invertebrates. Dev. Comp. Immunol. 29, 9–32. Cho, N. K., Keyes, L., Johnson, E., Heller, J., Ryner, L., Karim, F., and Krasnow, M. A. (2002). Developmental control of blood cell migration by the Drosophila VEGF pathway. Cell 108, 865–876. Chothia, C., Gelfand, I., and Kister, A. (1998). Structural determinants in the sequences of immunoglobulin variable domains. J. Mol. Biol. 278, 457–479. Cuthbertson, B. J., Shepard, E. F., Chapman, R. W., and Gross, P. S. (2002). Diversity of the penaeidin antimicrobial peptides in two shrimp species. Immunogenet 54, 442–445. Dehal, P., Satou, Y., Campbell, R. K., Chapman, J., Degnan, B., De Tomaso, A., Davidson, B., Di Gregorio, A., Gelpke, M., Goodstein, D. M., Harafuji, N., Hastings, K. E. M., Ho, I., Hotta, K., Huang, W., Kawashima, T., Lamaire, P., Martinez, D., Meinertzhagan, I. A., Necula, S., Nonaka, M., Putnam, N., Rash, S., Salga, H., Sataka, M., Terry, A., Yamada, L., Wang, H.-G., Awazu, S., Azumi, K., Boore, J., Branno, M., Chin-bow, S., DeSantis, R., Doyle, S., Francino, P., Keys, D. N., Haga, S., Hayashi, H., Hino, K., Imai, K. S., Inaba, K., Kano, S., Kobayashi, K., Kobayashi, M., Lee, B.-I., Makabe, K. W., Manohar, C., Matassi, G., Medina, M., Mochizuki, Y., Mount, S., Morishita, T., Miura, S., Nakayama, A., Nishizaka, S., Nomoto, H., Ohta, F., Oishi, K., Rigoutsos, I., Sano, M., Sasaki, A., Sasakura, Y., Shoguchi, E., Shin-i, T., Spagnuolo, A., Stainier, D., Suzuki, M. M., Tassy, O., Takatori, N., Tokuoka, M., Yagi, K., Yoshizaki, F., Wada, S., Zhang, C., Hyatt, P. D., Larimer, F., Detter, C., Doggett, N., Glavina, T., Hawkins, T., Richardson, P., Lucas, S., Kohara, Y., Levine, M., Satoh, N., and Rokhsar, D. S. (2002). The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins. Science 298, 2157–2160. Dietrich, J., Nakajima, H., and Colonna, M. (2000). Human inhibitory and activating Ig-like receptor which modulates the function of myeloid cells. Microbes. Infect. 2, 323–329.

novel immune mechanisms

233

Dimopoulos, G., Christophides, G. K., Meister, S., Schultz, J., White, K. P., Barillas-Mury, C., and Kafatos, F. C. (2002). Genome expression analysis of Anopheles gambiae: Responses to injury, bacterial challenge, and malaria infection. Proc. Natl. Acad. Sci. USA 99, 8814–8819. Donelson, J. E. (2003). Antigenic variation and the African trypanosome genome. Acta Tropica. 85, 391–404. Dziarski, R. (2003). Peptidoglycan recognition proteins (PGRPs). Mol. Immunol. 40, 877–886. Finstad, J., and Good, R. A. (1964). The evolution of the immune response. III. Immunologic responses in the lamprey. J. Exp. Med. 120, 1151–1168. Fuhrman, J. (1999). Marine viruses and their biogeochemical and ecological effects. Nature 399, 541–548. Good, R.A., Finstad, J. and Litman, G.W. (1972). ‘‘Immunology,’’ London, Academic Press, pp. 405–432. Hawke, N. A., Yoder, J. A., Haire, R. N., Mueller, M. G., Litman, R. T., Miracle, A. L., Stuge, T., Shen, L., Miller, N., and Litman, G. W. (2001). Extraordinary variation in a diversified family of immune-type receptor genes. Proc. Natl. Acad. Sci. USA 98, 13832–13837. Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S., Lipford, G., and Wagner, H. (2004). Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303, 1526–1529. Hernandez Prada, J. A., Haire, R. N., Cannon, J. P., Litman, G. W., and Ostrov, D. A. (2004). Crystallization and preliminary x-ray analysis of VCBP3 from Branchiostoma floridae. Acta Crystallogr. D. Biol. Crystallogr. 60, 2022–2024. Hiom, K., Melek, M., and Gellert, M. (1998). DNA transposition by the RAG1 and RAG2 proteins: A possible source of oncogenic translocations. Cell 94, 463–470. Hoffmann, J. A. (2003). The immune response of Drosophila. Nature 426, 33–38. Hoffmann, J. A., and Reichhart, J.-M. (2002). Drosophila innate immunity: An evolutionary perspective. Nature Immunol. 3, 121–126. Hornef, M. W., Pu¨ tsep, K., Karlsson, J., Refai, E., and Andersson, M. (2004). Increased diversity of intestinal antimicrobial peptides by covalent dimer formation. Nat. Immunol. 5, 836–843. Huttner, K. M., Selsted, M. E., and Ouellette, A. J. (1994). Structure and diversity of the murine cryptdin gene family. Genomics 19, 448–453. Jung, D., and Alt, F. W. (2004). Unraveling V(D)J recombination; insights into gene regulation. Cell 116, 299–311. Khakoo, S. I., Rajalingam, R., Shum, B. P., Weidenbach, K., Flodin, L., Muir, D. G., Canavez, F., Cooper, S. L., Valiante, N. M., Lanier, L. L., and Parham, P. (2000). Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans. Immunity 12, 687–698. Kilpatrick, D. C. (2002). Animal lectins: A historical introduction and overview. Biochimica. et. Biophysica. Acta 2002, 187–197. Knight, K. L., and Crane, M. A. (1994). Generating the antibody response in rabbit. Adv. Immunol. 56, 179–218. Kokubu, F., Litman, R., Shamblott, M. J., Hinds, K., and Litman, G. W. (1988). Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate. EMBO J. 7, 3413–3422. Kurtz, J., and Franz, K. (2003). Evidence for memory in invertebrate immunity. Nature 425, 37–38. Lazzaro, B. P., Sceurman, B. K., and Clark, A. G. (2004). Genetic basis of natural variation in D. melanogaster antibacterial immunity. Science 303, 1873–1876. Lebestky, T., Chang, T., Hartenstein, V., and Banerjee, U. (2000). Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science 288, 146–149.

234

g a ry w. li tm an

E T AL .

Lee, S. Y., and So¨ derha¨ ll, K. (2001). Characterization of a pattern recognition protein, a masquerade-like protein, in the freshwater crayfish Pacifastacus leniusculus. J. Immunol. 166, 7319–7326. Lehrer, R. I., and Ganz, T. (2002). Defensins of vertebrate animals. Curr. Opin. Immunol. 14, 96–102. Leonard, P. M., Adema, C. M., Zhang, S.-M., and Loker, E. S. (2001). Structure of two FREP genes that combine IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata. Gene 269, 155–165. Litman, G. W., Anderson, M. K., and Rast, J. P. (1999). Evolution of antigen binding receptors. Ann. Rev. Immunol. 17, 109–147. Litman, G. W., Hawke, N. A., and Yoder, J. A. (2001). Novel immune-type receptor genes. Immunol. Rev. 181, 250–259. Loker, E. S., Adema, C. M., Zhang, S.-M., and Kepler, T. B. (2004). Invertebrate immune systems not homogeneous, not simple, not well understood. Immunol. Rev. 198, 10–24. Maxwell, A. I., Morrison, G. M., and Dorin, J. R. (2003). Rapid sequence divergence in mammalian b-defensins by adaptive evolution. Mol. Immunol. 40, 413–421. Mayer, W. E., Uinuk-Ool, T., Tichy, H., Gartland, L. A., Klein, J., and Cooper, M. D. (2002). Isolation and characterization of lymphocyte-like cells from a lamprey. Proc. Natl. Acad. Sci. USA 99, 14350–14355. McDowell, J. M., and Woffenden, B. J. (2003). Plant disease resistance genes: Recent insights and potential applications. Trends Biotechnol. 21, 178–183. Medzhitov, R. (2001). Toll-like receptors and innate immunity. Nature Rev. 1, 135–145. Melchers, F., ten Boekel, E., Seidl, T., Kong, X. C., Yamagami, T., Ohnishi, K., Shimizu, T., Rolink, A. G., and Andersson, J. (2000). Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells. Immunol. Rev. 175, 33–46. Millet, A. C., and Ewbank, J. J. (2004). Immunity in Caenorhabditis elegans. Curr. Opin. Immunol. 16, 4–9. Nicholas, H. R., and Hodgkin, J. (2004). Responses to infection and possible recognition strategies in the innate immune system of Caenorhabditis elegans. Mol. Immunol. 41, 479–493. Ohnishi, K., and Melchers, F. (2003). The nonimmunoglobulin portion of l5 mediates cellautonomous pre-B cell receptor signaling. Nat. Immunol. 4, 849–856. Pancer, Z. (2000). Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin. Proc. Natl. Acad. Sci. USA 97, 13156–13161. Pancer, Z., Amemiya, C. T., Ehrhardt, R. A., Ceitlin, J., Gartland, G. L., and Cooper, M. D. (2004a). Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 430, 174–180. Pancer, Z., Mayer, W. E., Klein, J., and Cooper, M. D. (2004b). Prototypic T cell receptor and CD4-like coreceptor are expressed by lymphocytes in the agnathan sea lamprey. Proc. Natl. Acad. Sci. USA 101, 13273–13278. Pancer, Z., Rast, J. P., and Davidson, E. H. (1999). Origins of immunity: Transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes. Immunogenetics 49, 773–786. Pancer, Z., Skorokhod, A., Blumbach, B., and Muller, W. E. G. (1998). Multiple Ig-like featuring genes divergent within and among individuals of the marine sponge Geodia cydonium. Gene 207, 227–233. Pende, D., Parolini, S., Pessino, A., Sivori, S., Augugliaro, R., Morelli, L., Marcenaro, E., Accame, L., Malaspina, A., Biassoni, R., Bottino, C., Moretta, L., and Moretta, A. (1999). Identification

novel immune mechanisms

235

and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J. Exp. Med. 190, 1505–1516. Rast, J. P., Anderson, M. K., Strong, S. J., Luer, C., Litman, R. T., and Litman, G. W. (1997). a, b, g, and d T cell antigen receptor genes arose early in vertebrate phylogeny. Immunity 6, 1–11. Rast, J. P., Haire, R. N., Litman, R. T., Pross, S., and Litman, G. W. (1995). Identification and characterization of T-cell antigen receptor related genes in phylogenetically diverse vertebrate species. Immunogenetics 42, 204–212. Rast, J. P., Pancer, Z., and Davidson, E. H. (2000). New approaches towards an understanding of deuterostome immunity. Curr. Top. Microbiol. Immunol. 248, 3–16. Reynaud, C. A., Bertocci, B., Dahan, A., and Weill, J. C. (1994). Formation of the chicken B-cell repertoire - ontogeny, regulation of Ig gene rearrangement, and diversification by gene conversion. Adv. Immunol. 57, 353–378. Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000). Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101, 671–684. Shen, L., Stuge, T. B., Bengten, E., Wilson, M., Chinchar, V. G., Naftel, J. P., Bernanke, J. M., Clem, L. W., and Miller, N. W. (2004). Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus). Dev. Comp. Immunol. 28, 139–152. Shintani, S., Terzic, J., Sato, A., Saraga-Babic, M., O’hUigin, C., Tichy, H., and Klein, J. (2000). Do lampreys have lymphocytes? The Spi evidence. Proc. Natl. Acad. Sci. USA 97, 7417–7422. Smirnova, I., Hamblin, M. T., McBride, C., Beutler, B., and Di Rienzo, A. (2001). Excess of rare amino acid polymorphisms in the Toll-like receptor 4 in humans. Genetics 158, 1657–1664. Smirnova, I., Poltorak, a., Chan, E. K. L., McBride, C., and Beutler, B. (2000). Phylogenetic variation and polymorphism at the Toll-like receptor 4 locus (TLR4). Genome. Biology 1, 002.1–002.10. Strong, S. J., Mueller, M. G., Litman, R. T., Hawke, N. A., Haire, R. N., Miracle, A. L., Rast, J. P., Amemiya, C. T., and Litman, G. W. (1999). A novel multigene family encodes diversified variable regions. Proc. Natl. Acad. Sci. USA 96, 15080–15085. Suzuki, T., Shin-i, T., Kohara, Y., and Kasahara, M. (2004). Transcriptome analysis of hagfish leukocytes: A framework for understanding the immune system of jawless fishes. Dev. Comp. Immunol. 28, 993–1003. Suzuki, Y., Tasumi, S., Tsutsui, S., Okamoto, M., and Suetake, H. (2003). Molecular diversity of skin mucus lectins in fish. Comp. Biochem. Physiol. 136, 723–730. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302, 575–581. Trowsdale, J. (2001). Genetic and functional relationships between MHC and NK receptor genes. Immunity 15, 363–374. Uinuk-Ool, T., Mayer, W. E., Sato, A., Dongak, R., Cooper, M. D., and Klein, J. (2002). Lamprey lymphocyte-like cells express homologs of genes involved in immunologically relevant activities of mammalian lymphocytes. Proc. Natl. Acad. Sci. USA 99, 14356–14361. Vasta, G. R., Ahmed, H., and Odom, E. W. (2004). Structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates. Curr. Opin. Struct. Biol. 14, 617–630. Vilches, C., and Parham, P. (2002). KIR: Diverse, rapidly evolving receptors of innate and adaptive immunity. Ann. Rev. Immunol. 20, 217–251. Waltzer, L., Bataille, L., Peyrefitte, S., and Haenlin, M. (2002). Two isoforms of Serpent containing either one or two GATA zinc fingers have different roles in Drosophila haematopoiesis. EMBO J. 21, 5477–5486.

236

g a ry w. li tm an

E T AL .

White, S. N., Taylor, K. H., Abbey, C. A., Gill, C. A., and Womack, J. E. (2003). Haplotype variation in bovine Toll-like receptor 4 and computational prediction of a positively selected ligandbinding domain. Proc. Natl. Acad. Sci. USA 100, 10364–10369. Wojtowicz, W. M., Flanagan, J. J., Millard, S. S., Zipursky, S. L., and Clemens, J. C. (2004). Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding. Cell 118, 619–633. Wright, G. J., Puklavec, M. J., Wilson, A. C., Hoek, R. M., Sedgwick, J. D., Brown, M. H., and Barclay, A. N. (2000). Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity 13, 233–242. Wyllie, D. H., Kiss-Toth, E., Visintin, A., Smith, S. C., Boussouf, S., Segal, D. M., Duff, G. W., and Dower, S. K. (2000). Evidence for an accessory protein function for Toll-like receptor 1 in antibacterial responses. J. Immunol. 165, 7125–7132. Yoder, J. A., Litman, R. T., Mueller, M. G., Desai, S., Dobrinski, K. P., Montgomery, J. S., Buzzeo, M. P., Amemiya, C. T., Trede, N. S., Wei, S., Djeu, J. Y., Humphray, S., Jekosch, K., Hernandez Prada, J. A., Ostrov, D. A., and Litman, G. W. (2004). Resolution of the NITR gene cluster in zebrafish. Proc. Natl. Acad. Sci. USA 101, 15706–15711. Yoder, J. A., Mueller, M. G., Wei, S., Corliss, B. C., Prather, D. M., Willis, T., Litman, R. T., Djeu, J. Y., and Litman, G. W. (2001). Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian lymphocyte receptor cluster. Proc. Natl. Acad. Sci. USA 98, 6771–6776. Young, J. A. T., and Dillin, A. (2004). MAPping innate immunity. Proc. Natl. Acad. Sci. USA 101, 12781–12782. Zhang, S.-M., Adema, C. M., Kepler, T. B., and Loker, E. S. (2004). Diversification of Ig superfamily genes in an invertebrate. Science 305, 251–254. Zhang, S.-M., Leonard, P. M., Adema, C. M., and Loker, E. S. (2001). Parasite-responsive IgSF members in the snail Biomphalaria glabrata: Characterization of novel genes with tandemly arranged IgSF domains and a fibrinogen domain. Immunogenetics 53, 684–694. Zhang, S.-M., and Loker, E. S. (2004). Representation of an immune responsive gene family encoding fibrinogen-related proteins in the freshwater mollusc Biomphalaria glabrata, an intermediate host for Schistosoma mansoni. Gene 341, 255–266.

The Repair of DNA Damages/Modifications During the Maturation of the Immune System: Lessons from Human Primary Immunodeficiency Disorders and Animal Models Patrick Revy,* Dietke Buck,* Franc¸oise le Deist,*,{ and Jean-Pierre de Villartay*,{ *De´veloppement Normal et Pathologique du Syste`me Immunitaire, INSERM U429, Hoˆpital Necker, Paris, France { Assistance Publique–Hoˆpitaux de Paris (AP/HP), Paris, France Abstract............................................................................................................. 1. Introduction ....................................................................................................... 2. Fundamental Mechanisms of Lymphoid-Specific DNA Cleavage and Repair Mechanisms ....................................................................................... 3. Human Primary Immunodeficiency Disorders Associated with Defective DNA Repair..... 4. Human Primary Immunodeficiency Disorders Associated with Defective Cell Cycle Control Following DNA Damage ........................................................................... 5. Defective DNA Repair and Malignancies in the Immune System ................................. 6. Conclusions........................................................................................................ References .........................................................................................................

237 238 240 257 268 274 278 279

Abstract The immune system is the site of various genotoxic stresses that occur during its maturation as well as during immune responses. These DNA lesions/modifications are primarily the consequences of specific physiological processes such as the V(D)J recombination, the immunoglobulin class switch recombination (CSR), and the generation of somatic hypermutations (SHMs) within Ig variable domains. The DNA lesions can be introduced either by specific factors (RAG1 and RAG2 in the case of V(D)J recombination and AID in the case of CSR and SHM) or during the various phases of cellular proliferation and cellular activation. All these DNA lesions are taken care of by the diverse DNA repair machineries of the cell. Several animal models as well as human conditions have established the critical importance of these DNA lesions/modifications and their repair in the physiology of the immune system. Indeed their defects have consequences ranging from immune deficiency to development of immune malignancy. The survey of human pathology has been highly instrumental in the past in identifying key factors involved in the generation of DNA modifications (AID for the Ig CSR and generation of SHM) or the repair of specific DNA damages (Artemis for V(D)J recombination). Defects in factors involved in the cell cycle checkpoints following DNA damage also have deleterious consequences on the immune system. The continuous survey of human diseases characterized by primary immunodeficiency

237 advances in immunology, vol. 87 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)87007-5

238

pat ri ck r ev y

ET AL.

associated with increased sensitivity to ionizing radiation should help identify other important DNA repair factors essential for the development and maintenance of the immune system. 1. Introduction The immune system is the site of intense DNA damage and/or modifications, which result from programmed and specific mechanisms at play during its maturation, or as a consequence of nonspecific injuries inflicted during cellular proliferation and/or cellular activation (Fig. 1). The V(D)J recombination is a somatic DNA rearrangement process common to both B and T lymphocytes that is required for the ‘‘construction’’ of functional immunoglobulin (Ig) and T-cell receptor (TCR) genes (Tonegawa, 1983). This process is initiated by the lymphoid-specific RAG1 and RAG2 proteins through the specific generation of a DNA double strand break (DNA-dsb), a particularly harmful form of DNA damage (Bassing et al., 2002; Brandt and Roth, 2002; Gellert, 2002). The terminal maturation of B-lymphocytes, which occurs during an immune response in the germinal center of secondary lymphoid organs such as the spleen, is characterized by two important modifications of the rearranged immunoglobulin genes (Durandy et al., 2004). The isotype CSR and the generation of SHM ensure the production of efficient antibodies of various isotypes. These two B-cell-specific processes are triggered by the activationinduced cytidine deaminase (AID) factor through DNA modification within Ig genes (see Section 2.2, 2.3). Beside these three specific DNA-altering mechanisms, B and T lymphocytes are also exposed to general DNA injuries known to occur for example during DNA replication, as several waves of intense cellular proliferation accompany not only their maturation, but their expansion during immune responses. Lastly, one important aspect of an immune response relies on the inflammatory reaction during which several soluble factors and/or natural reactive metabolites are produced that can be considered as possible environmental causes of DNA damage. Altogether, this demonstrates that the lymphoid tissue is at particular high risk for mutagenic events inflicted through defective DNA repair machineries and, as such, represents a model of choice for the study of DNA repair mechanisms and pathways. DNA damage, regardless of its originating causes, activates a series of ordered and specific biochemical pathways, which involve separate groups of protein complexes (Sancar et al., 2004). Very briefly, the first phenotypic response of the injured cell to DNA damage is an arrest from cycling, whether the damage occurred in the G1, S, or G2 phase of the cell cycle. To accomplish these various checkpoints, the lesion is first recognized by specific DNA damage ‘‘sensors,’’ such as the ATM or ATR proteins, which trigger a specific

d na r e pa i r i n t h e i m m un e sy s t e m

239

Figure 1 Development and maturation of the immune system. Lymphocytes develop into three distinct cell lineages (B, T, and NK) from a putative common lymphoid progenitor (CLP) derived from the hematopoietic stem cell (HSC). DNA damages/modifications can occur during V(D)J recombination (B and T lymphocytes), CSR and SHM (Mature B lymphocytes) and possibly as a consequence of cell proliferation/activation in the three lineages.

activation cascade through the phosphorylation of DNA repair complexes (such as the Mre11/Rad50/Nbs1 or MRN complex), or ‘‘mediator’’ factors (e.g., BRCA1, H2AX, 53BP1, MDC1) leading to the activation of the two ‘‘transducer’’ kinases—Chk1 and Chk2. These two kinases will then phosphorylate the ‘‘effectors’’ P53 and Cdc25, directly responsible for cell cycle arrest. Once the extent of the DNA damage has been evaluated, two options can be followed: apoptosis if the cell is too heavily injured, or DNA repair if the cell can accommodate it. Depending on the DNA lesion, different DNA repair

240

pat ri ck r ev y

ET AL.

machineries are recruited. They include DNA double strand break repair by homologous recombination (HR) or nonhomologous end joining (NHEJ), base excision repair (BER), or nucleotide excision repair (NER). The reality is of course much more complicated than this oversimplified summary because some factors, depending on the multiprotein complexes they are recruited in, may participate in several steps of the DNA repair cascade. As we will see in this review, several of these mechanisms are at play in the immune system. Regardless of the DNA damage considered, or the DNA repair machinery employed to fix it, an incapacity to proceed with the repair of these damages will ultimately lead to genomic instability and its deleterious consequences such as tumor development. In the specific case of the immune system, this also leads to various degrees of immunodeficiency. Because our laboratory is historically inclined in the study of the various aspects of the immune system through the analysis of immune deficiency conditions in humans, we got involved in the field of ‘‘DNA repair and mutagenesis’’ through the analyses of a subgroup of these patients. Our review will therefore concentrate on the recent advances obtained pertaining to the understanding of DNA repair in relation with the lymphoid tissue. It is therefore evident that we will not entirely cover the field of DNA repair. We will first introduce the various lymphoid‐specific mechanisms resulting in programmed DNA damage/modifications and review the knowledge gained through the analysis of various human and animal models impaired in their ability to take care of these damages. We will then discuss a particular aspect of DNA repair represented by the controls of cell cycle checkpoints following DNA damage, the defects of which also lead to notable consequences on the immune system. Finally, we will discuss the impact that defects in DNA repair factors have on the onset of malignancies within the lymphoid tissue. 2. Fundamental Mechanisms of Lymphoid-Specific DNA Cleavage and Repair Mechanisms 2.1. V(D)J Recombination The vertebrate immune system has to face a virtually infinite number and variety of foreign antigens. While innate immunity provides the first line of defense against pathogens, the adaptive immune response plays a crucial role by generating an immune repertoire of soluble and membrane-bound antigen receptors expressed on B and T lymphocytes, respectively. Ig and TCR are composed of an invariant constant region (C), which mediates effector functions, linked to a polymorphic variable domain responsible for the

d na r e pa i r i n t h e i m m un e sy s t e m

241

Figure 2 Rearrangement and expression of TCR and Ig genes. (A) Ig and TCR loci are composed of several gene clusters encoding the Variable (V), diversity (D), and junction (J) segments of the variable domain scattered along the chromosome. A specific somatic DNA recombination process, which assembles a V, a D, and a J segment in a productive rearranged V(D)J unit is a prerequisite for the expression of an Ig or TCR encoding mRNA. The V(D)J unit is further spliced to the constant region (C). (B) the V(D)J recombination reaction is initiated by the recognition of recombination signal sequences (RSS) that flank all V, D, and J gene segments. RSS are composed of evolutionary conserved heptamers and A/T rich nonamers separated by variable spacer DNA sequences of either 12 or 23 bp. Recombination always occurs between segments having 12 and 23 bp spacers respectively (the 12/23 rule).

antigenic recognition. The variable domain is encoded by separate subgenic segments (Variable [V], Diversity [D], and Joining [J]) arranged in separate arrays on the chromosome. The V(D)J recombination is a site-specific DNA rearrangement process, which by a simple cut-and-paste reaction fuses one of each of these three subgenic segments to form a functional V(D)J rearranged exon (Fig. 2A) (Tonegawa, 1983). As virtually any V segment can rearrange to any D and J region, the purpose of this specialized mechanism is the generation of an immune repertoire as diversified as possible through combinatorial association. Each of the V, D, and J gene segments are flanked by Recombination Specific Sequences (RSS) composed of a highly conserved

242

pat ri ck r ev y

ET AL.

Figure 3 V(D)J recombination. The V(D)J recombination reaction can be divided into three steps. The lymphoid specific recombination activating genes RAG1 and RAG2 initiate the reaction by introducing a DNA‐dsb at the border of the RSS, leaving hairpin‐sealed coding ends and blunt signal ends excised from the chromosome. The subsequent phases are meant to repair this DNA lesion through nonhomologous end joining (NHEJ) DNA repair pathway. During the second phase the Ku70‐80/DNA‐PK complex recognizes the lesion and signals it to the DNA repair machinery. DNA‐PKcs recruits and phosphorylates Artemis, activating its DNA endonuclease activity required for hairpin opening. The XRCC4/DNA‐LigaseIV complex finally repairs the DNA‐dsb. Although all these factors have been demonstrated to be critically required for V(D)J recombination, several recent studies indicate that additional factors are most certainly necessary to complete the reaction.

palindromic heptamer, which is separated from an A/T-rich nonamer by a spacer DNA sequence of either 12bp or 23bp (Fig. 2B). Rearrangements occur exclusively between segments, which are flanked by RSSs of different spacer length, a restriction known as the ‘‘12/23 rule’’ (Hiom and Gellert, 1998). The V(D)J recombination reaction can be roughly divided into three steps (Fig. 3), the initiation of which being specific to immature B and T lymphocytes. 2.1.1. Initiation of the Reaction: Introducing a DNA Double Strand Break The initiation of the V(D)J recombination is performed by the products of the recombination activating genes RAG1 and RAG2 (Oettinger et al., 1990; Schatz et al., 1989), whose lymphoid restricted expression renders this phase specific to immature lymphocytes. The RAG1 and RAG2 proteins are necessary and sufficient to initiate V(D)J rearrangement of accessible

d na r e pa i r i n t h e i m m un e sy s t e m

243

antigen-receptor loci, although high mobility (HMG) proteins facilitate their action (Bassing et al., 2002; Brandt and Roth, 2002; Gellert, 2002). As a result of the development of cell-free reaction systems, this step has been investigated extensively. The RAG1/2 proteins guide recombination events to RSS and introduce a DNA single strand break (nick) at the boundary of the heptamer and the adjacent coding segment. The resulting 30 OH then performs a nucleophilic attack on the phosphodiester bond on the opposite strand. Subsequent to this transesterification reaction, four DNA intermediates are produced: two hairpin-sealed coding ends on the broken chromosome and two blunt 50 -phosphorylated signal ends excised from the chromosome (McBlane et al., 1995; Roth et al., 1992; Schlissel et al., 1993). The RAG1/2 proteins remain bound to the generated coding and signal ends in a ‘‘post-cleavage complex,’’ which possibly stabilizes and directs the adequate joining of the cleaved ends in the following steps (see Section 5.2). The RAG postcleavage complex may ensure avoiding inappropriate chromosomal rearrangements seen in immune malignancies, as recently proposed by D. Roth (Lee et al., 2004). The critical role of both RAG1 and RAG2 during V(D)J recombination, and more generally in the development of the immune system, was first demonstrated by the lack of mature B and T cells in mice with a targeted inactivation of either ones (Mombaerts et al., 1992; Shinkai et al., 1992). This was further confirmed by the analysis of human severe combined immunodeficiency (SCID) conditions (see Section 3). 2.1.2. Identifying and Resealing the Break Two major DNA repair pathways are utilized by mammalian cells to repair DNA injuries (Haber, 2000; Sancar et al., 2004). The homologous recombination (HR) is a precise mechanism that repairs the damage by copying the DNA on the noninjured homologous DNA template. The nonhomologous end-joining (NHEJ) pathway is homology independent and imprecise. Whereas HR is mainly used in yeast, NHEJ is the principal DNA repair pathway in mammalian cells and is the one required to repair the RAG1/2 generated DNA-dsb during V(D)J recombination. The first link between V(D)J recombination and DNA repair came from the study of the severe combined immunodeficient (scid) mice, identified in 1980 as a natural mutant strain lacking B and T lymphocytes (Bosma and Carroll, 1991). Several studies demonstrated an increased sensitivity of cells from these mice to ionizing radiations as a result of a general defect in DNA-dsb repair (Biedermann et al., 1991; Fulop and Phillips, 1990; Hendrickson et al., 1991). During V(D)J recombination, this defect translates into an incapacity to reseal the RAG1/2 generated DNA-dsb, leading to an accumulation of hairpin-sealed coding ends (Roth

244

pat ri ck r ev y

ET AL.

et al., 1992). Additional evidence for the functional overlap between the general DNA repair machinery of the cell and the V(D)J recombination process was provided by the characterization of other mutants that display defects in both V (D)J recombination and DNA-dsb repair (Pergola et al., 1993; Taccioli et al., 1993). A large panel of x-ray sensitive Chinese hamster ovary (CHO) cell lines was tested for its ability to perform V(D)J recombination on extrachromosomal substrates after the introduction of the RAG1/2 genes. Four ‘‘x-ray cross-complementation groups’’ (termed XRCC4 through XRCC7) of cells were identified that displayed not only impaired repair of radiation-induced DNA-dsb, but also showed defects in V(D)J recombination. Except for murine scid (XRCC7), all mutants are defective in both signal and coding joint formation. The XRCC5, XRCC6, and XRCC7 factors define the DNA-PKcs/Ku70/Ku80 complex (review in (Meek et al., 2004)). The Ku heterodimer is composed of 70-kDa and 86kDa subunits and is able to bind to altered DNA structures such as broken ends, single stranded gaps, and hairpins. Ku possesses helicase activity and functions as the DNA-binding component within the DNA-dependent protein kinase (DNA-PK) complex. The group of XRCC7 mutants, including the murine scid, lacks DNA-PK activity. The murine scid is caused by an intragenic mutation at the 30 end of the DNA-PK catalytic subunit (DNA-PKcs) encoding gene, which results in the weak expression of a truncated, catalytically inactive protein (Blunt et al., 1996; Boubnov and Weaver, 1995; Kirchgessner et al., 1995). DNAPKcs gene targeting in mice confirmed that DNA-PKcs is critical for codingjoint formation but dispensable for signal-joint formation (Gao et al., 1998; Taccioli et al., 1998). The defect in XRCC4, a 38 kDa nuclear phosphoprotein, was identified in the x-ray sensitive CHO cell line XR1 via a complementation cloning method (Li et al., 1995). XRCC4 interacts with (Critchlow et al., 1997; Grawunder et al., 1998a), and stabilizes (Bryans et al., 1999) the DNALigaseIV enzyme. The XRCC4/DNA-LigaseIV complex plays a major role during NHEJ both in yeast and in mammalian cells. Gene targeting of either one of these NHEJ factors in mice ultimately results in immunodeficient animals devoid of mature B and T lymhocytes owing to a major defect in the V(D)J recombination process (review in (de Villartay et al., 2003)). Beside this general presentation, these mice display quite different phenotypes with regard to cellular proliferation and neuronal development. While targeted inactivation of Ku70, Ku80, or DNA-PKcs results in viable animals, mice lacking XRCC4 or DNA-LigaseIV die at late stages of embryogenesis because of extensive apoptotic death of newly generated postmitotic neurons. As discussed in Section 3.1.2., this last characteristic is of particular importance when surveying human conditions for possible molecular defects in one of these two genes.

d na r e pa i r i n t h e i m m un e sy s t e m

245

2.1.3. Artemis DNA-PKcs deficient situations (scid model or KO mice) differ from the other NHEJ-deficient mutants in that the V(D)J recombination signal joint formation is not (or poorly) affected, a characteristic also expressed by cells from human radiosensitive SCID (RS-SCID) patients (Nicolas et al., 1998) from which the Artemis gene was identified (see Section 3.1.1 for a presentation of RS-SCID patients) as well as Artemis-deficient murine cells (Rooney et al., 2002, 2003). Like other NHEJ mutant animals, Artemisdeficient human patients and mice are devoid of mature B and T lymphocytes, and their cells harbor increased sensitivity to ionizing radiations caused by a general defect in DNA-dsb repair machinery. The major difference between coding- and signal-joint formation being the prerequisite for hairpin opening before DNA re-ligation in the former, it was hypothesized early on that Artemis and DNA-PKcs may be specifically involved in coding-joint formation during V(D)J recombination, possibly through the necessary hairpin opening activity (Moshous et al., 2001). Artemis does in fact interact with DNA-PKcs in vitro and in vivo and, in these circumstances, exerts a DNA endonuclease activity capable of processing 50 and 30 DNA single strand overhangs as well as opening RAG1/2 generated hairpins at coding ends (Ma et al., 2002). The predominant site of hairpin opening occurs 30 to the tip and leads to the generation of a 30 overhang. This observation is reminiscent of early work by M. Schlissel showing that nonhairpin coding ends recovered from lymphoid progenitors exhibit 30 overhangs (Schlissel, 1998). On its own (i.e., in the absence of DNA-PKcs), Artemis possesses a 50 to 30 DNA exonuclease activity (Ma et al., 2002). The role of ‘‘hairpin opener’’ for Artemis was further reinforced by the observation of hairpin accumulation in thymocytes from Artemis KO mice (Rooney et al., 2002), similar to the situation previously described in DNA-PKcs deficient scid mice (Roth et al., 1992). It is worth noting that in the absence of Artemis, some alternative factors may be capable of hairpin opening activity on coding ends as suggested by both the leakiness observed in some Artemis KO mice (Rooney et al., 2002) and the residual V(D)J coding-joint formation recently demonstrated in fibroblasts from human Artemis deficient RS-SCID patients (Poinsignon et al., 2004b). Several candidates (the RAG themselves and Mre11) have been proposed in the past to accomplish this task (as discussed in (Schlissel, 2002)). Artemis is a nuclear protein of 78kDa encoded for by 692 amino acids (Fig. 4) (Moshous et al., 2001). The entire cDNA sequence comprises 2354bp. The genomic organization of the human Artemis gene on the short arm of chromosome 10 shows the presence of 14 exons, an organization conserved in the murine Artemis gene. Artemis is ubiquitously expressed,

246

pat ri ck r ev y

ET AL.

Figure 4 Artemis is a metallo‐b‐lactamase enzyme. The association of the metallo‐b‐lactamase homology domain (bLact) and the bCASP region constitutes the catalytic core of Artemis. (A) The bLact domain of Artemis contains four of the five conserved residues defining the signature of this protein family. The fifth catalytic residue is present in the evolutionary conserved bCASP region, always appended to the bLact in proteins of this family involved in the processing of nucleic acids (DNA repair or RNA processing). (B) In vitro mutagenesis studies demonstrated the absolute requirement of these conserved residues for the full Artemis endonuclease activity required for V(D)J recombination. The loss of function in these mutants is not the result of an absence of interaction or phosphorylation of Artemis with DNA‐PKcs.

and no overexpression was detected in the sites of V(D)J recombination (bone marrow and thymus) or meiotic recombination (ovary, testis). Moreover, Artemis KO mice are fertile, which precludes any essential role for Artemis during meiosis (Rooney et al., 2002). Close sequence analysis of the Artemis protein suggested the presence of three regions. The first 155 amino acids encoded for by exons 1 to 6 present a loose although significant homology with proteins of the metallo-b-lactamase family (Pfam00753), which was confirmed through secondary structure prediction by hydrophobic cluster analysis (Callebaut et al., 2002). In particular, four out of the five highly conserved anchor residues that define the metallo-b-lactamase signature are present in Artemis (Fig. 4). This signature is mainly composed of aspartic acid and histidine residues critical for the catalytic active site of this class of enzyme. Metallo-b-lactamases (also known as class B b-lactamases) were first identified in bacteria where they are responsible for the degradation of certain antibiotics through the cleavage of their b-lactam ring. The metallo-b-lactamase fold was subsequently identified in a wide range of proteins in all living organisms (Aravind, 1999). No clear common characteristics exist among the broad spectrum of substrates for which they are specific except for an ester bond and a general negative charge. The structures of several of these prototype

d na r e pa i r i n t h e i m m un e sy s t e m

247

enzymes have been solved (Wang et al., 1999). It consists of a four-layered b-sandwich with two mixed b-sheets flanked by a-helices, with a catalytic site constituted of two metal-binding sites located at one edge of the b–sandwich. The binuclear Zn(II) center, used to perform the cleavage reaction, is located at the bottom of a wide shallow groove. The fifth anchor residue that defines the metallo-b-lactamase signature is sometimes difficult to identify precisely as it can be separated from the others on the primary protein sequence by a spacer of various length (Aravind, 1999; Daiyasu et al., 2001). In searching for this motif in the Artemis sequence, a new region was identified (Fig. 4, residues 155–385) that is always appended to the metallo-b-lactamase domain in a particular group of proteins that includes the cleavage and polyadenylation factor of RNA (CPSF), the murine SNM1 DNA repair protein and its yeast homolog PSO2, and for this reason was designated b-CASP (Callebaut et al., 2002). Of note, metallo-b-lactamase domain containing proteins with a b-CASP domain appear to have in common involvement in the metabolism of nucleic acids (RNA processing and DNA repair) and thus define a particular entity within this class of enzymes. Recent evidences suggested that the blact/bCASP domain containing factor CPSF-73 indeed carries the 30 processing endonuclease activity of the multi-subunit CPSF complex (Ryan et al., 2004). As b-CASP does not exist as an individual domain, but is always appended to a metallo-blactamase region, it was proposed that both domains probably participate together in the building of the catalytic site in these enzymes (Callebaut et al., 2002). Two recent studies (Fig. 4B) confirmed the functional link between Artemis and metallo-b-lactamase enzymes by showing that in vitro directed mutagenesis of aspartic acids and histidines within the conserved metallo-blactamase signature strongly impairs the DNA endonuclease activity of Artemis in vitro and its activity in V(D)J recombination in vivo (Pannicke et al., 2004; Poinsignon et al., 2004b). This suggests that the structure of the Artemis catalytic pocket may resemble the one described for bacterial metallo-blactamases (Fig. 5). Surprisingly, however, the exonuclease activity is not affected in these mutants (Pannicke et al., 2004). These studies also established that the association of the metallo-b-lactamase and the b-CASP domains constitutes the minimal catalytic core of Artemis required for its activity during V(D)J recombination (Poinsignon et al., 2004b). If the role of Artemis during V(D)J recombination has been established, its precise function in more general aspects of DNA repair, such as following ionizing radiations, remains elusive. It is probably fair to say that, based on its demonstrated various nuclease activities, Artemis is probably involved in some sort of DNA ends processing required for accurate DNA double strand break repair. However, in contrast to other NHEJ factors (Ku70/80, DNA-PKcs, and XRCC4), Artemis is not required for transposition in mammalian cells using

248

pat ri ck r ev y

ET AL.

Figure 5 Hypothesis for the Artemis 3D structure. Hypothetical structure of the Artemis catalytic site based on homology with B. cereus and S. maltophilia metallo‐b‐lactamases and in vitro– directed mutagenesis studies.

the sleeping beauty element, which proceed through the generation of a DNA dsb during excision of the transposable element but not hairpin formation (Izsvak et al., 2004). The dissociation of function of Artemis in V(D)J recombination and irradiation-induced DNA damage noted through in vitro mutagenesis (Poinsignon et al., 2004b) is further strengthened by the very peculiar observation that Artemis-/- and DNA-PKcs-/- embryonic stem cells (ES), in contrast to murine embryonic fibroblasts (MEFs), are not overly sensitive to ionizing radiation, although both types of cells are profoundly V(D)J defective (Gao et al., 1998; Rooney et al., 2003). As another indication of its probable implication in general DNA-dsb repair, we recently found that Artemis is specifically phosphorylated in response to various DNA damages (Poinsignon et al., 2004a), a characteristic that is shared by many other known DNA repair factors. 2.2. Class Switch Recombination (CSR) During humoral responses, two major events leading to antibody diversification occur. These events correspond to two distinct DNA modification reactions during which DNA repair factors are required. These events are namely SHM and CSR. Somatic hypermutation consists of the introduction of mutations, mainly single nucleotide changes and more rarely insertions and deletions, into the variable region exons of Ig genes. SHM can generate immunoglobulins with high affinity for antigens and thus permits the positive selection of B cells bearing such Ig on their surface. The CSR lies in the replacement of the Ig gene portion coding for the constant region by another one (IgG, IgA, or IgE). This process is achieved by a DNA recombination event that joins two Ig switch regions (S) by deletion of intervening sequences

d na r e pa i r i n t h e i m m un e sy s t e m

249

Figure 6 Model for Ig CSR. (1) Transcription of Ig locus produces sterile transcripts that form R‐ loop structures with DNA from S region. (1A) AID targets both DNA strands of S regions and deaminates cytidines. (1B) UNG recognizes and deglycosylates uridines produced by AID, thus creating DNA abasic sites. Abasic sites are processed by an AP‐endonuclease, resulting in DNA nicks on both strands. (2) The DNA‐dsb created in S regions leads to the deletion of intervening sequences. (3) The DNA‐dsb is processed and ligated to produce the new isotype expressing Ig gene (here, IgE) and a CSR excision circle. Known factors as participating during the different steps are mentioned.

(Fig. 6). CSR enables Ig to change its effector function (i.e., localization, receptor, and complement interaction) without modifying its variable region, thus its antigen specificity. CSR and SHM are induced in a same spatial and temporal window (i.e., in germinal centers of secondary lymphoid organs after B-cell stimulation through antigen receptor [BCR] and CD40). Although CSR and SHM are induced in a same environment, they are not necessarily linked since some unswitched B cells (bearing IgM) carry out SHM in their V region, and conversely, switched B cells (bearing Ig of another isotype than IgM) can be unmutated in their V region (Jacob and Kelsoe, 1992; Kaartinen et al., 1983; Liu et al., 1996; Weller et al., 2004, 2001). 2.2.1. CSR Specific DNA-dsb (CSR-DNA-dsb) To complete CSR, S regions must pass through a DNA-dsb step (Wuerffel et al., 1997) initiated by a B-cell specific enzyme, activation-induced cytidine deaminase (AID), which is specifically expressed in activated B cells undergoing CSR (Muramatsu et al., 1999, 2000; Revy et al., 2000). AID is essential

250

pat ri ck r ev y

ET AL.

for CSR and SHM since B cells from AID-deficient patients and mice are unable to produce SHM and to carry out CSR (Muramatsu et al., 2000; Revy et al., 2000). Hence, AID deficiency leads to a hyper IgM syndrome (HIGM2) characterized by a normal or high amount of serum IgM, contrasting with the absence of Ig of other isotypes (G, A, E). Patients suffering from this syndrome are highly susceptible to bacterial infections and must be substituted by intravenous Ig. AID bears sequence similarity with the RNA-editing enzyme APOBEC-1. Although the precise role of AID is still controversial, some works propose a RNA-editing activity of AID on a yet unknown RNA substrate (Begum et al., 2004b; Doi et al., 2003; Honjo et al., 2004). However, increasing data support a DNA-editing activity of AID. Indeed, AID most likely acts directly on specific DNA regions (switch regions [S] for CSR) by modifying cytidine residues to uridine by a deamination process (Fig. 6) (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Nambu et al., 2003; Petersen-Mahrt et al., 2002; Pham et al., 2003; Sohail et al., 2003). The AID-dependent cytidine deamination requires transcription of the target regions (i.e., I exon, S region, and C exon). This transcription leads to the production of germline transcripts that interact with the DNA coding strand of S region, thus creating DNA/RNA hybrid structures named R-loops (Reaban and Griffin, 1990; Reaban et al., 1994; Shinkura et al., 2003; Yu et al., 2003). These stable structures displace the nontemplate G rich as a single strand DNA, which is located in S regions and represents a target for AID. AID modifies cytidine to uridine residues by its cytidine deaminase activity (Chaudhuri et al., 2003). Although AID preferentially modifies the G-rich single strand, the template strand can also be deaminated by AID but to a much lower extent (Chaudhuri et al., 2003; Dickerson et al., 2003; Pham et al., 2003; Ramiro et al., 2003; Sohail et al., 2003) (Fig. 6, step1A). Uridine residues introduced by AID in both strands of S regions are further processed into staggered DNA-dsb. If not tightly regulated, AID could be a wide DNA mutator due to its cytidine deaminase activity. Indeed, the constitutive and ubiquitous expression of AID in transgenic mice leads to the development of T-cell lymphomas and lung microadenomas (Okazaki et al., 2003). Physiologically, AID expression is confined to activated B cells, and its cellular localization also seems to be regulated. Recent studies demonstrated that AID possesses a nuclear export signal (NES) excluding it from the nucleus (Brar et al., 2004; Ito et al., 2004; McBride et al., 2004). One can thus hypothesize that AID is actively translocated to (or specifically retained in) the nucleus only when B cells are triggered to undergo CSR (and/or SHM), but the mechanism controlling this regulation is still unknown. Moreover, the efficiency of AID activity depends on its phosphorylation status, thus adding another level of complexity to its regulation (Chaudhuri et al., 2004).

d na r e pa i r i n t h e i m m un e sy s t e m

251

Although AID-induced cytidine deamination appears to be the first step of DNA alteration leading to CSR-specific DNA-dsb, other proteins are required. The uridine residues created by AID on both strands of S regions have to be modified to lead to DNA-dsb. The illegitimate uridine residues are recognized and removed, by the Uracil N glycosylase (UNG), thereby producing an abasic site (Fig. 6, steps 1B and 1C). The essential role of UNG in CSR has been demonstrated by the study of UNG knockout mice, which present a CSR defect (Rada et al., 2002). In humans, deleterious mutations in the UNG encoding gene lead to a block in CSR resulting in a hyper IgM syndrome (Imai et al., 2003b). Presumably, abasic sites produced by UNG are modified by an apyrimidinic endonuclease (APE), which creates a nick (SSB) (Fig. 6, steps 1B and 1C). The exact nature of the factor providing this endonuclease activity is still undefined. When nicks are created in the two strands of the same S region, it leads to DNA-dsb and thus allows CSR (Fig. 6, step 2). The absolute requirement for AID and UNG activities to produce CSR-specific DNA-dsb in Switch regions of Ig genes has been demonstrated in B cells from both AID-deficient and UNG-deficient patients and mice. Indeed, UNG- and AID-deficient activated B cells are unable to produce DNA-dsb in S regions (Catalan et al., 2003; Imai et al., 2003b; Petersen et al., 2001; Rush et al., 2004). However, the requirement of UNG activity for the production of CSR-induced DNA-dsb in vitro has recently been contested (Begum et al., 2004a). 2.2.2. CSR Specific DNA-dsb Resolution After the production of CSR-specific DNA-dsb, the opened DNA ends in the two S regions have to be ligated. The different stages of CSR-specific DNAdsb solving are not known. Peculiar AID mutations leading to the truncation of the last AID amino acids have been found in some hyper-IgM patients. Surprisingly, these mutations do not alter AID cytidine deaminase activity (and thus permit CSR-specific DNA-dsb), but cells are unable to carry out CSR to completion (Ta et al., 2003). This suggests that the C terminal region of AID might be a docking domain necessary to recruit specific cofactors involved in the repair of CSR-specific DNA-dsb. Moreover, a novel hyperIgM syndrome (HIGM4), very similar to AID deficiency has been described, in which the generation of CSR-specific DNA-dsb is not affected. One can imagine that HIGM4 is caused by the defect of an AID cofactor required to resolve these DNA-dsb (Imai et al., 2003a). 2.2.2.1. Homologous Recombination System (HR). The molecules specifically involved in DNA repair through homologous recombination, such as RAD51, RAD52, and RAD54, do not seem to be required for CSR. Although RAD51 is overexpressed in activated B cells undergoing CSR (Li et al., 1996;

252

pat ri ck r ev y

ET AL.

Peakman and Maizels, 1998), it is not recruited to the site of CSR-specific DNA-dsb (Petersen et al., 2001). Moreover, RAD54 knockout mice do not present a CSR defect (Bross et al., 2003). 2.2.2.2. Nonhomologous End Joining System (NHEJ). Some NHEJ factors are clearly involved in the resolution of CSR-specific DNA-dsb. Mice devoid of Ku70 or Ku80 do not produce B and T lymphocytes because of a V(D)J recombination defect (see Section 2.1.2). To reconstitute the B-cell compartment in Ku deficient mice, pre-rearranged Ig genes were introduced into the Ku deficient background. CSR induction is absent in such Ku70 or Ku80 deficient, Ig reconstituted, mice (Casellas et al., 1998; Manis et al., 1998; ReinaSan-Martin et al., 2003). The absence of CSR is caused by a post-cleavage DNA repair defect, since CSR-specific DNA-dsb are normally produced in activated B cells from these animals (Casellas et al., 1998). Concerning the role of DNA-PKcs in CSR, results are less clear. Scid mice expressing prerearranged Ig genes are able to class switch with efficiency similar to control animals (Bosma et al., 2002; Cook et al., 2003). In contrast, CSR is abolished except for IgG1 isotype in DNA-PKcs knockout mice (Manis et al., 2002). These studies suggest that the presence of DNA-PKcs, but not its kinase activity, is required during CSR. Even if not totally understood, the ability of DNA-PKcs knockout mice to undergo CSR to IgG1 may reflect structural differences in the IgG1 S region sequences compared to other S regions (Manis et al., 2002). 2.2.2.3. Mismatch Repair (MMR) Proteins. The mismatch repair system is critical to ensure genome integrity through the elimination of spontaneously occurring mutations. When base-pair mismatch happens, a cascade of DNA repair events takes place. Mismatch repair proteins (MLH1, PMS2, MSH2, MSH6) recognize the mismatched bases, and additional factors (PCNA, EXO1, DNA polymerase) are recruited to excise and restore the unmutated DNA sequence. The study of mice deficient in MSH2, MSH6, PMS2, or MLH1 revealed that these factors are required to carry out efficient CSR since the isotype switching is substantially reduced in these mice (Ehrenstein and Neuberger, 1999; Martin et al., 2003; Schrader et al., 1999). Similarly, mice deficient in Exo1, an exonuclease that interacts with MSH2 and MLH1, also have decreased CSR (Bardwell et al., 2004; Schmutte et al., 2001). Conversely, mice devoid of Msh3 can undergo CSR as efficiently as wildtype mice (Li et al., 2004; Martomo et al., 2004). 2.2.2.4. Error-Prone DNA Polymerases. Sequencing analysis of the S-S junctions produced by CSR has revealed the presence of deletions, duplications, and SHM-like mutations. It was proposed that protruding DNA ends created

d na r e pa i r i n t h e i m m un e sy s t e m

253

in S regions during CSR could be filled by error prone DNA polymerases to produce blunt-ended DNA-dsb before ligation (Chen et al., 2001). However, patients with xeroderma pigmentosum variant (XP-V) disease, whose errorprone DNA polymerase eta is defective, do not present a CSR defect even if mutations introduced in S-S junctions are different (Faili et al., 2004; Zeng et al., 2004). However, until now the role of the different error-prone DNA polymerases during CSR has not been studied in details. 2.2.2.5. Other DNA Repair Factors. Other factors that are recruited and activated upon DNA damage participate in CSR. ATM, a serine/threonine kinase related to the phosphoinositide 3 kinase (PI3K), is activated by DNAdsb and phosphorylates many downstream proteins involved in DNA repair (e.g., Chk1, Chk2, NBS1) (Bakkenist and Kastan, 2003; Shiloh, 2003). ATMdeficient patients (A-T, see Section 4.2) display variable hyper-IgM conditions ([Chun and Gatti, 2004], and our unpublished observations). These observations demonstrate that, although not absolutely required, ATM contributes to CSR. S-S junctions resulting from CSR in B cells from A-T patients present anomalies characterized by the predominance of long stretches of sequence homology (Pan et al., 2002). A multimeric complex formed of Mre11, Rad50, and Nbs1 molecules (MRN complex) is also recruited to DNA lesions and activated by ATM. Nbs1 is localized at CSR-specific DNA-dsb (Petersen et al., 2001). Nbs1-deficient (Nijmegen breakage syndrome), and Mre11-deficient (A-T like deficiency, ATLD) patients present HIGM syndromes and/or abnormal S-S junctions similar to those found in A-T patients (Chun and Gatti, 2004; Lahdesmaki et al., 2004; Pan et al., 2002). This suggests that the MRN complex and ATM participate in the modification of DNA ends produced during CSR and are necessary to facilitate S-S regions ligation. H2AX is an H2A histone variant specifically phosphorylated in chromatin flanking DNA-dsb (Paull et al., 2000; Rogakou et al., 1998, 1999). Many roles of phosphorylated H2AX (gH2AX) have been proposed, including the recruitment of DNA repair factors (such as the MRN complex) and anchorage of broken DNA ends to keep them in close proximity (Bassing and Alt, 2004; Redon et al., 2002). The ser/thr kinases ATM, ATR, and DNA-PK, all related to PI3K, can phosphorylate H2AX upon DNA lesion (Brown and Baltimore, 2003; Burma et al., 2001; Furuta et al., 2003; Park et al., 2003; Paull et al., 2000; Ward and Chen, 2001). Many DNA repair proteins such as Nbs1, MDC1/NFBD1, and 53BP1 can interact with gH2AX (Goldberg et al., 2003; Stewart et al., 2003; Ward et al., 2003; Xu and Stern, 2003). gH2AX and Nbs1 colocalize within the Ig locus during CSR (Petersen et al., 2001). H2AX knockout mice are viable but present increased genomic instability and impaired CSR (Celeste et al., 2002; Reina-San-Martin et al.,

254

pat ri ck r ev y

ET AL.

2003). When CSR occurs in these mice, S-S junctions are normal, suggesting that H2AX is not necessary to the DNA end modifications before ligation. It was proposed that the CSR defect in H2AX mice reflects the role of H2AX in keeping CSR DNA-dsb close to each other, thus facilitating their ligation (Reina-San-Martin et al., 2003). Similar to H2AX, the DNA damage mediator 53bp1 (Wang et al., 2002) is also required to efficiently repair CSR-DNA-dsb, since 53bp1 knockout mice exhibit a profound block in CSR (Manis et al., 2004; Ward et al., 2004). Taking into account the presumed role of the aforementioned molecules during CSR, an integrated model can be proposed (Fig. 6): (1) transcription through S regions leads to the production of germline transcripts, which create stable RNA/DNA hybrids known as R loop structures; (2) single-strand DNA nicks are produced on both strands of S regions by the successive activities of AID, UNG (and/or MMR), and AP-endonuclease. These nicks lead to staggered CSR-DNA-dsb; (3) the introduced CSR-DNA-dsb are gap-filled, or nibbled, to produce blunted DNA-dsb. Error-prone DNA polymerases, mismatch repair enzymes (including MSH2, MSH6, PML1, MLH1, and EXO1), the MRN complex, and ATM are suspected to be involved during this step; (4) CSR-induced DNA-dsb would be kept in close proximity by gH2AX, Mre11, Rad50, Nbs1, ATM, and 53bp1; (5) Lastly, ligation and production of S-S junctions could be ensured by NHEJ factors such as Ku70/ Ku80, DNA-PKcs, and other yet undefined proteins. It is most likely, as suggested by HIGM4 findings, that additional factors of the DNA repair machinery participate in CSR. One can expect, for example, that MDC1/NFBD1, which shares many functional characteristics with 53bp1, plays a role in CSR process (Goldberg et al., 2003; Peng and Chen, 2003; Shang et al., 2003; Stewart et al., 2003; Xu and Stern, 2003). The analysis of knockout mouse models and the study of new hyper IgM syndromes in humans will probably shed new light on the precise mechanisms and molecules involved in CSR. 2.3. Somatic Hypermutation (SHM) As previously mentioned, AID is essential to carry out SHM (Muramatsu et al., 2000; Revy et al., 2000). An interaction between Replication Protein A (RPA) and the phosphorylated form of AID was recently described (Chaudhuri et al., 2004). This association increases the deamination of SHM DNA targets. This observation, together with the description of UNG-deficient mice and humans that present defective CSR and perturbed SHM (with almost essentially transition mutations (i.e., G–A; C–T mutations) at dC/dG pairs (Imai et al., 2003b; Rada et al., 2002), demonstrates the importance of abasic site formation

d na r e pa i r i n t h e i m m un e sy s t e m

255

to produce varied SHM. Rada et al. (2002) have proposed a model in which the activity of AID in Ig V regions produces U-G mismatches. If replication occurs, the following mutations could be produced: C–T and G–A on the other strand (Fig. 7A, phase IA). If UNG removes the dU created by AID and an AP-endonuclease cuts the abasic site, a single strand DNA nick is produced. This nick could either be repaired by base excision repair (BER) (without producing mutations) or be bypassed by error-prone polymerases, which will introduce mutations (Fig. 7A, phase IB). Finally, if U-G mismatches are not processed by UNG, mismatch repair may excise dU that could be replaced by an error-prone DNA polymerase (with introduction of mutations) (Fig. 7A, phase II). However, this model does not take into account the observation of DNA-dsb in V regions of activated B cells (Bross et al., 2000, 2002; Catalan et al., 2003; Papavasiliou and Schatz, 2000, 2002; Zan et al., 2003). Moreover, these DNA-dsb found in V regions are produced independently of AID during SHM generation, which represents a major difference with what can be observed for DNA-dsb generated during CSR (see previous discussion) (Bross et al., 2002; Catalan et al., 2003; Papavasiliou and Schatz, 2002; Zan et al., 2003). Hence, conversely to CSR, AID may not be necessary to produce DNA-dsb in V regions during SHM but to modify these DNA-dsb. Indeed, Zan et al. (2003) showed that DNA-dsb found in V regions in the absence of AID are in majority blunt, while staggered DNA ends are generated in the presence of AID. AID appears to resect blunt DNA-dsb to create protruding ends that are gap-filled by error-prone DNA polymerases and MMR proteins that introduce mutations during DNA repair (Fig. 7B). Surprisingly, DNA-dsb that are specifically introduced in V regions are not only found in hypermutating B cells but also in other proliferating cells such as fibroblasts or T cells (Catalan et al., 2003; Zan et al., 2003). The reason why V regions are prone to breakage in proliferating cells is still unknown. However, even if the mechanism producing SHM is still obscure and controversial, several factors have been demonstrated to be involved in this process. 2.3.1. Homologous Recombination and NHEJ Although the RAD52 and RAD51 proteins are recruited to the protruding ends of DNA-dsb in V regions of hypermutating B cells, no direct evidence of their role in SHM has been provided (Zan et al., 2003). RAD54/RAD54Bdeficient mice normally generate SHM (Bross et al., 2003) as well as XRCC2/ XRCC3- (which are RAD51 paralogs) deficient DT40 cells (Sale et al., 2001). These observations suggest that the role of HR factors during SHM is not essential. The role of NHEJ factors during SHM production has not been

256

pat ri ck r ev y

ET AL.

Figure 7 Two different models for the generation of Ig SHM generation. During SHM production, the Ig‐V exon is the site of intensive transcription. (A) Model originally proposed by Rada et al. (2002). AID‐RPA complex targets RGYW motifs found in V region and deaminates cytidines to uridines, thus creating U‐G mismatches. DNA replication through G‐U mismatches will result in C–T mutations (phase IA). If UNG deglycosylates uracils, the subsequent abasic sites will be replaced by any bases during DNA replication (phase IB). If G‐U mismatches or MMR proteins and error-prone DNA polymerases recognize abasic sites, mutations on A/T base pairs will be generated (phase II). (B) Model originally proposed by Zan et al. (2003). AID independent, blunt DNA‐dsb occurs in Ig‐V regions of proliferating B cells. The successive interventions of AID‐RPA and UNG‐APE complexes lead to resected DNA‐dsb. Protruding DNA ends are gap‐filled by error‐prone DNA polymerases, which introduce mutations. DNA ends are subsequently religated by yet unknown factors.

d na r e pa i r i n t h e i m m un e sy s t e m

257

studied in detail. Nonetheless, SHM are produced normally in scid mice (Bemark et al., 2000). 2.3.2. Mismatch Repair (MMR) Proteins The MMR system is required to keep the genome free of mutations (mesappariement) created during replication, homologous recombination, and after cytidine deamination (Harfe and Jinks-Robertson, 2000). However, the immune system has diverted the primary MMR function to permit efficient SHM generation. Indeed, mouse models in which MMR coding genes such as PMS2, MSH2, MLH1, MSH6, or Exo1 are invalidated by gene targeting present an abnormally low level of SHM. Moreover, SHM are shifted toward transitions and more often targeted on dC and dG residues in these mice (Bardwell et al., 2004; Cascalho et al., 1998; Jacobs et al., 1998; Kong and Maizels, 1999; Phung et al., 1998; Rada et al., 1998; Vora et al., 1999; Wiesendanger et al., 2000; Winter et al., 1998). Hence, MMR appears to participate in SHM, possibly by recruiting error-prone DNA polymerases to the DNA lesions localized in V genes. 2.3.3. Error-Prone DNA Polymerases Among the increasing number of identified DNA polymerases, some of them have been found involved in the SHM process. Mice deficient for pol d (Longacre et al., 2003); polb (Esposito et al., 2000); pol k, pol l, pol m (Bertocci et al., 2002; Schenten et al., 2002); and pol i (McDonald et al., 2003) bear normal frequency and pattern of SHM. On the other hand, the DNA polymerases pol x (Diaz et al., 2001; Zan et al., 2001) and pol Z participate in the production of SHM (Faili et al., 2004; Yavuz et al., 2002; Zeng et al., 2001, 2004). Deficiency of either one of these molecules leads to lower frequency of SHM and/or skewed pattern. 3. Human Primary Immunodeficiency Disorders Associated with Defective DNA Repair 3.1. Nonhomologous End Joining (NHEJ) Repair Defects Severe combined immunodeficiencies (SCID) are the most severe forms of primary immunodeficiency disorders (1 in 75,000–100,000 births) and are characterized by a profound block of T lymphocyte differentiation, variably associated with abnormal development of other lymphocyte lineages (i.e., B or NK lymphocytes or more rarely of the myeloid lineage) (Fischer, 2000). V(D)J recombination defects cause 20% of SCID disorders in humans and must be divided into two groups. The group showing mutations in the RAG1 or RAG2

258

pat ri ck r ev y

ET AL.

gene (Schwarz et al., 1996), a defect that is responsible for an early block of V(D)J recombination, is not a subject of this review. The other group displays mutations in the Artemis gene (Moshous et al., 2001), resulting in a general NHEJ repair defect, responsible for an inability to resolve the RAG1/2 generated DNA-dsb during V(D)J recombination (see Section 3.1.1). Besides Artemis, only one other NHEJ factor, DNA ligase IV, is associated with a human pathology that is characterized by immunodeficiency, increased radiosensitivity, and microcephaly (see Section 3.1.2). Finally, there are patients with a V(D)J recombination defect associated to increased radiosensitivity for whom no molecular cause has been identified so far (Section 3.1.3). 3.1.1. Artemis Defect (RS-SCID) These patients are characterized by increased cellular radiosensitivity and SCID and are therefore referred to as RS-SCID. This condition represents the first human pathology associating deficient V(D)J recombination with a general DNA-dsb repair defect. 3.1.1.1. Clinical and Cellular Characteristics. In RS-SCID, which has autosomal recessive inheritance, mature T and B lymphocytes are absent, while functional NK cells are detectable. The clinical presentation is similar to the other SCID forms: early onset of infections, mainly of the respiratory tract and gut. Oral candidiasis, persistent diarrhea with failure to thrive, and/or interstitial pneumonitis are the most frequent infectious manifestations leading to diagnosis (Stephan et al., 1993). There is a preponderance of opportunistic infections such as Pneumocystis carinii. A characteristic feature of RS-SCID is the frequent incidence of severe oral and/or genital ulcers (Kwong et al., 1999; O’Marcaigh et al., 2001). In most cases, cervical lymph nodes and tonsils are undetectable, and the thymus shadow is absent on chest x-ray. The distinctive cellular feature of RS-SCID is the increased sensitivity to ionizing radiation tested in bone marrow cells and skin fibroblasts (Nicolas et al., 1996). To date, stem cell or bone marrow transplantation (BMT) represents the only curative treatment for RS-SCID that is otherwise fatal in infancy. The cure rate applying genoidentical BMT in SCID in general has been greater than 95% in recent years due to a virtual absence of GVHD. However, around 4.5 years following T-cell-depleted nonidentical BMT, survival with T-cell engraftment/function is significantly lower for patients with B
SCID (35%) than for those with BþSCID (60%) (Bertrand et al., 1999). 3.1.1.2. Identification of Artemis. In 1993, skin fibroblasts and bone marrow precursor cells of a subset of T-B-SCID patients were found to display increased sensitivity to ionizing radiation (Cavazzana-Calvo et al., 1993).

d na r e pa i r i n t h e i m m un e sy s t e m

259

These patients were devoid of mutations in the RAG1 or RAG2 gene, but their fibroblasts were clearly deficient in V(D)J recombination in vitro, as coding joints were absent, while signal joints were normally generated (Nicolas et al., 1998). This condition is reminiscent of the SCID mouse profile, but RS-SCID patients’ fibroblasts did not lack DNA-PK activity (Nicolas et al., 1996). Simultaneously to these findings, a genome-wide survey revealed a linkage of the SCIDA (T-B-SCID with a high prevalence among Athabascan-speaking Navajo and Apache Native Americans) locus to markers on the short arm of chromosome 10 (Li et al., 1998b). Subsequent analysis showed that SCIDA patients’ fibroblasts are impaired in V(D)J coding joint formation, and that RS-SCID is linked to the same region of chromosome 10p as SCIDA (Moshous et al., 2000). In 2001, the responsible gene Artemis was identified and cloned (Moshous et al., 2001). Functional complementation studies and mutation analysis have proven that Artemis deficiency is in fact the underlying cause for RS-SCID and SCIDA (Kobayashi et al., 2003; Li et al., 2002; Moshous et al., 2001; Noordzij et al., 2003). 3.1.1.3. Mutations of Artemis. A variety of mutations have been identified in RS-SCID patients (Fig. 8). Nearly all of them are located within the catalytic domain (b-Lact/bCASP) of the Artemis coding gene and are loss-of-function mutations that comprise genomic deletions of several 50 exons, the expression of severely truncated proteins, and rarely missense mutations (Kobayashi et al., 2003; Li et al., 2002; Moshous et al., 2001; Noordzij et al., 2003). The protein truncations result from deletions or insertions, nonsense mutations in the

Figure 8 Artemis mutations in RS‐SCID patients. Nearly all of the Artemis mutations in RS‐SCID patients are located within the catalytic domain (b‐Lact/bCASP, see fig. 4)–including genomic deletions (horizontal arrows), splice site mutations (vertical arrows), micro deletions or insertions, nonsense substitutions, as well as some missense substitutions ‐ leading to null alleles or severe truncations.

260

pat ri ck r ev y

ET AL.

coding region, or mutations in splice sites. Hypomorphic mutations in the Artemis coding gene have been detected in four patients presenting a syndrome of combined immunodeficiency associated with a predisposition to Bcell lymphoma (Moshous et al., 2003). Three patients from one family were compound heterozygotes, harboring a genomic deletion of exons 1-3 on one allele (null allele) and a deletion of seven nucleotides in exon 14 (T1348A1390), resulting in a frameshift at D451 followed by a premature stop codon 10 amino acids downstream. In the fourth patient, a homozygous deletion of 17 nucleotides in exon 14 (A1328-T1344) was identified leading to a frameshift at T432 and a premature stop codon 15 amino acids downstream. These mutations, located in the last exon of the Artemis gene, result in protein truncations, leaving intact the b-Lact/bCASP domain and allowing a residual activity. All patients presented with severe T and B lymphocytopenia and hypogammaglobulinemia involving IgG and IgA. They had recurrent infections including candidiasis, respiratory tract infections, and persistent diarrhea with failure to thrive. One patient developed a cerebral abscess caused by toxoplasma gondii, and a second suffered from cryptosporidium infection, which to cholangitis and liver disease. Patients’ skin fibroblasts showed increased sensitivity to ionizing radiation. Introduction of one of the patients’ form (DT1348-A1390) in a fully Artemis-deficient cell line partially complemented the increased radiosensitivity. Ex vivo analysis of TCR-b V(D)J junctions from lymphocytes showed a virtual absence of N nucleotide addition, while the TCR-Vb repertoire was diversified. This profile is found in TdT KO mice and, interestingly, was also described in Ku80 KO mice (Bogue et al., 1997; Purugganan et al., 2001). V(D)J tests using an extrachromosomal recombination substrate in patients’ fibroblasts revealed an absence (compound heterozygous patients) or decrease (homozygous patient) of coding joint formation. Of highest interest, underlining the role of Artemis as a genomic caretaker (see Section 5.3), is the observation that two of the four patients developed lethal aggressive EBV-associated B-cell lymphomas. Arguments for the malignant origin are the clonality of B-cell proliferation in both cases, clonal chromosomal alteration (clonal trisomy of chromosome 9) found in lymphoma cells of one patient, and the finding of general chromosomal instability in activated T cells of all four patients. 3.1.2. DNA Ligase IV Defect To date, five patients have been described with a DNA ligase IV defect. The first patient was identified in 1999 due to a highly increased sensitivity to radiotherapy in the context of leukemia (Riballo et al., 1999). Two years later, a series of four patients was reported with a quite distinct phenotype-comprising

d na r e pa i r i n t h e i m m un e sy s t e m

261

immunodeficiency and developmental and growth delay, including microcephaly (O’Driscoll et al., 2001). Finally, we have unpublished data on two siblings with a DNA ligase IV defect presenting an atypical form of radiosensitive severe combined immunodeficiency and microcephaly. 3.1.2.1. Clinical and Cellular Characteristics. The clinical presentation of the first reported patient (Table 1, patient 1) differs from those subsequently described. Apparently this patient did not display overt immunodeficiency and developed normally until the age of 12 years, when acute T-cell lymphoblastic leukemia was diagnosed and an over-response to radiotherapy led to death following radiation morbidity (Badie et al., 1997; Plowman et al., 1990). On the basis of the DNA repair deficiency, reflected by increased radiosensitivity of the patient, a screen of NHEJ factors revealed the defect in DNA ligase IV (Riballo et al., 1999). The clinical features of the four patients described by O’Driscoll et al. (Table 1, patients 2 to 5) (O’Driscoll et al., 2001) resemble the phenotype of patients with the DNA damage response disorder ‘‘Nijmegen breakage syndrome’’ (NBS): immunodeficiency, short stature, marked microcephaly, and typical facial features (Section 4.3). In contrast to NBS, none of the four patients has developed cancer, although two of them were in their mid-40s when reported. The immunodeficiency is not described in detail and does not seem to play a major role in these patients. The affected siblings we have identified, however, had marked lymphopenia with global hypogammablobulinemia. A son of this non-consanguineous family of Moroccan origin had died of ‘‘pneumonitis’’ at the age of 11 months, and a daughter (Table 1, patient 6) suffered from repeated infections since the third month of life—mostly otitis, bronchiolitis, and pneumonia, including a pneumococcal septicemia. Additionally, the girl was microcephalic and showed length and weight retardation, but she had no dysmorphic features. Lymphocyte count was markedly low with maintained proliferation of the few remaining T lymphocytes to mitogen and a virtual absence of B lymphocytes. The Vb repertoire was diversified, and ex vivo analysis of V(D)J junctions was normal for several Vb-Jb and Va-Ja regions. The bone marrow showed increased radiosensitivity. In the context of this atypical, radiosensitive severe combined immunodeficiency, the patient was bone marrow transplanted at the age of 19 months and died of an EBV-associated lymphoproliferative syndrome 50 days after transplantation. A second girl in this family (Table 1, patient 7) was affected by the same syndrome and was transplanted at 2 months of age. The immunodeficiency seen in patients with a DNA ligase IV defect may be a cause of incomplete lymphocyte maturation in the bone marrow or thymus due to the V(D)J recombination defect, but one could also imagine a defective DNA damage repair during cellular proliferation and/or cellular activation that

Table 1. Phenotypic Characteristics of LIG4 Patients

Patients

262

1 2 3/4 5 6/7

Mutations R278H A3V, T9I, R278H R580X, R814X G469E, R814X Q280R, K424del5

Homozygous Homozygous Heterozygous Heterozygous Compound heterozygous

Microcephaly

Facial dysmorphia

Immunodeficiency

Cancer

Reference


þ þ þ þ


þ þ þ



þ þ þ þ

þ





Riballo et al., 1999 O’Driscoll et al., 2001 O’Driscoll et al., 2001 O’Driscoll et al., 2001 Our unpublished data

d na r e pa i r i n t h e i m m un e sy s t e m

263

leads to progressive immunodeficiency. The second striking clinical feature reported for all but one DNA ligase IV patient is marked microcephaly. Endogenous DNA damage in the nervous system probably occurs during cellular proliferation or oxidative damage from cellular metabolism (Abner and McKinnon, 2004). An insufficient DNA ligase IV activity could lead to an increased amount of unresolved DNA-dsb activating the p53 pathway. P53 serves to integrate the DNA damage with the apoptotic machinery, and it is known that the developing nervous system is highly susceptible to DNA damage-induced apoptosis (Abner and McKinnon, 2004). Furthermore, as already mentioned, DNA ligase IV knockout mice die during embryogenesis due to massive apoptosis of developing neurons (Frank et al., 1998). The clinical phenotype found in most DNA ligase IV patients is thus the association of immunodeficiency, growth retardation and marked microcephaly, often accompanied by facial dysmorphia. The typical cellular phenotype found in all DNA ligase IV patients is markedly increased radiosensitivity and normal cell cycle checkpoint control following exposure to ionizing radiation. The radiosensitivity is more pronounced than in fibroblasts from Artemis deficient patients. Normal cell cycle control is an important feature to distinguish these patients from NBS patients, who are otherwise phenotypically similar. There is a single patient with deficiency of DNA ligase I with a partially overlapping phenotype: severe immunodeficiency, small stature, and sun sensitivity (cells are sensitive to ionizing radiation and ultraviolet light) (Barnes et al., 1992). The analysis of V(D)J recombination in DNA ligase IV deficient patients’ fibroblasts using extrachromosomal recombination substrates revealed a small decrease in rejoining frequency. Most interestingly, however, is the constant finding of pronounced infidelity of formed signal junctions (Smith et al., 2003). Altogether, the reduced level of DNA ligase IV activity seems to be sufficient to carry out V(D)J recombination efficiently (although with impaired fidelity of generated signal joints), but it is insufficient for properly repairing the large number of breaks induced by ionizing radiation as manifested by increased radiosensitivity. 3.1.2.2. Molecular Basis and Mutation Analysis. The human DNA ligase IV gene is localized on the large arm of chromosome 13 and consists of a single exon coding for a 911 amino acid protein. It has a ligase domain at its N-terminus comprising an active site that is highly conserved within mammalian DNA ligases and includes the active site lysine at amino acid position 273 and a unique large C-terminal region with a tandem repeat of BRCT (BRCA1 carboxyl terminus) domains (Tomkinson and Mackey, 1998) (Fig. 9). Initially identified in the breast cancer susceptibility protein BRCA1, BRCT domains

264

pat ri ck r ev y

ET AL.

Figure 9 DNA ligase IV mutations in LIG4 patients. The DNA ligase IV protein has a catalytic domain at its N‐terminus comprising an active site that is highly conserved within mammalian DNA ligases and a tandem repeat of BRCT domains at its C‐terminal region. The identified mutations in Lig4 patients include missense and nonsense mutations as well as a small deletion that leads to a frameshift and a premature stop codon (see also Table 1).

are commonly found in proteins involved in DNA repair and DNA damage signaling and are thought to be implicated in protein-protein interactions (Callebaut and Mornon, 1997; Koonin et al., 1996). DNA ligase IV interacts with, and is catalytically stimulated by, the XRCC4 protein (Critchlow et al., 1997; Grawunder et al., 1997). Surprisingly, the XRCC4 binding motif of DNA ligase IV is situated between, rather than within, the two BRCT domains (Grawunder et al., 1998b). Crystallographic analysis has revealed the structure of the XRCC4/DNA ligase IV complex that consists of a single ligase chain binding asymmetrically to an XRCC4 dimer (Sibanda et al., 2001). In accordance to the fact that the complete absence of the DNA ligase IV protein is embryonic lethal in DNA ligase IV knockout mice, all known mutations are hypomorphic (i.e., maintaining a residual function of the protein). Patient 1, who developed acute lymphoblastic leukaemia and who apparently did not show any overt immunodeficiency or developmental abnormality, has a homozygous missense mutation in the DNA ligase IV encoding gene at amino acid position 278 (substitution of a histidine for an arginine – R278H) (Riballo et al., 1999). This mutation lies within the region encoding the highly conserved catalytic motif of ATP-dependent DNA ligases. The stability of the protein is not impaired, but adenylation and ligation activities are diminished (Riballo et al., 2001). The responsible mutations reported by O’Driscoll et al. are situated in a region that encodes either the catalytic- or the XRCC4binding domain. Patient 2 has the same homozygous mutation as the leukemia patient (R278H) but shows a quite distinct clinical presentation (immunodeficiency, global developmental delay, microcephaly, and facial dysmorphia [Table 1]). One possible explanation is that two additional homozygous N-terminal substitutions (leading to amino acid substitutions A3V and T9I) in the immunodeficient microcephalic patient may contribute to the clinical

d na r e pa i r i n t h e i m m un e sy s t e m

265

difference, although these are polymorphic changes since they have been observed in healthy individuals. Furthermore, they have been associated with a reduced risk of developing multiple myeloma (Roddam et al., 2002). Using recombinant mutant proteins, Girard et al. recently showed that the ligase activity of the R278H mutation (10% of wild-type activity) is further reduced to a nearly undetectable level after combination with the two polymorphisms (A3V þ T9I þ R278H LIG4) (Girard et al., 2004). These results propose that the distinct clinical features of patients 1 and 2 are due to the presence of the two polymorphisms. The R580X mutation in siblings 3 and 4 leads to a truncated protein with largely decreased stability, impaired interaction with XRCC4, and impaired localization into the nucleus (Girard et al., 2004). The nonsense mutation on the other allele causes a stop codon at amino acid position 814 (R814X) and impairs the interaction with XRCC4. The nonsense mutation in patient 5 leads to the amino acid change G469E, which impairs ligation activity. Finally, we identified compound heterozygous mutations in siblings 6 and 7 (our unpublished data). The missense mutation on one allele leads to the amino acid change Q280R close to the active site; the other allele harbors a 5 base pair deletion leading to a frameshift at amino acid position K424, which causes a premature stop codon a few positions downstream. 3.1.3. Additional NHEJ Defects? Several facts suggest the existence of yet other so far unidentified factors of the V(D)J recombination/NHEJ process, whose defects may cause human pathologies comprising immunodeficiency: first, the V(D)J recombination reaction cannot be accomplished in vitro by using the known purified proteins and second, there are SCID patients showing a V(D)J recombination defect that does not seem to be caused by any of the known factors. Dai et al. reported such a T-B-SCID patient with increased cellular radiosensitivity and a V(D)J recombination defect in the patients’ fibroblasts affecting both signal and coding joint formation, which could not be complemented by any of the known V(D)J/NHEJ proteins suggesting the existence of additional activities for V(D)J recombination/NHEJ in mammals (Dai et al., 2003). Furthermore, Maraschio et al. reported a patient with an NBS/DNA ligase IV like clinical phenotype including progressive immunodeficiency, growth retardation, microcephaly and facial dysmorphia without NBS1 or DNA ligase IV mutations (Maraschio et al., 2003). The cellular phenotype showed increased radiosensitivity, chromosome instability and normal cell cycle control. Moreover, they reviewed the literature of reported NBS-like patients. Unfortunately, only Maraschio et al. had performed cell cycle control experiments and none of the groups reported any information on V(D)J function in these patients.

266

pat ri ck r ev y

ET AL.

Finally, we have unpublished data on five patients, who are phenotypically similar to patients with a DNA ligase IV defect (i.e., combined immunodeficiency, microcephaly, profound radiosensitivity and normal cell cycle control). However, neither DNA-ligase IV nor the cofactor Xrcc4 is deficient in these patients. In our extrachromosomal V(D)J recombination assay, the fibroblasts show a defect that is characterized by an impaired fidelity of signal joints. Thus, one could imagine the involvement of one of the remaining known factors of the NHEJ, for which no human pathology has been described so far, but also the existence of a hitherto undescribed additional factor of the NHEJ pathway responsible for this pathology. 3.2. Class Switch Recombination/Somatic Hypermutation Defects Hyper IgM syndromes (HIGM) are primary immunodeficiencies characterized by normal or increased IgM serum levels contrasting with low or absent IgG, IgA, and IgE. Patients suffer from recurrent bacterial infections and need immunoglobulin substitution. HIGM are caused by a defect in the class switch recombination process (Durandy et al., 2004). The absolute requirement of CD40-mediated B-cell activation to induce CSR has been demonstrated by the description of HIGM1 and HIGM3. HIGM1 is an X-linked HIGM caused by deleterious mutations found in the CD40 ligand (CD154) coding gene (Allen et al., 1993; Aruffo et al., 1993; DiSanto et al., 1993; Korthauer et al., 1993). CD154 is expressed on activated CD4þ T lymphocytes and is the ligand of CD40 that is constitutively expressed on B cells, monocytes, and dendritic cells. Due to the absence of CD154-CD40 interaction, B cells from HIGM1 patients cannot proliferate, form germinal centers, and undergo CSR and SHM. In addition to the humoral defect, the inability of CD154-defective T cells to activate monocytes and dendritic cells also leads to a susceptibility to opportunistic infections. B cells from HIGM1 patients do not have an intrinsic defect since they can carry out CSR after in vitro activation with CD40 agonists and appropriate cytokines (Durandy et al., 1993). Few HIGM1 have been reported in female patients associated with a nonrandom pattern of X chromosome inactivation (de Saint Basile et al., 1999). HIGM3 is an autosomal recessive syndrome that presents an identical phenotype to that described in HIGM1. HIGM3 is caused by mutations located in the CD40-coding gene. Until now, four HIGM3 cases have been reported (Ferrari et al., 2001; Kutukculer et al., 2003). The two other molecularly defined HIGM syndromes (HIGM2 and UNG deficiency) are caused by defects in the production of the DNA lesions necessary to undergo CSR and SHM (see previous discussion). Deleterious mutations in the AID gene cause autosomal recessive HIGM2 (Revy et al., 2000). AID mutations have been found throughout the gene

d na r e pa i r i n t h e i m m un e sy s t e m

267

and lead to profound CSR defect associated to the absence of SHM production (Minegishi et al., 2000; Quartier et al., 2004; Revy et al., 2000; Ta et al., 2003; Zhu et al., 2003). Since AID is only expressed in activated B cells, HIGM2 is caused by an intrinsic functional B-cell deficiency. In addition to the classical HIGM phenotype, HIGM2 patients frequently display an enlargement of the lymphoid organs due to an abnormal proliferation of B cells known as germinal center founder cells in the germinal centers (coexpressing IgM, IgD, and CD38) (Lebecque et al., 1997; Revy et al., 2000). It has been proposed that the enhanced B-cell proliferation is due to constant antigen stimulation since, in the absence of AID, no efficient antibody maturation occurs (Fagarasan et al., 2002). Even if peripheral blood B cells from HIGM2 patients carry a normal frequency of the CD27 marker (known as B-cell memory marker, (Klein et al., 1998) ), they very rarely display SHM (Revy et al., 2000). The HIGM2 phenotype is very similar to what is observed in the AID deficient murine model (Muramatsu et al., 2000). To date three patients have been reported to be affected by a severe HIGM associated to skewed pattern of SHM caused by deleterious mutations of the Uracil-N-glycosylase (UNG) gene (Imai et al., 2003b). CSR was found defective both in vivo and in vitro, and hyperplasia was detected in two of the three patients. As HIGM2, UNG deficient B cells proliferate normally but are unable to produce CSR-specific DNA-dsb (Imai et al., 2003b). These observations have reinforced the model in which S regions are consecutively targeted by AID, UNG, and APE to create CSR-DNA-dsb (Rada et al., 2002). The main difference observed between UNG and HIGM2 deficiencies is the normal frequency of SHM found in the V region of Ig from UNG-deficient CD27þ B cells (Imai et al., 2003b). This suggests that UNG is not required to produce SHM. However, the pattern of SHM is abnormal, resulting in almost exclusively transitions at dG/dC residues (Imai et al., 2003b). Even if CSR defect is milder in UNG-deficient mice, the global phenotype is similar to that observed in human UNG deficiency (Rada et al., 2002). No HIGM syndrome associated to a defect in a DNA repair factor specifically involved in CSR has ever been described. However, it has recently been observed that peculiar AID mutations, leading to deletion of the last amino acids, perturb neither AID-cytidine deaminase activity nor SHM production, but result in overall CSR deficiency. This strongly suggests that the C terminal region of AID is a docking domain that can recruit molecules important in CSR-DNA-dsb repair. In addition, a new HIGM condition has been reported (HIGM4). HIGM4 is phenotypically similar to HIGM2 (no CSR, normal B-cell proliferation) to the exception of normal SHM and CSR-DNA-dsb production (Imai et al., 2003a). The molecular defect causing HIGM4 is not yet defined but likely implicates a

268

pat ri ck r ev y

ET AL.

CSR-DNA-dsb repair factor and possibly one of those that interact with the C-terminus region of AID. 4. Human Primary Immunodeficiency Disorders Associated with Defective Cell Cycle Control Following DNA Damage 4.1. DNA Damages and Safeguard Genome Integrity Sequence alterations in DNA arise from three main causes: (1) environmental agents such as the UV irradiation, ionizing radiation, and genotoxic agents; (2) normal cellular metabolism constituents, including reactive oxygen species derived from oxidative respiration and products of lipid peroxydation (superoxyde anions, hydroxyl radicals, hydrogen peroxide); and (3) spontaneous and physiological nucleotide alterations (hydrolysis or deamination). The DNA damages are multiple, including covalent changes in the DNA structure processed by DNA repair and recombination pathways and noncovalent changes processed by mismatch repair pathways (Sancar et al., 2004). All the consequences of DNA damages are referred to as ‘‘DNA damage responses,’’ including the biochemical events that delay or arrest cell cycle progression in order to allow the actual DNA damage repair, and consequently, to ensure the genome integrity. Conceptually, the DNA damage response requires three steps: DNA damage recognition, transduction of cell-cycle regulation signals, and, finally, the actual cell cycle arrest. This activation pathway has three components: sensors, signal transducers, and effectors (Fig. 10). The first step is under control of DNA damage sensors, which, with the aid of mediators, transduce the signal to transducers, which in turn activate or inactivate effectors that control the inhibition of G1/S transition, S-phase progression, and G2/M transition. Two groups of proteins have been described as sensors (i.e., the phosphoinositide 3-kinase [PI3-K]-like kinase family members ATM and ATR) (Durocher and Jackson, 2001) and the RFC/PCNA- related Rad 17-RFC/91-1 complex (Melo and Toczyski, 2002). Moreover, some repair proteins such as BRCA1 and Nbs1 may also function as DNA damage sensors. The mediators associate with sensors and transducers according to cell cycle phases and, consequently, provide signal transduction specificity. In humans, four proteins are known to play the role of mediators: the p53 binding protein (53bp1) (Wang et al., 2002), the topoisomerase binding protein (TopBP1) (Yamane et al., 2002), BRCA1, and the mediator of DNA damage checkpoint 1 (MDC-1) (Goldberg et al., 2003; Lou et al., 2003; Stewart et al., 2003). The two serine/threonine kinases Chk1 and Chk2 are known as transducers. The signal of DNA-dsb, sensed by ATM, is transduced by Chk2 (Chaturvedi

d na r e pa i r i n t h e i m m un e sy s t e m

269

Figure 10 DNA‐dsb response in mammalian cells. Upon ionizing radiation or other DNA‐dsb– inducing agents, the DNA‐dsb is detected by sensors that transfer this information to the Chk2 transducer with the help of mediators. The Chk2 transducer then activates cell‐cycle checkpoint effectors, leading to G1/S transition, S‐phase progression, and G2/M transition arrests.

et al., 1999; Kaneko et al., 1999) while UV-damage signals, sensed by ATR, are transduced by Chk1 (Pichierri and Rosselli, 2004). The main substrates of these kinases consist of the three phosphotyrosine phosphatases, Cdc25 A, B, and C, which represent the effectors (Chen et al., 2003; Walworth, 2001; Xiao et al., 2003). Cdc25 promotes the G1/S and G2/M transitions by dephosphorylating Cdk2 and Cdc2, respectively, which act on factors directly involved in cell cycle transition (Bartek and Lukas, 2001). The Cdc25 phosphorylation creates a binding site for the adaptator 14-3-3 (Lopez-Girona et al., 1999) and inhibits its activity in the promotion of cell cycle phase transition. Several chromosomal breakage syndromes in humans are characterized by genome instability, defects in DNA damage recognition and/or repair, and are accompanied by a high predisposition to develop cancer. Some of them, including ataxia-telangiectasia (A-T) and the Nijmegen breakage syndrome (NBS) are also associated with various degrees of immunodeficiency. Ataxiatelangiectasia-like disorder (ATLD) is caused by hypomorhic mutations in hMRE11, and there are only six cases reported until today, showing normal

270

pat ri ck r ev y

ET AL.

levels of total IgG, IgA and IgM; in some cases, levels of specific antibodies are reduced (Stewart et al., 1999; Taylor et al., 2004). However, La¨ hdesmaki et al. recently reported on a partial reduction of isotype subgroups accompanied by altered switch junction DNA sequences in these patients (Lahdesmaki et al., 2004). Although these syndromes are defined by specific gene defects, they share many clinical and biological features, illustrating the interplays among numerous DNA repair pathways. 4.2. Ataxia-Telangiectasia In 1926, Syllaba and Henner first described three members of a single family that presented progressive choreoathetosis and ocular telangiectasia. Fifteen years later, the association of progressive cerebellar ataxia and cutaneous telangiectasia was described as Louis Bar syndrome. Two groups identified the association of progressive ataxia, oculocutaneous telangiectasia, and frequent pulmonary infections in children as a familial distinct disease (Biemond, 1957; Boder and Sedgwick, 1958). The humoral deficiency, thymic hypoplasia, and tendency to develop malignancies were subsequently described in A-T (Peterson et al., 1964). A-T is autosomal recessive with full penetrance and is a complex, multisystem disorder of childhood onset, characterized by progressive cerebellar ataxia, ocular and cutaneous telangiectasia, variable immunodeficiency with susceptibility to sinopulmonary infections, impaired organ maturation, radiosensitivity, and a predisposition to malignancy. According to the type of inheritance, A-T is represented equally in male and female patients. A-T is reported in all ethnic groups throughout the world and its estimated incidence is 1 case in 40,000 to 100,000 births (Boder and Sedgwick, 1970; Swift et al., 1986; Woods and Taylor, 1992). The frequency of A-T mutant allele heterozygosity was reported to be 1.4–2% in the general population (Su and Swift, 2000; Swift et al., 1986). 4.2.1. ATM Protein The disease was first thought to be heterogeneous, both clinically and genetically, as shown by the existence of four complementation groups (A, C, D, E). However, the responsible gene in all groups was mapped to chromosome 11q22-23 and codes for the same protein, ataxia-telangiectasia mutated (ATM), a member of the phosphatidylinositol-3-kinase–related family involved in cell cycle control, intracellular protein transport, and DNA damage response. This protein family includes ATM and Ataxia telangiectasia and Rad-3 related (ATR), the DNA-dependent protein kinase (DNA-PKcs), the target of rapamycine (mTOR), and ATX/hSMG-1 (McMahon et al., 2000;

d na r e pa i r i n t h e i m m un e sy s t e m

271

Nikiforov et al., 2002). ATM is a 350 kDa-oligomeric protein, which lacks lipid kinase activity, but exerts Ser/Thr kinase activity. Agents known to induce DNA-dsb activate its protein kinase activity. ATM is a sensor of DNA-dsb, which tranduces this information to enzymes involved in DNA repair and cellcycle checkpoints. In response to ionizing radiation, ATM phosphorylates many proteins including p53, Chk2, the proteins of the M/R/N complex (Mre11, Rad50, Nbs1), BRCA1, H2AX, FANCD2, SMC1, and itself (Shiloh, 2003), which all localize within ionizing radiation-induced foci (IRIFs) at the site of the DNA damage. Autophosphorylation of ATM converts the oligomeric form into monomers, the active species of the enzyme responsible for the checkpoint response. The ATM-dependent Chk2 phosphorylation leads to Cdc25 phosphorylation and, consequently, its inactivation through nuclear exclusion and degradation, thus initiating the G1/S transition arrest, the S phase progression, as well as the G2 arrests. P53, directly phosphorylated by ATM as well as by phosphorylation-dependent activated Chk2, accumulates in the nucleus, maintaining the G1/S arrest (Agarwal et al., 1995). These complexes initiate a pathway, which plays a crucial role in DNA repair as well as in S-phase checkpoint. ATM therefore represents a key element to control DNA damage checkpoint and DNA repair. Moreover, recent data support a role of the MRN complex in the activation of ATM when DNA damages occur (Horejsi et al., 2004; Lee and Paull, 2004; Uziel et al., 2003). 4.2.2. Genetics The ATM gene spans approximately 150 kb of genomic DNA and encodes a large transcript consisting in 66 exons (Uziel et al., 1996). With the exception of a founder mutation identified among patients of Moroccan-Jewish origin, most mutations described in A-T are unique and consequently, the majority of nonconsanguineous patients are compound heterozygous. The mutations involve the whole gene without ‘‘hot spots.’’ However, more than 80% of ATM mutations are predicted to result in truncated proteins. In these cases, as well as in most cases of missense mutations, the ATM protein is unstable and not detectable (Chun and Gatti, 2004). These mutations can thus be considered as null alleles. Some rare missense mutations within the kinase domain (V2716A, R2849P, G2867R) lead to a protein with a dominant-negative effect, evidenced by the interference with normal protein after transfection in normal cells. An especially interesting short in-frame deletion (7636del9) was observed in British/Irish A-T patients. The loss of three amino acids within the HEAT motif leads to a catalytically inactive ATM protein, presumably because of a modification in the protein conformation or its capacity to interact with other partners. Moreover, this protein also exerts a dominant-negative effect

272

pat ri ck r ev y

ET AL.

on normal ATM, inhibiting the radiation-induced kinase activity. Heterozygous patients for this deletion, or homozygous for a single truncating mutation, have significantly poorer prognosis than those heterozygous for a splice site or missense mutation, or heterozygous for two truncating mutations (Li and Swift, 2000). Of note, the knock-in mouse for this mutation differs from the ATM-/- mice in terms of longevity and tumor occurrence (Spring et al., 2001). This is in accordance with the phenotype heterogeneity seen in A-T patients (Becker-Catania et al., 2000). 4.2.3. Cellular Characteristics One common characteristic of the disease is the abnormal sensitivity of A-T cells to ionizing radiation and radiomimetic drugs, but not to UV irradiation. This threefold–fourfold increase in radiosensitivity is not confined to a particular cell lineage. This could account for the adverse responses of patients treated by x-rays during therapy of neoplasias. This radiosensitivity is associated with a subtle defect in DNA repair, with a residual unrepaired radiation-induced DNA-dsb (Cornforth and Bedford, 1985), leading to chromosome and chromatid breaks. Nonrandom rearrangements involving TCR and heavy-chain Ig genes characterize chromosome breaks observed in lymphocytes from A-T patients. Indeed, in the mouse model, the occurrence of lymphoma that involves alleles of the TCR-a/d locus, is dependent on RAG activity (Liao and Van Dyke, 1999; Petiniot et al., 2000). Moreover, the presence of ATM at DNAdsb associated with V(D)J recombination indicates that ATM could have a role of DNA-dsb sensor during TCR and Ig gene rearrangements (Perkins et al., 2002). The defect of ATM as sensor of radiation-induced DNA-dsb leads to defective cell cycle checkpoints in A-T cells, which fail to activate either the G1/ S or G2/M arrests in response to DNA damages (Beamish and Lavin, 1994). The mechanisms responsible for neurological disease, thymus aplasia, telangiectasias, growth retardation, and impaired organ maturation have not been elucidated, but are most likely linked to accelerated telomere loss (Qi et al., 2003). ATM was shown to be pivotal for neurodevelopment, especially for stem cell differentiation, as well as for elimination of damaged postmitotic cells (Lee et al., 2001). A general disturbance in tissue differentiation accounts for the almost constant elevation of alpha-fetoprotein (AFP), a fetal serum protein of hepatic origin that indicates dedifferentiation of liver cells. A-T may be associated with dysregulation of the immunoglobulin gene superfamily, which includes genes for T-cell receptors (Kirsch, 1994; Peterson and Funkhouser, 1989). CSR is defective in some A-T patients, resulting in variable HIGM, and the same may apply to the switch from immature T cells that express the gamma/delta rather than the alpha/beta receptors

d na r e pa i r i n t h e i m m un e sy s t e m

273

(Pan et al., 2002). A microcephaly associated with the classical A-T phenotype has also been reported in very rare A-T cases. This peculiar phenotype (known as A-TFresno syndrome) associating NBS and A-T features is still not understood (Chun and Gatti, 2004 and our unpublished observations). 4.3. Nijmegen Breakage Syndrome (NBS) NBS is a rare autosomal recessive disorder that is clinically characterized by microcephaly, a distinct facial appearance, short stature, immunodeficiency, radiation sensitivity, and a strong predisposition to lymphoid malignancy. This syndrome was described in 1981 in Dutch patients (Weemaes et al., 1981) even though the majority of patients are of Slavic origin. Further investigations revealed that cells derived from patients with NBS display characteristic abnormalities similar to those observed in A-T in vitro. Therefore, NBS has long been considered as a variant of A-T. However, the clinical manifestations are distinct. More than 130 cases have been identified worldwide, including 68 in Poland and 26 in the Czech Republic. NBS has been observed in other European countries, in Morocco, the United States, New Zealand, and Russia. Patients with NBS seem to be overwhelmingly of Slavic origin. Mutations in the NBS1 gene, located in chromosome 8q21, are responsible for NBS (Carney et al., 1998; Matsuura et al., 1998; Varon et al., 1998). 4.3.1. Nbs1 Protein The NBS1 gene product, nibrin (or Nbs1), is a 754 amino acid protein and is a component of the MRN complex. Three functional domains can be identified: (1) a FHA/BRCT domain at the N terminus that allows protein-protein interactions, especially the binding of gH2AX required for IRIF formation (Tauchi et al., 2002); (2) an ATM-phosphorylated serine residue at a central place; and (3) the Mre11-binding domain at the C-terminus. Nbs1 is involved in processing DNA-dsb induced by either external agents or physiological processes such as meiotic recombination and V(D)J rearrangements in maturing lymphocytes. This is consistent with the observation that lymphocytes from NBS1 mutant mice display increased interchromosomal V(D)J recombination events in comparison with control mice (Kang et al., 2002). However, the role of Nbs1 in V(D)J recombination is not clear since its frequency and quality is not impaired in Nbs1-deficient cells (Harfst et al., 2000). As discussed previously, Nbs1 is a substrate for ATM phosphorylation. However, this phosphorylation is dispensable for the recruitment in IRIFs, which is also the case for Mre11 (Kobayashi et al., 2002). Petrini et al. proposed that Mre11 acts as a sensor of DNA-dsb (Petrini and Stracker, 2003). However, an expression study of the

274

pat ri ck r ev y

ET AL.

murine NBS1 gene during mouse development provides evidence that, apart from sites of physiological DNA-dsb in the testis, thymus, and spleen, NBS1 expression is also evident in several tissues and organs in which rejoining of DNA-dsb is not known to occur. 4.3.2. Genetics The NBS1 gene is located on human chromosome 8 (8q21) and spans more than 50 kb. It encodes for a 2277 bp transcript that consists of 16 exons (Carney et al., 1998; Matsuura et al., 1998; Varon et al., 1998). More than 90% of patients are homozygous for a major founder mutation of Slavic origin, a 5 base pair (bp) deletion (657del5) in exon 6. A high frequency of this allele (1 case per 177 individuals) was found in the Czech Republic, Poland, and Ukraine, a factor that may contribute to the frequency of cancers in those countries. Seven other truncating mutations, located between nucleotides 657 and 1142, have been identified, each occurring in isolated families of different origins (Maraschio et al., 2003). Some patients had compound heterozygosity for 657del5 and a second mutation. All the mutations hitherto described lead to a truncated protein. In all cases, the truncation involves the BHA/BRCT domain, but a residual activity of the protein is preserved. Indeed, Nbs1 knockout mice die early in the development in contrast to Nbs1 mutant mice (Zhu et al., 2001). Murine models of NBS have recently been derived. Nbs1(m/m) mice, which reproduce the mutation of NBS patients, are viable, growth retarded, hypersensitive to ionizing radiation, exhibit multiple lymphoid developmental defects, and rapidly develop thymic lymphoma (Williams et al., 2002). 4.3.3. Cellular Characteristics NBS shares cellular features with A-T. These include characteristic chromosomal rearrangements in cultured lymphocytes, radiosensitivity, chromosome breakage involving chromosomes 7 and 14, and cell cycle checkpoint defects. However, the radiosensitivity seems to correlate with a defect in DNA-dsb repair rather than an impaired DNA damage checkpoint (Girard et al., 2000). As in A-T cells, the CSR defect in NBS patients can be explained by a role of the NBS1 protein in DNA repair (Featherstone and Jackson, 1998), signal transduction, cell cycle regulation, or apoptosis (van Engelen et al., 2001). 5. Defective DNA Repair and Malignancies in the Immune System Lymphoid tumors are very often associated with chromosomal rearrangements that involve tumor suppressor genes or cellular oncogenes, such as c-myc, and immunoglobulin or TCR genes, suggesting that they are somehow related to

d na r e pa i r i n t h e i m m un e sy s t e m

275

the V(D)J recombination process (Vanasse et al., 1999) or CSR (Ramiro et al., 2004). Neoplastic transformation can also be envisioned as resulting from defects in both of the two major groups of proteins belonging to the tumor suppressor genes, the ‘‘gatekeepers,’’ and the ‘‘caretakers’’ (Kinzler and Vogelstein, 1997). Gatekeepers control cellular proliferation and cell death. As previously discussed, they regulate the cell cycle progression and, upon introduction of DNA damage into the cell, they induce apoptosis or cell cycle arrest to allow appropriate DNA repair by the different repair systems. In the particular case of the immune system, one has to take into account several levels at which neoplastic transformation can occur: (1) the specificity of the RAG1/2 generated DNA break; (2) a possible deregulation of the RAG1/ 2 activity such as a transposition reaction; (3) the B cell specificity of the AID initiated DNA break during CSR; and (4) a defect in the DNA repair machinery. 5.1. Lymphomas/Leukemias as a Result of Illegitimate V(D)J and CSR Recombination In the physiological situation, the V(D)J recombination process concerns exclusively the V, D, and J gene segments of the immunoglobulin and TCR gene loci. In vertebrates, millions of lymphoid precursors undergo V(D)J rearrangement each day. The fact that the RSS is the unique element in cis that is necessary and sufficient to initiate the site-specific reconnaissance by the V(D)J recombinase represents a considerable danger with regard to the stability of the genome. RSS-like sequences might be an erroneous aim for the V(D)J recombinase machinery, leading to illegitimate recombination events followed by genomic deletions and inversions or chromosomal translocations. If these pseudo-RSS, the number of which is estimated at about 10 million in the whole genome, are located near proto-oncogenes, an illegitimate rearrangement event may result in the inappropriate activation of these oncogenes. The restricted expression of RAG1/2 genes to immature lymphoid cells directs these oncogenic mechanisms toward the immune system, explaining why specific chromosomal translocations between gene segments of the TCR/Ig and cellular proto-oncogenes are a characteristic feature of lymphoid neoplasms. In acute lymphoblastic T-cell leukemia (T-ALL), the translocations of LMO2, TAL1, and TAL2 are compatible with an illegitimate recombination event between a TCR locus and a locus of a proto-oncogene containing a fortuitous but functional RSS (Marculescu et al., 2002). In the case of LMO2, the cryptic RSS functions with an efficacy only slightly reduced when compared to a consensus RSS-12 (Raghavan et al., 2001). In the case of nonHodgkin’s B-cell lymphomas (B-NHL), the translocations concern the genes

276

pat ri ck r ev y

ET AL.

BCL1 and BCL2. Only the breakpoints at the IgH locus seem to be induced by an illegitimate V(D)J recombination process, whereas the DNA-dsb on the side of the proto-oncogene may be initiated by other mechanisms. However, the RAG1/2 complex nicks the major breakpoint region (Mbr) of the BCL2 gene in vitro and in vivo on extrachromosomal substrates very efficiently despite the absence of RSS in this region (Raghavan and Lieber, 2004; Raghavan et al., 2004). The transformation events leading to the development of Bcell lymphomas occur mainly at two distinct developmental stages: during V (D)J recombination in B-cell precursors in the bone marrow and during the maturation of B cells in the germinal center following T-cell dependent immune responses (Kuppers et al., 1999). The recent finding that AID is essential for the generation of c-myc/IgH translocation onset in a model of generation of B-cell lymphomas in Il6 transgenic mice indicates that ‘‘illegitimate CSR’’ may participate in mature B-cell neoplasms (Ramiro et al., 2004). Lastly, mice deficient in the UNG uracil-DNA glycosylase frequently developed B-cell lymphomas beyond 1 year of age. The molecular basis underlying lymphomagenesis in these mice is not yet known (Nilsen et al., 2003). 5.2. RAG1/2: A Harmful Transposase? The initial phases of the V(D)J recombination manifest many characteristics of DNA transposition, in particular, the transesterification reaction and hairpin formation that follows the RAG1/2 mediated initial DNA nick (Gellert, 2002; Roth and Craig, 1998). The Rag complex indeed has transposase activity in vitro (Agrawal et al., 1998; Hiom et al., 1998), and drugs known to block the function of the HIV integrase also inhibit V(D)J recombination (Melek et al., 2002). This analogy of RAG1/2 and transposases led to the proposal that the V(D)J recombination process may have evolved from an ancient transposable element (Agrawal et al., 1998; Hiom et al., 1998; Roth and Craig, 1998). Whether RAG1/2-mediated transposition can really occur in vivo became an important issue given the deleterious consequences it would have. Up to now only one study has reported on such an event (Messier et al., 2003). The integration of signal ends from the TCR-a locus within the HPRT locus was identified in T cells from two different individuals and was probably mediated through a bona fide transposition event. Although it was thought that the signal joint formation would represent a safe dead-end product to avoid the attack from the free 30 hydroxyl group at signal ends, several new studies demonstrated that signal joints can be recleaved by RAG1/2 and thus may well be utilized as donors in transposition reactions. Fortunately, cells have evolved several mechanisms to avoid transposition from occurring, as previously

d na r e pa i r i n t h e i m m un e sy s t e m

277

discussed (Roth, 2003). Analyzing RAG1/2 mutants defective in coding joint formation (nick-only mutants), Lee et al. demonstrated that the RAG1/2 postcleavage complex is rather unstable in these circumstances and that RAG1/2generated nicks can efficiently promote homologous recombination (Lee et al., 2004). RAG1/2-generated nicks at pseudo-RSS could well represent the initiating lesion responsible for some oncogenic translocations via homologous recombination. In turn, this study also suggests that one important role of the Rag post-cleavage complex is therefore to guide the DNA ends to the proper and safe NHEJ DNA repair pathway, acting as a molecular shepherd, but ‘‘a shepherd in wolves’ clothing’’ (D. Roth, unpublished observation). 5.3. NHEJ of the V(D)J Recombination Are Genomic Caretakers Defects of NHEJ components with alteration of the V(D)J recombination predispose the deficient mice in a selective way to develop lymphoid neoplasia. This link has been initially established by the observation that scid mice display the delayed apparition of thymic lymphomas with moderate incidence of about 15% (Bosma and Carroll, 1991). These lymphomas reveal translocations in the majority of cases. To prevent illegitimate rearrangement events, the V(D)J recombination process is highly regulated and submitted to tight surveillance. Despite the fact that the ‘‘caretaker’’ ATM is not required for the V(D)J recombination process, it seems to be important for the surveillance of the reaction, especially with regard to efficient prevention of aberrant V(D)J recombination events. ATM has been shown to be recruited at DNA-dsb during V(D)J recombination of TCR-a loci in thymocytes (Perkins et al., 2002). ATM may carry out its ‘‘caretaker’’ function by preventing oncogenic translocations that may lead to the development of neoplasms. The same may be true for Nbs1 and gH2AX, which although dispensable for V(D)J recombination, are present at the RAG1/2-mediated DNA-dsb (Chen et al., 2000). The targeted disruption of NHEJ genes has contributed considerably to the understanding of the mechanisms leading to the onset of lymphomas/leukemias. The first evidence for a link between a defect in this class of factors and tumorigenesis was provided by the observation that Ku70-/- mice develop T-cell lymhomas (Li et al., 1998a) and that as mentioned previously, DNA-PKcs deficiency predisposes scid mice to the apparition of thymic lymphoblastic lymphomas (Jhappan et al., 1997). Additional evidence for this correlation was supplied through the analysis of crosses between NHEJ-deficient mice and p53 or ATM-mutant mice (Ferguson and Alt, 2001). Whereas p53-/- mice generally develop thymic lymphomas at approximately 5 months of age, doublemutant mice develop early-onset pro–B-cell lymphomas. These lymphomas are in most cases accompanied by characteristic t(12;15) translocations

278

pat ri ck r ev y

ET AL.

involving the IgH locus and c-myc. In contrast to the thymic lymphomas arising spontaneously in scid mice, the B-cell lymphomas in the case of NHEJ deficiency on p53-/- can be suppressed by inactivation of the Rag genes, suggesting that the mechanism of tumorigenesis in the case of NHEJ deficiency may indeed be initiated by inappropriate repair of DNA-dsb generated by the V(D)J recombinase (Difilippantonio et al., 2002; Zhu et al., 2002). Interestingly, in the case of Artemis/p53 double mutants, most B-cell lymphomas that arise harbor translocations and gene amplification that involve N-myc rather than c-myc, although the reason for this difference is not presently known (Rooney et al., 2004). The development of B-cell lymphomas in Artemis/p53 double-mutant mice argued in favor of Artemis being a genomic caretaker like the other NHEJ factors. This was also documented through the analysis of a series of four patients presenting with hypomorphic Artemis mutations (see Section 3.1.1.3), two of whom developed EBV-associated, very aggressive B-cell lymphomas (Moshous et al., 2003). In cases of DNA-ligase IV deficiency in humans, one of the patients described thus far has developed acute lymphoblastic leukemia (see Section 3.1.2.1) (Riballo et al., 1999). The infidelity of signal joint formation seen in this patient may have been an important parameter in the underlying changes inducing oncogenesis. There is evidence that error-prone pathways for the repair of DNA double strand breaks, most likely from the NHEJ pathway, may be critically involved in the generation of chromosomal instability and leukemia (Rassool, 2003). 6. Conclusions The immune system is the only biological system that needs irreversible somatic DNA modifications to efficiently develop and mature. This special feature implies that some factors (i.e., RAG1/2 and AID) that induce DNA changes (i.e., V[D]J recombination, CSR, and SHM) are uniquely expressed in specialized immune cells (i.e., T- and B-lymphocytes). In addition, a large set of ubiquitously expressed DNA repair factors, comprising DNA damage sensors, mediators, and effectors, are also required to ensure the normal development of the immune system. The study of inherited human immunodeficiencies and murine models has been highly instrumental in understanding the DNA damage/DNA repair events that occur during the recombination processes of Ig and TCR genes. The defects in other DNA repair factors, not necessarily directly involved in the DNA-dsb repair process, but rather in the surveillance of the genome integrity, can also result in variable immunodeficiency (53BP1, UNG deficiencies) sometimes associated with various developmental defects (often neurological impairment) as observed in A-T and NBS. The connection between DNA repair factors and the immune system is also

d na r e pa i r i n t h e i m m un e sy s t e m

279

highlighted by the increased occurrence of lymphomas/leukemias observed in several DNA repair deficiencies as a consequence of illegitimate recombinations (i.e., translocation) during V(D)J recombination and CSR. The description of human conditions characterized by the association of microcephaly, increased radiosensitivity, and a variable degree of immunodeficiency sometimes associated with defective CSR-specific DNA-dsb repair of yet unknown molecular origin(s) clearly indicates that additional DNA repair factors contribute to the development and the maturation of the immune system. The molecular dissection of these human conditions, together with the development of new animal models, will contribute to a better knowledge of the complex relations between DNA metabolism and the immune system. References Abner, C. W., and McKinnon, P. J. (2004). The DNA double-strand break response in the nervous system. DNA Repair (Amst) 3, 1141–1147. Agarwal, M. L., Agarwal, A., Taylor, W. R., and Stark, G. R. (1995). p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts. Proc. Natl. Acad. Sci. USA 92, 8493–8497. Agrawal, A., Eastman, Q. M., and Schatz, D. G. (1998). Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394, 744–751. Allen, R. C., Armitage, R. J., Conley, M. E., Rosenblatt, H., Jenkins, N. A., Copeland, N. G., Bedell, M. A., Edelhoff, S., Disteche, C. M., Simoneaux, D. K., Fanslow, W. C., Belmont, J., and Spriggs, M. K. (1993). CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science 259, 990–993. Aravind, L. (1999). An evolutionary classification of the metallo-beta-lactamase fold proteins. In. Silico. Biol. 1, 69–91. Aruffo, A., Farrington, M., Hollenbaugh, D., Li, X., Milatovich, A., Nonoyama, S., Bajorath, J., Grosmaire, L. S., Stenkamp, R., Neubauer, M., Roberts, R. L., Noelle, R. J., Ledbetter, J. A., Francke, U., and Ochs, H. D. (1993). The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome. Cell 72, 291–300. Badie, C., Goodhardt, M., Waugh, A., Doyen, N., Foray, N., Calsou, P., Singleton, B., Gell, D., Salles, B., Jeggo, P., Arlett, C. F., and Malaise, E. P. (1997). A DNA double-strand break defective fibroblast cell line (180BR) derived from a radiosensitive patient represents a new mutant phenotype. Cancer Res. 57, 4600–4607. Bakkenist, C. J., and Kastan, M. B. (2003). DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506. Bardwell, P. D., Woo, C. J., Wei, K., Li, Z., Martin, A., Sack, S. Z., Parris, T., Edelmann, W., and Scharff, M. D. (2004). Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice. Nat. Immunol. 5, 224–229. Barnes, D. E., Tomkinson, A. E., Lehmann, A. R., Webster, A. D., and Lindahl, T. (1992). Mutations in the DNA ligase I gene of an individual with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. Cell 69, 495–503. Bartek, J., and Lukas, J. (2001). Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr. Opin. Cell. Biol. 13, 738–747.

280

pat ri ck r ev y

ET AL.

Bassing, C. H., and Alt, F. W. (2004). H2AX may function as an anchor to hold broken chromosomal DNA ends in close proximity. Cell Cycle 3, 149–153. Bassing, C. H., Swat, W., and Alt, F. W. (2002). The mechanism and regulation of chromosomal V(D)J recombination. Cell 109, S45–S55. Beamish, H., and Lavin, M. F. (1994). Radiosensitivity in ataxia-telangiectasia: Anomalies in radiation-induced cell cycle delay. Int. J. Radiat. Biol. 65, 175–184. Becker-Catania, S. G., Chen, G., Hwang, M. J., Wang, Z., Sun, X., Sanal, O., BernatowskaMatuszkiewicz, E., Chessa, L., Lee, E. Y., and Gatti, R. A. (2000). Ataxia-telangiectasia: Phenotype/genotype studies of ATM protein expression, mutations, and radiosensitivity. Mol. Genet. Metab. 70, 122–133. Begum, N. A., Kinoshita, K., Kakazu, N., Muramatsu, M., Nagaoka, H., Shinkura, R., Biniszkiewicz, D., Boyer, L. A., Jaenisch, R., and Honjo, T. (2004a). Uracil DNA glycosylase activity is dispensable for immunoglobulin class switch. Science 305, 1160–1163. Begum, N. A., Kinoshita, K., Muramatsu, M., Nagaoka, H., Shinkura, R., and Honjo, T. (2004b). De novo protein synthesis is required for activation-induced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination. Proc. Natl. Acad. Sci. USA 101, 13003–13007. Bemark, M., Sale, J. E., Kim, H. J., Berek, C., Cosgrove, R. A., and Neuberger, M. S. (2000). Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) or recombination-activating gene (RAG)1 activity. J. Exp. Med. 192, 1509–1514. Bertocci, B., De Smet, A., Flatter, E., Dahan, A., Bories, J. C., Landreau, C., Weill, J. C., and Reynaud, C. A. (2002). Cutting edge: DNA polymerases mu and lambda are dispensable for Ig gene hypermutation. J. Immunol. 168, 3702–3706. Bertrand, Y., Landais, P., Friedrich, W., Gerritsen, B., Morgan, G., Fasth, A., Cavazzana-Calvo, M., Porta, F., Cant, A., Espanol, T., Muller, S., Veys, P., Vossen, J., Haddad, E., and Fischer, A. (1999). Influence of severe combined immunodeficiency phenotype on the outcome of HLA non-identical, T-cell-depleted bone marrow transplantation: A retrospective European survey from the European group for bone marrow transplantation and the european society for immunodeficiency. J. Pediatr. 134, 740–748. Biedermann, K. A., Sun, J. R., Giaccia, A. J., Tosto, L. M., and Brown, J. M. (1991). scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc. Natl. Acad. Sci. USA 88, 1394–1397. Biemond, A. (1957). Acute ataxia in children. Ned. Tijdschr. Geneeskd. 101, 529–532. Blunt, T., Gell, D., Fox, M., Taccioli, G. E, Lehmann, A. R., Jackson, S. P., and Jeggo, P. A. (1996). Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc. Natl. Acad. Sci. USA 93, 10285–10290. Boder, E., and Sedgwick, R. P. (1958). Ataxia-telangiectasia; a familial syndrome of progressive cerebellar ataxia, oculocutaneous telangiectasia and frequent pulmonary infection. Pediatrics 21, 526–554. Boder, E., and Sedgwick, R. P. (1970). Ataxia-telangiectasia. (Clinical and immunological aspects). Psychiatr. Neurol. Med. Psychol. Beih. 13–14, 8–16. Bogue, M. A., Wang, C., Zhu, C., and Roth, D. B. (1997). V(D)J recombination in Ku86-deficient mice: Distinct effects on coding, signal, and hybrid joint formation. Immunity 7, 37–47. Bosma, G. C., Kim, J., Urich, T., Fath, D. M., Cotticelli, M. G., Ruetsch, N. R., Radic, M. Z., and Bosma, M. J. (2002). DNA-dependent protein kinase activity is not required for immunoglobulin class switching. J. Exp. Med. 196, 1483–1495. Bosma, M. J., and Carroll, A. M. (1991). The SCID mouse mutant: Definition, characterization, and potential uses. Ann. Rev. Immunol. 9, 323–350.

d na r e pa i r i n t h e i m m un e sy s t e m

281

Boubnov, N. V., and Weaver, D. T. (1995). scid cells are deficient in Ku and replication protein A phosphorylation by the DNA-dependent protein kinase. Mol. Cell. Biol. 15, 5700–5706. Brandt, V. L., and Roth, D. B. (2002). A recombinase diversified: New functions of the RAG proteins. Curr. Opin. Immunol. 14, 224–229. Bransteitter, R., Pham, P., Scharff, M. D., and Goodman, M. F. (2003). Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100, 4102–4107. Brar, S., Watson, M., and Diaz, M. (2004). Activation-induced cytosine deaminase, AID, is actively exported out of the nucleus but retained by the induction of DNA breaks. J. Biol. Chem. 279, 26395–26401. Bross, L., Fukita, Y., McBlane, F., Demolliere, C., Rajewsky, K., and Jacobs, H. (2000). DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermutation. Immunity 13, 589–597. Bross, L., Muramatsu, M., Kinoshita, K., Honjo, T., and Jacobs, H. (2002). DNA double-strand breaks: Prior to but not sufficient in targeting hypermutation. J. Exp. Med. 195, 1187–1192. Bross, L., Wesoly, J., Buerstedde, J. M., Kanaar, R., and Jacobs, H. (2003). Somatic hypermutation does not require Rad54 and Rad54B-mediated homologous recombination. Eur. J. Immunol. 33, 352–357. Brown, E. J., and Baltimore, D. (2003). Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes. Dev. 17, 615–628. Bryans, M., Valenzano, M. C., and Stamato, T. D. (1999). Absence of DNA ligase IV protein in XR-1 cells: Evidence for stabilization by XRCC4. Mutat. Res. 433, 53–58. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A., and Chen, D. J. (2001). ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem. 276, 42462–42467. Callebaut, I., and Mornon, J. P. (1997). From BRCA1 to RAP1: A widespread BRCT module closely associated with DNA repair. FEBS. Lett. 400, 25–30. Callebaut, I., Moshous, D., Mornon, J. P., and de Villartay, J. P. (2002). Metallo-beta-lactamase fold within nucleic acids processing enzymes: The beta-CASP family. Nucleic Acids Res. 30, 3592–3601. Carney, J. P., Maser, R. S., Olivares, H., Davis, E. M., Le Beau, M., Yates, J. R. 3rd, Hays, L., Morgan, W. F., and Petrini, J. H. (1998). The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: Linkage of double-strand break repair to the cellular DNA damage response. Cell 93, 477–486. Cascalho, M., Wong, J., Steinberg, C., and Wabl, M. (1998). Mismatch repair co-opted by hypermutation. Science 279, 1207–1210. Casellas, R., Nussenzweig, A., Wuerffel, R., Pelanda, R., Reichlin, A., Suh, H., Qin, X. F., Besmer, E., Kenter, A., Rajewsky, K., and Nussenzweig, M. C. (1998). Ku80 is required for immunoglobulin isotype switching. EMBO J 17, 2404–2411. Catalan, N., Selz, F., Imai, K., Revy, P., Fischer, A., and Durandy, A. (2003). The block in immunoglobulin class switch recombination caused by activation-induced cytidine deaminase deficiency occurs prior to the generation of DNA double strand breaks in switch mu region. J. Immunol. 171, 2504–2509. Cavazzana-Calvo, M., Le Deist, F., De Saint Basile, G., Papadopoulo, D., De Villartay, J. P., and Fischer, A. (1993). Increased radiosensitivity of granulocyte macrophage colony-forming units and skin fibroblasts in human autosomal recessive severe combined immunodeficiency. J. Clin. Invest. 91, 1214–1218. Celeste, A., Petersen, S., Romanienko, P. J., Fernandez-Capetillo, O., Chen, H. T., Sedelnikova, O. A., Reina-San-Martin, B., Coppola, V., Meffre, E., Difilippantonio, M. J., Redon, C., Pilch, D. R., Olaru, A., Eckhaus, M., Camerini-Otero, R. D., Tessarollo, L., Livak, F., Manova, K.,

282

pat ri ck r ev y

ET AL.

Bonner, W. M., Nussenzweig, M. C., and Nussenzweig, A. (2002). Genomic instability in mice lacking histone H2AX,. Science 296, 922–927. Chaturvedi, P., Eng, W. K., Zhu, Y., Mattern, M. R., Mishra, R., Hurle, M. R., Zhang, X., Annan, R. S., Lu, Q., Faucette, L. F., Scott, G. F., Li, X., Carr, S. A., Johnson, R. K., Winkler, J. D., and Zhou, B. B. (1999). Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Oncogene 18, 4047–4054. Chaudhuri, J., Khuong, C., and Alt, F. W. (2004). Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature 430, 992–998. Chaudhuri, J., Tian, M., Khuong, C., Chua, K., Pinaud, E., and Alt, F. W. (2003). Transcriptiontargeted DNA deamination by the AID antibody diversification enzyme. Nature 422, 726–730. Chen, H. T., Bhandoola, A., Difilippantonio, M. J., Zhu, J., Brown, M. J., Tai, X., Rogakou, E. P., Brotz, T. M., Bonner, W. M., Ried, T., and Nussenzweig, A. (2000). Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX. Science 290, 1962–1965. Chen, M. S., Ryan, C. E., and Piwnica-Worms, H. (2003). Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding. Mol. Cell. Biol. 23, 7488–7497. Chen, X., Kinoshita, K., and Honjo, T. (2001). Variable deletion and duplication at recombination junction ends: Implication for staggered double-strand cleavage in class-switch recombination. Proc. Natl. Acad. Sci. USA 98, 13860–13865. Chun, H. H., and Gatti, R. A. (2004). Ataxia-telangiectasia, an evolving phenotype. DNA Repair (Amst) 3, 1187–1196. Cook, A. J., Oganesian, L., Harumal, P., Basten, A., Brink, R., and Jolly, C. J. (2003). Reduced switching in SCID B cells is associated with altered somatic mutation of recombined S regions. J. Immunol. 171, 6556–6564. Cornforth, M. N., and Bedford, J. S. (1985). On the nature of a defect in cells from individuals with ataxia-telangiectasia. Science 227, 1589–1591. Critchlow, S. E., Bowater, R. P., and Jackson, S. P. (1997). Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr. Biol. 7, 588–598. Dai, Y., Kysela, B., Hanakahi, L. A., Manolis, K., Riballo, E., Stumm, M., Harville, T. O., West, S. C., Oettinger, M. A., and Jeggo, P. A. (2003). Nonhomologous end joining and V(D)J recombination require an additional factor. Proc. Natl. Acad. Sci. USA 100, 2462–2467. Daiyasu, H., Osaka, K., Ishino, Y., and Toh, H. (2001). Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold. FEBS. Lett. 503, 1–6. de Saint Basile, G., Tabone, M. D., Durandy, A., Phan, F., Fischer, A., and Le Deist, F. (1999). CD40 ligand expression deficiency in a female carrier of the X-linked hyper-IgM syndrome as a result of X chromosome lyonization. Eur. J. Immunol. 29, 367–373. de Villartay, J. P., Poinsignon, C., de Chasseval, R., Buck, D., Le Guyader, G., and Villey, I. (2003). Human and animal models of V(D)J recombination deficiency. Curr. Opin. Immunol. 15, 592–598. Diaz, M., Verkoczy, L. K., Flajnik, M. F., and Klinman, N. R. (2001). Decreased frequency of somatic hypermutation and impaired affinity maturation but intact germinal center formation in mice expressing antisense RNA to DNA polymerase zeta. J. Immunol. 167, 327–335. Dickerson, S. K., Market, E., Besmer, E., and Papavasiliou, F. N. (2003). AID mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197, 1291–1296. Difilippantonio, M. J., Petersen, S., Chen, H. T., Johnson, R., Jasin, M., Kanaar, R., Ried, T., and Nussenzweig, A. (2002). Evidence for replicative repair of DNA double-strand breaks leading to oncogenic translocation and gene amplification. J. Exp. Med. 196, 469–480. DiSanto, J. P., Bonnefoy, J. Y., Gauchat, J. F., Fischer, A., and de Saint Basile, G. (1993). CD40 ligand mutations in x-linked immunodeficiency with hyper-IgM. Nature 361, 541–543.

d na r e pa i r i n t h e i m m un e sy s t e m

283

Doi, T., Kinoshita, K., Ikegawa, M., Muramatsu, M., and Honjo, T. (2003). De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination. Proc. Natl. Acad. Sci. USA 100, 2634–2638. Durandy, A., Revy, P., and Fischer, A. (2004). Human models of inherited immunoglobulin class switch recombination and somatic hypermutation defects (hyper-IgM syndromes). Adv. Immunol. 82, 295–330. Durandy, A., Schiff, C., Bonnefoy, J. Y., Forveille, M., Rousset, F., Mazzei, G., Milili, M., and Fischer, A. (1993). Induction by anti-CD40 antibody or soluble CD40 ligand and cytokines of IgG, IgA and IgE production by B cells from patients with X-linked hyper IgM syndrome. Eur. J. Immunol. 23, 2294–2299. Durocher, D., and Jackson, S. P. (2001). DNA-PK, ATM and ATR as sensors of DNA damage: Variations on a theme? Curr. Opin. Cell. Biol. 13, 225–231. Ehrenstein, M. R., and Neuberger, M. S. (1999). Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation. EMBO J. 18, 3484–3490. Esposito, G., Texido, G., Betz, U. A., Gu, H., Muller, W., Klein, U., and Rajewsky, K. (2000). Mice reconstituted with DNA polymerase beta-deficient fetal liver cells are able to mount a T celldependent immune response and mutate their Ig genes normally. Proc. Natl. Acad. Sci. USA 97, 1166–1171. Fagarasan, S., Muramatsu, M., Suzuki, K., Nagaoka, H., Hiai, H., and Honjo, T. (2002). Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora. Science 298, 1424–1427. Faili, A., Aoufouchi, S., Weller, S., Vuillier, F., Stary, A., Sarasin, A., Reynaud, C. A., and Weill, J. C. (2004). DNA Polymerase {eta} Is Involved in Hypermutation Occurring during Immunoglobulin Class Switch Recombination. J. Exp. Med. 199, 265–270. Featherstone, C., and Jackson, S. P. (1998). DNA repair: The Nijmegen breakage syndrome protein. Curr. Biol. 8, R622–R625. Ferguson, D. O., and Alt, F. W. (2001). DNA double strand break repair and chromosomal translocation: Lessons from animal models. Oncogene 20, 5572–5579. Ferrari, S., Giliani, S., Insalaco, A., Al-Ghonaium, A., Soresina, A. R., Loubser, M., Avanzini, M. A., Marconi, M., Badolato, R., Ugazio, A. G., Levy, Y., Catalan, N., Durandy, A., Tbakhi, A., Notarangelo, L. D., and Plebani, A. (2001). Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM. Proc. Natl. Acad. Sci. USA 98, 12614–12619. Fischer, A. (2000). Severe combined immunodeficiencies (SCID). Clin. Exp. Immunol. 122, 143–149. Frank, K. M., Sekiguchi, J. M., Seidl, K. J., Swat, W., Rathbun, G. A., Cheng, H. L., Davidson, L., Kangaloo, L., and Alt, F. W. (1998). Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 396, 173–177. Fulop, G. M., and Phillips, R. A. (1990). The scid mutation in mice causes a general defect in DNA repair. Nature 347, 479–482. Furuta, T., Takemura, H., Liao, Z. Y., Aune, G. J., Redon, C., Sedelnikova, O. A., Pilch, D. R., Rogakou, E. P., Celeste, A., Chen, H. T., Nussenzweig, A., Aladjem, M. I., Bonner, W. M., and Pommier, Y. (2003). Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J. Biol. Chem. 278, 20303–20312. Gao, Y., Chaudhuri, J., Zhu, C., Davidson, L., Weaver, D. T., and Alt, F. W. (1998). A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination. Immunity 9, 367–376. Gellert, M. (2002). V(D)J recombination: Rag proteins, repair factors, and regulation. Annu. Rev. Biochem. 71, 101–132.

284

pat ri ck r ev y

ET AL.

Girard, P. M., Foray, N., Stumm, M., Waugh, A., Riballo, E., Maser, R. S., Phillips, W. P., Petrini, J., Arlett, C. F., and Jeggo, P. A. (2000). Radiosensitivity in Nijmegen Breakage Syndrome cells is attributable to a repair defect and not cell cycle checkpoint defects. Cancer Res. 60, 4881–4888. Girard, P. M., Kysela, B., Harer, C. J., Doherty, A. J., and Jeggo, P. A. (2004). Analysis of DNA ligase IV mutations found in LIG4 syndrome patients: The impact of two linked polymorphisms. Hum. Mol. Genet 13, 2369–2376. Goldberg, M., Stucki, M., Falck, J., D’Amours, D., Rahman, D., Pappin, D., Bartek, J., and Jackson, S. P. (2003). MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421, 952–956. Grawunder, U., Wilm, M., Wu, X., Kulesza, P., Wilson, T. E., Mann, M., and Lieber, M. R. (1997). Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 388, 492–495. Grawunder, U., Zimmer, D., Kulesza, P., and Lieber, M. R. (1998a). Requirement for an interaction of XRCC4 with DNA ligase IV for wild-type V(D)J recombination and DNA double-strand break repair in vivo. J. Biol. Chem. 273, 24708–24714. Grawunder, U., Zimmer, D., and Leiber, M. R. (1998b). DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains. Curr. Biol. 8, 873–876. Haber, J. E. (2000). Partners and pathways repairing a double-strand break. Trends Genet. 16, 259–264. Harfe, B. D., and Jinks-Robertson, S. (2000). DNA mismatch repair and genetic instability. Annu. Rev. Genet. 34, 359–399. Harfst, E., Cooper, S., Neubauer, S., Distel, L., and Grawunder, U. (2000). Normal V(D)J recombination in cells from patients with Nijmegen breakage syndrome. Mol. Immunol. 37, 915–929. Hendrickson, E. A., Qin, X. Q., Bump, E. A., Schatz, D. G., Oettinger, M., and Weaver, D. T. (1991). A link between double-strand break-related repair and V(D)J recombination: The scid mutation. Proc. Natl. Acad. Sci. USA 88, 4061–4065. Hiom, K., and Gellert, M. (1998). Assembly of a 12/23 paired signal complex: A critical control point in V(D)J recombination. Mol. Cell 1, 1011–1019. Hiom, K., Melek, M., and Gellert, M. (1998). DNA transposition by the RAG1 and RAG2 proteins: A possible source of oncogenic translocations. Cell 94, 463–470. Honjo, T., Muramatsu, M., and Fagarasan, S. (2004). AID: How does it aid antibody diversity? Immunity 20, 659–668. Horejsi, Z., Falck, J., Bakkenist, C. J., Kastan, M. B., Lukas, J., and Bartek, J. (2004). Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation. Oncogene 23, 3122–3127. Imai, K., Catalan, N., Plebani, A., Marodi, L., Sanal, O., Kumaki, S., Nagendran, V., Wood, P., Glastre, C., Sarrot-Reynauld, F., Hermine, O., Forveille, M., Revy, P., Fischer, A., and Durandy, A. (2003a). Hyper-IgM syndrome type 4 with a B lymphocyte-intrinsic selective deficiency in Ig class-switch recombination. J. Clin. Invest. 112, 136–142. Imai, K., Slupphaug, G., Lee, W. I., Revy, P., Nonoyama, S., Catalan, N., Yel, L., Forveille, M., Kavli, B., Krokan, H. E., Ochs, H. D., Fischer, A., and Durandy, A. (2003b). Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nat. Immunol. 4, 1023–1028. Ito, S., Nagaoka, H., Shinkura, R., Begum, N., Muramatsu, M., Nakata, M., and Honjo, T. (2004). Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1. Proc. Natl. Acad. Sci. USA 101, 1975–1980.

d na r e pa i r i n t h e i m m un e sy s t e m

285

Izsvak, Z., Stuwe, E. E., Fiedler, D., Katzer, A., Jeggo, P. A., and Ivics, Z. (2004). Healing the wounds inflicted by sleeping beauty transposition by double-strand break repair in mammalian somatic cells. Mol. Cell. 13, 279–290. Jacob, J., and Kelsoe, G. (1992). In situ studies of the primary immune response to (4-hydroxy-3nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers. J. Exp. Med. 176, 679–687. Jacobs, H., Fukita, Y., van der Horst, G. T., de Boer, J., Weeda, G., Essers, J., de Wind, N., Engelward, B. P., Samson, L., Verbeek, S., de Murcia, J. M., de Murcia, G., te Riele, H., and Rajewsky, K. (1998). Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice. J. Exp. Med. 187, 1735–1743. Jhappan, C., Morse, H. C. 3rd, Fleischmann, R. D., Gottesman, M. M., and Merlino, G. (1997). DNA-PKcs: A T-cell tumour suppressor encoded at the mouse scid locus. Nat. Genet. 17, 483–486. Kaartinen, M., Griffiths, G. M., Markham, A. F., and Milstein, C. (1983). mRNA sequences define an unusually restricted IgG response to 2-phenyloxazolone and its early diversification. Nature 304, 320–324. Kaneko, Y. S., Watanabe, N., Morisaki, H., Akita, H., Fujimoto, A., Tominaga, K., Terasawa, M., Tachibana, A., Ikeda, K., and Nakanishi, M. (1999). Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase. Oncogene 18, 3673–3681. Kang, J., Bronson, R. T., and Xu, Y. (2002). Targeted disruption of NBS1 reveals its roles in mouse development and DNA repair. EMBO J. 21, 1447–1455. Kinzler, K. W., and Vogelstein, B. (1997). Cancer-susceptibility genes. Gatekeepers and caretakers. Nature 386, 761–763. Kirchgessner, C. U., Patil, C. K., Evans, J. W., Cuomo, C. A., Fried, L. M., Carter, T., Oettinger, M. A., and Brown, J. M. (1995). DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science 267, 1178–1183. Kirsch, I. R. (1994). V(D)J recombination and ataxia-telangiectasia: A review. Int. J. Radiat. Biol. 66, S97–S108. Klein, U., Rajewsky, K., and Kuppers, R. (1998). Human immunoglobulin (Ig)MþIgDþ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J. Exp. Med. 188, 1679–1689. Kobayashi, J., Tauchi, H., Sakamoto, S., Nakamura, A., Morishima, K., Matsuura, S., Kobayashi, T., Tamai, K., Tanimoto, K., and Komatsu, K. (2002). NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr. Biol. 12, 1846–1851. Kobayashi, N., Agematsu, K., Sugita, K., Sako, M., Nonoyama, S., Yachie, A., Kumaki, S., Tsuchiya, S., Ochs, H. D., Sugita, K., Fukushima, Y., and Komiyama, A. (2003). Novel Artemis gene mutations of radiosensitive severe combined immunodeficiency in Japanese families. Hum. Genet. 112, 348–352. Kong, Q., and Maizels, N. (1999). PMS2-deficiency diminishes hypermutation of a lambda1 transgene in young but not older mice. Mol. Immunol. 36, 83–91. Koonin, E. V., Altschul, S. F., and Bork, P. (1996). BRCA1 protein products . . . Functional motifs. Nat. Genet. 13, 266–268. Korthauer, U., Graf, D., Mages, H. W., Briere, F., Padayachee, M., Malcolm, S., Ugazio, A. G., Notarangelo, L. D., Levinsky, R. J., and Kroczek, R. A. (1993). Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature 361, 539–541. Kuppers, R., Klein, U., Hansmann, M. L., and Rajewsky, K. (1999). Cellular origin of human B-cell lymphomas. N. Engl. J. Med. 341, 1520–1529.

286

pat ri ck r ev y

ET AL.

Kutukculer, N., Aksoylar, S., Kansoy, S., Cetingul, N., and Notarangelo, L. D. (2003). Outcome of hematopoietic stem cell transplantation in hyper-IgM syndrome caused by CD40 deficiency. J. Pediatr. 143, 141–142. Kwong, P. C., O’Marcaigh, A. S., Howard, R., Cowan, M. J., and Frieden, I. J. (1999). Oral and genital ulceration: a unique presentation of immunodeficiency in Athabascan-speaking American Indian children with severe combined immunodeficiency. Arch. Dermatol. 135, 927–931. Lahdesmaki, A., Taylor, A. M., Chrzanowska, K. H., and Pan-Hammarstrom, Q. (2004). Delineation of the role of the Mre11 complex in class switch recombination. J. Biol. Chem. 279, 16479–16487. Lebecque, S., de Bouteiller, O., Arpin, C., Banchereau, J., and Liu, Y. J. (1997). Germinal center founder cells display propensity for apoptosis before onset of somatic mutation. J. Exp. Med. 185, 563–571. Lee, G. S., Neiditch, M. B., Salus, S. S., and Roth, D. B. (2004). RAG proteins shepherd doublestrand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination. Cell 117, 171–184. Lee, J. H., and Paull, T. T. (2004). Direct activation of the ATM protein kinase by the Mre11/ Rad50/Nbs1 complex. Science 304, 93–96. Lee, S. A., Dritschilo, A., and Jung, M. (2001). Role of ATM in oxidative stress-mediated c-Jun phosphorylation in response to ionizing radiation and CdCl2. J. Biol. Chem. 276, 11783–11790. Li, A., and Swift, M. (2000). Mutations at the ataxia-telangiectasia locus and clinical phenotypes of A-T patients. Am. J. Med. Genet. 92, 170–177. Li, G. C., Ouyang, H., Li, X., Nagasawa, H., Little, J. B., Chen, D. J., Ling, C. C., Fuks, Z., and Cordon-Cardo, C. (1998a). Ku70: A candidate tumor suppressor gene for murine T cell lymphoma. Mol. Cell 2, 1–8. Li, L., Drayna, D., Hu, D., Hayward, A., Gahagan, S., Pabst, H., and Cowan, M. J. (1998b). The gene for severe combined immunodeficiency disease in Athabascan-speaking Native Americans is located on chromosome 10p. Am. J. Hum. Genet. 62, 136–144. Li, L., Moshous, D., Zhou, Y., Wang, J., Xie, G., Salido, E., Hu, D., de Villartay, J. P., and Cowan, M. J. (2002). A founder mutation in Artemis, an SNM1-like protein, causes SCID in Athabascanspeaking Native Americans. J. Immunol. 168, 6323–6329. Li, M. J., Peakman, M. C., Golub, E. I., Reddy, G., Ward, D. C., Radding, C. M., and Maizels, N. (1996). Rad51 expression and localization in B cells carrying out class switch recombination. Proc. Natl. Acad. Sci. USA 93, 10222–10227. Li, Z., Otevrel, T., Gao, Y., Cheng, H. L., Seed, B., Stamato, T. D., Taccioli, G. E., and Alt, F. W. (1995). The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 83, 1079–1089. Li, Z., Scherer, S. J., Ronai, D., Iglesias-Ussel, M. D., Peled, J. U., Bardwell, P. D., Zhuang, M., Lee, K., Martin, A., Edelmann, W., and Scharff, M. D. (2004). Examination of Msh6- and Msh3deficient Mice in Class Switching Reveals Overlapping and Distinct Roles of MutS Homologues in Antibody Diversification. J. Exp. Med. 200, 47–59. Liao, M. J., and Van Dyke, T. (1999). Critical role for Atm in suppressing V(D)J recombinationdriven thymic lymphoma. Genes. Dev. 13, 1246–1250. Liu, Y. J., Malisan, F., de Bouteiller, O., Guret, C., Lebecque, S., Banchereau, J., Mills, F. C., Max, E. E., and Martinez-Valdez, H. (1996). Within germinal centers, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation. Immunity 4, 241–250. Longacre, A., Sun, T., Goldsby, R. E., Preston, B. D., and Storb, U. (2003). Ig gene somatic hypermutation in mice defective for DNA polymerase delta proofreading. Int. Immunol. 15, 477–481.

d na r e pa i r i n t h e i m m un e sy s t e m

287

Lopez-Girona, A., Furnari, B., Mondesert, O., and Russell, P. (1999). Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature 397, 172–175. Lou, Z., Minter-Dykhouse, K., Wu, X., and Chen, J. (2003). MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways. Nature 421, 957–961. Ma, Y., Pannicke, U., Schwarz, K., and Lieber, M. R. (2002). Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108, 781–794. Manis, J. P., Dudley, D., Kaylor, L., and Alt, F. W. (2002). IgH class switch recombination to IgG1 in DNA-PKcs-deficient B cells. Immunity 16, 607–617. Manis, J. P., Gu, Y., Lansford, R., Sonoda, E., Ferrini, R., Davidson, L., Rajewsky, K., and Alt, F. W. (1998). Ku70 is required for late B cell development and immunoglobulin heavy chain class switching. J. Exp. Med. 187, 2081–2089. Manis, J. P., Morales, J. C., Xia, Z., Kutok, J. L., Alt, F. W., and Carpenter, P. B. (2004). 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination. Nat. Immunol. 5, 481–487. Maraschio, P., Spadoni, E., Tanzarella, C., Antoccia, A., Di Masi, A., Maghnie, M., Varon, R., Demuth, I., Tiepolo, L., and Danesino, C. (2003). Genetic heterogeneity for a Nijmegen breakage-like syndrome. Clin. Genet. 63, 283–290. Marculescu, R., Le, T., Simon, P., Jaeger, U., and Nadel, B. (2002). V(D)J-mediated translocations in lymphoid neoplasms: A functional assessment of genomic instability by cryptic sites. J. Exp. Med. 195, 85–98. Martin, A., Li, Z., Lin, D. P., Bardwell, P. D., Iglesias-Ussel, M. D., Edelmann, W., and Scharff, M. D. (2003). Msh2 ATPase activity is essential for somatic hypermutation at a-T basepairs and for efficient class switch recombination. J. Exp. Med. 198, 1171–1178. Martomo, S. A., Yang, W. W., and Gearhart, P. J. (2004). A role for msh6 but not msh3 in somatic hypermutation and class switch recombination. J. Exp. Med. 200, 61–68. Matsuura, S., Tauchi, H., Nakamura, A., Kondo, N., Sakamoto, S., Endo, S., Smeets, D., Solder, B., Belohradsky, B. H., Der Kaloustian, V. M., Oshimura, M., Isomura, M., Nakamura, Y., and Komatsu, K. (1998). Positional cloning of the gene for Nijmegen breakage syndrome. Nat. Genet. 19, 179–181. McBlane, J. F., van Gent, D. C., Ramsden, D. A., Romeo, C., Cuomo, C. A., Gellert, M., and Oettinger, M. A. (1995). Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83, 387–395. McBride, K. M., Barreto, V., Ramiro, A. R., Stavropoulos, P., and Nussenzweig, M. C. (2004). Somatic Hypermutation Is Limited by CRM1-dependent Nuclear Export of Activation-induced Deaminase. J. Exp. Med. 199, 1235–1244. McDonald, J. P., Frank, E. G., Plosky, B. S., Rogozin, I. B., Masutani, C., Hanaoka, F., Woodgate, R., and Gearhart, P. J. (2003). 129-derived strains of mice are deficient in DNA polymerase iota and have normal immunoglobulin hypermutation. J. Exp. Med. 198, 635–643. McMahon, S. B., Wood, M. A., and Cole, M. D. (2000). The essential cofactor TRRAP recruits the histone acetyltransferase hGCN5 to c-Myc. Mol. Cell Biol. 20, 556–562. Meek, K., Gupta, S., Ramsden, D. A., and Lees-Miller, S. P. (2004). The DNA-dependent protein kinase: The director at the end. Immunol. Rev. 200, 132–141. Melek, M., Jones, J. M., O’Dea, M. H., Pais, G., Burke, T. R. Jr., Pommier, Y., Neamati, N., and Gellert, M. (2002). Effect of HIV integrase inhibitors on the RAG1/2 recombinase. Proc. Natl. Acad. Sci. USA 99, 134–137. Melo, J., and Toczyski, D. (2002). A unified view of the DNA-damage checkpoint. Curr. Opin. Cell. Biol. 14, 237–245.

288

pat ri ck r ev y

ET AL.

Messier, T. L., O’Neill, J. P., Hou, S. M., Nicklas, J. A., and Finette, B. A. (2003). In vivo transposition mediated by V(D)J recombinase in human T lymphocytes,. EMBO J. 22, 1381–1388. Minegishi, Y., Lavoie, A., Cunningham-Rundles, C., Bedard, P. M., Hebert, J., Cote, L., Dan, K., Sedlak, D., Buckley, R. H., Fischer, A., Durandy, A., and Conley, M. E. (2000). Mutations in activation-induced cytidine deaminase in patients with hyper IgM syndrome. Clin. Immunol. 97, 203–210. Mombaerts, P., Iacomini, J., Johnson, R. S., Herrup, K., Tonegawa, S., and Papaioannou, V. E. (1992). RAG-1 deficient mice have no mature B and T lymphocytes. Cell 68, 869–877. Moshous, D., Callebaut, I., de Chasseval, R., Corneo, B., Cavazzana-Calvo, M., Le Deist, F., Tezcan, I., Sanal, O., Bertrand, Y., Philippe, N., Fischer, A., and de Villartay, J. P. (2001). Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell 105, 177–186. Moshous, D., Li, L., Chasseval, R., Philippe, N., Jabado, N., Cowan, M. J., Fischer, A., and de Villartay, J. P. (2000). A new gene involved in DNA double-strand break repair and V(D)J recombination is located on human chromosome. 10p. Hum. Mol. Genet. 9, 583–588. Moshous, D., Pannetier, C., Chasseval, Rd. R, Deist, F. F., Cavazzana-Calvo, M., Romana, S., Macintyre, E., Canioni, D., Brousse, N., Fischer, A., Casanova, J. L., and Villartay, J. P. (2003). Partial T and B lymphocyte immunodeficiency and predisposition to lymphoma in patients with hypomorphic mutations in Artemis. J. Clin. Invest. 111, 381–387. Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563. Muramatsu, M., Sankaranand, V. S., Anant, S., Sugai, M., Kinoshita, K., Davidson, N. O., and Honjo, T. (1999). Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J. Biol. Chem. 274, 18470–18476. Nambu, Y., Sugai, M., Gonda, H., Lee, C. G., Katakai, T., Agata, Y., Yokota, Y., and Shimizu, A. (2003). Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 302, 2137–2140. Nicolas, N., Finnie, N. J., Cavazzana-Calvo, M., Papadopoulo, D., Le Deist, F., Fischer, A., Jackson, S. P., and de Villartay, J. P. (1996). Lack of detectable defect in DNA double-strand break repair and DNA-dependent protein kinase activity in radiosensitive human severe combined immunodeficiency fibroblasts. Eur. J. Immunol. 26, 1118–1122. Nicolas, N., Moshous, D., Cavazzana-Calvo, M., Papadopoulo, D., de Chasseval, R., Le Deist, F., Fischer, A., and de Villartay, J. P. (1998). A human severe combined immunodeficiency (SCID) condition with increased sensitivity to ionizing radiations and impaired V(D)J rearrangements defines a new DNA recombination/repair deficiency. J. Exp. Med. 188, 627–634. Nikiforov, M. A., Chandriani, S., Park, J., Kotenko, I., Matheos, D., Johnsson, A., McMahon, S. B., and Cole, M. D. (2002). TRRAP-dependent and TRRAP-independent transcriptional activation by Myc family oncoproteins. Mol. Cell. Biol. 22, 5054–5063. Nilsen, H., Stamp, G., Andersen, S., Hrivnak, G., Krokan, H. E., Lindahl, T., and Barnes, D. E. (2003). Gene-targeted mice lacking the Ung uracil-DNA glycosylase develop B-cell lymphomas. Oncogene 22, 5381–5386. Noordzij, J. G., Verkaik, N. S., van der Burg, M., van Veelen, L. R., de Bruin-Versteeg, S., Wiegant, W., Vossen, J. M., Weemaes, C. M., de Groot, R., Zdzienicka, M. Z., van Gent, D. C., and van Dongen, J. J. (2003). Radiosensitive SCID patients with Artemis gene mutations show a complete B-cell differentiation arrest at the pre-B-cell receptor checkpoint in bone marrow. Blood 101, 1446–1452.

d na r e pa i r i n t h e i m m un e sy s t e m

289

O’Driscoll, M., Cerosaletti, K. M., Girard, P. M., Dai, Y., Stumm, M., Kysela, B., Hirsch, B., Gennery, A., Palmer, S. E., Seidel, J., Gatti, R. A., Varon, R., Oettinger, M. A., Neitzel, H., Jeggo, P. A., and Concannon, P. (2001). DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency. Mol. Cell. 8, 1175–1185. O’Marcaigh, A. S., DeSantes, K., Hu, D., Pabst, H., Horn, B., Li, L., and Cowan, M. J. (2001). Bone marrow transplantation for T-B- severe combined immunodeficiency disease in Athabascan-speaking native Americans. Bone Marrow Transplant 27, 703–709. Oettinger, M. A., Schatz, D. G., Gorka, C., and Baltimore, D. (1990). RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248, 1517–1523. Okazaki, I. M., Hiai, H., Kakazu, N., Yamada, S., Muramatsu, M., Kinoshita, K., and Honjo, T. (2003). Constitutive expression of AID leads to tumorigenesis. J. Exp. Med. 197, 1173–1181. Pan, Q., Petit-Frere, C., Lahdesmaki, A., Gregorek, H., Chrzanowska, K. H., and Hammarstrom, L. (2002). Alternative end joining during switch recombination in patients with ataxia-telangiectasia. Eur. J. Immunol. 32, 1300–1308. Pannicke, U., Ma, Y., Hopfner, K. P., Niewolik, D., Lieber, M. R., and Schwarz, K. (2004). Functional and biochemical dissection of the structure-specific nuclease ARTEMIS. EMBO J. 23, 1987–1997. Papavasiliou, F. N., and Schatz, D. G. (2000). Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes. Nature 408, 216–221. Papavasiliou, F. N., and Schatz, D. G. (2002). The activation-induced deaminase functions in a postcleavage step of the somatic hypermutation process. J. Exp. Med. 195, 1193–1198. Park, E. J., Chan, D. W., Park, J. H., Oettinger, M. A., and Kwon, J. (2003). DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner. Nucleic Acids Res. 31, 6819–6827. Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., and Bonner, W. M. (2000). A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895. Peakman, M. C., and Maizels, N. (1998). Localization of splenic B cells activated for switch recombination by in situ hybridization with Igamma1 switch transcript and Rad51 probes. J. Immunol. 161, 4008–4015. Peng, A., and Chen, P. L. (2003). NFBD1, like 53BP1, is an early and redundant transducer mediating Chk2 phosphorylation in response to DNA damage. J. Biol. Chem. 278, 8873–8876. Pergola, F., Zdzienicka, M. Z., and Lieber, M. R. (1993). V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair. Mol. Cell. Biol. 13, 3464–3471. Perkins, E. J., Nair, A., Cowley, D. O., Van Dyke, T., Chang, Y., and Ramsden, D. A. (2002). Sensing of intermediates in V(D)J recombination by ATM. Genes Dev. 16, 159–164. Petersen, S., Casellas, R., Reina-San-Martin, B., Chen, H. T., Difilippantonio, M. J., Wilson, P. C., Hanitsch, L., Celeste, A., Muramatsu, M., Pilch, D. R., Redon, C., Ried, T., Bonner, W. M., Honjo, T., Nussenzweig, M. C., and Nussenzweig, A. (2001). AID is required to initiate Nbs1/ gamma-H2AX focus formation and mutations at sites of class switching. Nature 414, 660–665. Petersen-Mahrt, S. K., Harris, R. S., and Neuberger, M. S. (2002). AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–103. Peterson, R. D., and Funkhouser, J. D. (1989). Speculations on ataxia-telangiectasia: defective regulation of the immunoglobulin gene superfamily. Immunol. Today 10, 313–314. Peterson, R. D., Kelly, W. D., and Good, R. A. (1964). Ataxia-telangiectasia. Its association with a defective thymus, immunological-deficiency disease, and malignancy. Lancet 13, 1189–1193. Petiniot, L. K., Weaver, Z., Barlow, C., Shen, R., Eckhaus, M., Steinberg, S. M., Ried, T., Wynshaw-Boris, A., and Hodes, R. J. (2000). Recombinase-activating gene (RAG) 2-mediated

290

pat ri ck r ev y

ET AL.

V(D)J recombination is not essential for tumorigenesis in Atm-deficient mice. Proc. Natl. Acad. Sci. USA 97, 6664–6669. Petrini, J. H., and Stracker, T. H. (2003). The cellular response to DNA double-strand breaks: Defining the sensors and mediators. Trends Cell. Biol. 13, 458–462. Pham, P., Bransteitter, R., Petruska, J., and Goodman, M. F. (2003). Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424, 103–107. Phung, Q. H., Winter, D. B., Cranston, A., Tarone, R. E., Bohr, V. A., Fishel, R., and Gearhart, P. J. (1998). Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein. J. Exp. Med. 187, 1745–1751. Pichierri, P., and Rosselli, F. (2004). The DNA crosslink-induced S-phase checkpoint depends on ATR-CHK1 and ATR-NBS1-FANCD2 pathways. EMBO J. 23, 1178–1187. Plowman, P. N., Bridges, B. A., Arlett, C. F., Hinney, A., and Kingston, J. E. (1990). An instance of clinical radiation morbidity and cellular radiosensitivity, not associated with ataxia-telangiectasia. Br. J. Radiol. 63, 624–628. Poinsignon, C., de Chasseval, R., Soubeyrand, S., Moshous, D., Fischer, A., Hache, R. J., and de Villartay, J. P. (2004a). Phosphorylation of Artemis following irradiation induced DNA damage. Eur. J. Immunol. 34, 3146–3155. Poinsignon, C., Moshous, D., Callebaut, I., de Chasseval, R., Villey, I., and de Villartay, J. P. (2004b). The Metallo-{beta}-Lactamase/{beta}-CASP Domain of Artemis Constitutes the Catalytic Core for V(D)J Recombination. J. Exp. Med. 199, 315–321. Purugganan, M. M., Shah, S., Kearney, J. F., and Roth, D. B. (2001). Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 29, 1638–1646. Qi, L., Strong, M. A., Karim, B. O., Armanios, M., Huso, D. L., and Greider, C. W. (2003). Short telomeres and ataxia-telangiectasia mutated deficiency cooperatively increase telomere dysfunction and suppress tumorigenesis. Cancer Res. 63, 8188–8196. Quartier, P., Bustamante, J., Sanal, O., Plebani, A., Debre, M., Deville, A., Litzman, J., Levy, J., Fermand, J. P., Lane, P., Horneff, G., Aksu, G., Yalcin, I., Davies, G., Tezcan, I., Ersoy, F., Catalan, N., Imai, K., Fischer, A., and Durandy, A. (2004). Clinical, immunologic and genetic analysis of 29 patients with autosomal recessive hyper-IgM syndrome due to Activation-Induced Cytidine Deaminase deficiency. Clin. Immunol. 110, 22–29. Rada, C., Ehrenstein, M. R., Neuberger, M. S., and Milstein, C. (1998). Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting. Immunity 9, 135–141. Rada, C., Williams, G. T., Nilsen, H., Barnes, D. E., Lindahl, T., and Neuberger, M. S. (2002). Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNGdeficient mice. Curr. Biol. 12, 1748–1755. Raghavan, S. C., Kirsch, I. R., and Lieber, M. R. (2001). Analysis of the V(D)J recombination efficiency at lymphoid chromosomal translocation breakpoints. J. Biol. Chem. 276, 29126–29133. Raghavan, S. C., and Lieber, M. R. (2004). Chromosomal translocations and non-B DNA structures in the human genome. Cell Cycle 3, 762–768. Raghavan, S. C., Swanson, P. C., Wu, X., Hsieh, C. L., and Lieber, M. R. (2004). A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex. Nature 428, 88–93. Ramiro, A. R., Jankovic, M., Eisenreich, T., Difilippantonio, S., Chen-Kiang, S., Muramatsu, M., Honjo, T., Nussenzweig, A., and Nussenzweig, M. C. (2004). AID is required for c-myc/IgH chromosome translocations in vivo. Cell 118, 431–438.

d na r e pa i r i n t h e i m m un e sy s t e m

291

Ramiro, A. R., Stavropoulos, P., Jankovic, M., and Nussenzweig, M. C. (2003). Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nat. Immunol. 4, 452–456. Rassool, F. V. (2003). DNA double strand breaks (DSB) and nonhomologous end joining (NHEJ) pathways in human leukemia. Cancer Lett. 193, 1–9. Reaban, M. E., and Griffin, J. A. (1990). Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region. Nature 348, 342–344. Reaban, M. E., Lebowitz, J., and Griffin, J. A. (1994). Transcription induces the formation of a stable RNA.DNA hybrid in the immunoglobulin alpha switch region. J. Biol. Chem. 269, 21850–21857. Redon, C., Pilch, D., Rogakou, E., Sedelnikova, O., Newrock, K., and Bonner, W. (2002). Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 12, 162–169. Reina-San-Martin, B., Difilippantonio, S., Hanitsch, L., Masilamani, R. F., Nussenzweig, A., and Nussenzweig, M. C. (2003). H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation. J. Exp. Med. 197, 1767–1778. Revy, P., Muto, T., Levy, Y., Geissmann, F., Plebani, A., Sanal, O., Catalan, N., Forveille, M., Dufourcq-Labelouse, R., Gennery, A., Tezcan, I., Ersoy, F., Kayserili, H., Ugazio, A. G., Brousse, N., Muramatsu, M., Notarangelo, L. D., Kinoshita, K., Honjo, T., Fischer, A., and Durandy, A. (2000). Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575. Riballo, E., Critchlow, S. E., Teo, S. H., Doherty, A. J., Priestley, A., Broughton, B., Kysela, B., Beamish, H., Plowman, N., Arlett, C. F., Lehmann, A. R., Jackson, S. P., and Jeggo, P. A. (1999). Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient. Curr. Biol. 9, 699–702. Riballo, E., Doherty, A. J., Dai, Y., Stiff, T., Oettinger, M. A., Jeggo, P. A., and Kysela, B. (2001). Cellular and biochemical impact of a mutation in DNA ligase IV conferring clinical radiosensitivity. J. Biol. Chem. 276, 31124–31132. Roddam, P. L., Rollinson, S., O’Driscoll, M., Jeggo, P. A., Jack, A., and Morgan, G. J. (2002). Genetic variants of NHEJ DNA ligase IV can affect the risk of developing multiple myeloma, a tumour characterised by aberrant class switch recombination. J. Med. Genet. 39, 900–905. Rogakou, E. P., Boon, C., Redon, C., and Bonner, W. M. (1999). Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916. Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., and Bonner, W. M. (1998). DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868. Rooney, S., Alt, F. W., Lombard, D., Whitlow, S., Eckersdorff, M., Fleming, J., Fugmann, S., Ferguson, D. O., Schatz, D. G., and Sekiguchi, J. (2003). Defective DNA repair and increased genomic instability in Artemis-deficient murine cells. J. Exp. Med. 197, 553–565. Rooney, S., Sekiguchi, J., Whitlow, S., Eckersdorff, M., Manis, J. P., Lee, C., Ferguson, D. O., and Alt, F. W. (2004). Artemis and p53 cooperate to suppress oncogenic N-myc amplification in progenitor B cells. Proc. Natl. Acad. Sci. USA 101, 2410–2415. Rooney, S., Sekiguchi, J., Zhu, C., Cheng, H. L., Manis, J., Whitlow, S., DeVido, J., Foy, D., Chaudhuri, J., Lombard, D., and Alt, F. W. (2002). Leaky scid phenotype associated with defective V(D)J coding end processing in artemis-deficient mice. Mol. Cell. 10, 1379–1390. Roth, D. B. (2003). Restraining the V(D)J recombinase. Nat. Rev. Immunol. 3, 656–666. Roth, D. B., and Craig, N. L. (1998). VDJ recombination: A transposase goes to work. Cell 94, 411–414.

292

pat ri ck r ev y

ET AL.

Roth, D. B., Menetski, J. P., Nakajima, P. B., Bosma, M. J., and Gellert, M. (1992). V(D)J recombination: Broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell 70, 983–991. Rush, J. S., Fugmann, S. D., and Schatz, D. G. (2004). Staggered AID-dependent DNA double strand breaks are the predominant DNA lesions targeted to S mu in Ig class switch recombination. Int. Immunol. 16, 549–557. Ryan, K., Calvo, O., and Manley, J. L. (2004). Evidence that polyadenylation factor CPSF-73 is the mRNA 30 processing endonuclease. Rna 10, 565–573. Sale, J. E., Calandrini, D. M., Takata, M., Takeda, S., and Neuberger, M. S. (2001). Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412, 921–926. Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., and Linn, S. (2004). Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints. Annu. Rev. Biochem. 73, 39–85. Schatz, D. G., Oettinger, M. A., and Baltimore, D. (1989). The V(D)J recombination activating gene, RAG-1. Cell 59, 1035–1048. Schenten, D., Gerlach, V. L., Guo, C., Velasco-Miguel, S., Hladik, C. L., White, C. L., Friedberg, E. C., Rajewsky, K., and Esposito, G. (2002). DNA polymerase kappa deficiency does not affect somatic hypermutation in mice. Eur. J. Immunol. 32, 3152–3160. Schlissel, M., Constantinescu, A., Morrow, T., Baxter, M., and Peng, A. (1993). Double-strand signal sequence breaks in V(D)J recombination are blunt, 50 -phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 7, 2520–2532. Schlissel, M. S. (1998). Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination. Mol. Cell. Biol. 18, 2029–2037. Schlissel, M. S. (2002). Does artemis end the hunt for the hairpin-opening activity in V(D)J recombination? Cell 109, 1–4. Schmutte, C., Sadoff, M. M., Shim, K. S., Acharya, S., and Fishel, R. (2001). The interaction of DNA mismatch repair proteins with human exonuclease I. J. Biol. Chem. 276, 33011–33018. Schrader, C. E., Edelmann, W., Kucherlapati, R., and Stavnezer, J. (1999). Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes. J. Exp. Med. 190, 323–330. Schwarz, K., Gauss, G. H., Ludwig, L., Pannicke, U., Li, Z., Lindner, D., Friedrich, W., Seger, R. A., Hansen-Hagge, T. E., Desiderio, S., Lieber, M. R., and Bartram, C. R. (1996). RAG mutations in human B cell-negative SCID. Science 274, 97–99. Shang, Y. L., Bodero, A. J., and Chen, P. L. (2003). NFBD1, a novel nuclear protein with signature motifs of FHA and BRCT, and an internal 41-amino acid repeat sequence, is an early participant in DNA damage response. J. Biol. Chem. 278, 6323–6329. Shiloh, Y. (2003). ATM and related protein kinases: Safeguarding genome integrity. Nat. Rev. Cancer 3, 155–168. Shinkai, Y., Rathbun, G., Lam, K. P., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron., J., Datta, M., Young, F., Stall, A. M., and Alt, F. W. (1992). RAG-2 deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855–867. Shinkura, R., Tian, M., Smith, M., Chua, K., Fujiwara, Y., and Alt, F. W. (2003). The influence of transcriptional orientation on endogenous switch region function. Nat. Immunol. 4, 435–441. Sibanda, B. L., Critchlow, S. E., Begun, J., Pei, X. Y., Jackson, S. P., Blundell, T. L., and Pellegrini, L. (2001). Crystal structure of an Xrcc4-DNA ligase IV complex. Nat. Struct. Biol. 8, 1015–1019. Smith, J., Riballo, E., Kysela, B., Baldeyron, C., Manolis, K., Masson, C., Lieber, M. R., Papadopoulo, D., and Jeggo, P. (2003). Impact of DNA ligase IV on the fidelity of end joining in human cells. Nucleic Acids Res. 31, 2157–2167.

d na r e pa i r i n t h e i m m un e sy s t e m

293

Sohail, A., Klapacz, J., Samaranayake, M., Ullah, A., and Bhagwat, A. S. (2003). Human activationinduced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations. Nucleic Acids Res. 31, 2990–2994. Spring, K., Cross, S., Li, C., Watters, D., Ben-Senior, L., Waring, P., Ahangari, F., Lu, S. L., Chen, P., Misko, I., Paterson, C., Kay, G., Smorodinsky, N. I., Shiloh, Y., and Lavin, M. F. (2001). Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype. Cancer Res. 61, 4561–4568. Stephan, J. L., Vlekova, V., Le Deist, F., Blanche, S., Donadieu, J., De Saint-Basile, G., Durandy, A., Griscelli, C., and Fischer, A. (1993). Severe combined immunodeficiency: A retrospective single-center study of clinical presentation and outcome in 117 patients. J. Pediatr. 123, 564–572. Stewart, G. S., Maser, R. S., Stankovic, T., Bressan, D. A., Kaplan, M. I., Jaspers, N. G., Raams, A., Byrd, P. J., Petrini, J. H., and Taylor, A. M. (1999). The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99, 577–587. Stewart, G. S., Wang, B., Bignell, C. R., Taylor, A. M., and Elledge, S. J. (2003). MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421, 961–966. Su, Y., and Swift, M. (2000). Mortality rates among carriers of ataxia-telangiectasia mutant alleles. Ann. Intern. Med. 133, 770–778. Swift, M., Morrell, D., Cromartie, E., Chamberlin, A. R., Skolnick, M. H., and Bishop, D. T. (1986). The incidence and gene frequency of ataxia-telangiectasia in the United States. Am. J. Hum. Genet. 39, 573–583. Ta, V. T., Nagaoka, H., Catalan, N., Durandy, A., Fischer, A., Imai, K., Nonoyama, S., Tashiro, J., Ikegawa, M., Ito, S., Kinoshita, K., Muramatsu, M., and Honjo, T. (2003). AID mutant analyses indicate requirement for class-switch-specific cofactors. Nat. Immunol. 4, 843–848. Taccioli, G. E., Amatucci, A. G., Beamish, H. J., Gell, D., Xiang, X. H., Torres Arzayus, M. I., Priestley, A., Jackson, S. P., Marshak Rothstein, A., Jeggo, P. A., and Herrera, V. L. (1998). Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity. Immunity 9, 355–366. Taccioli, G. E., Rathbun, G., Oltz, E., Stamato, T., Jeggo, P. A., and Alt, F. W. (1993). Impairment of V(D)J recombination in double-strand break repair mutants. Science 260, 207–210. Tauchi, H., Matsuura, S., Kobayashi, J., Sakamoto, S., and Komatsu, K. (2002). Nijmegen breakage syndrome gene, NBS1, and molecular links to factors for genome stability. Oncogene 21, 8967–8980. Taylor, A. M., Groom, A., and Byrd, P. J. (2004). Ataxia-telangiectasia-like disorder (ATLD)—its clinical presentation and molecular basis. DNA Repair (Amst) 3, 1219–1225. Tomkinson, A. E., and Mackey, Z. B. (1998). Structure and function of mammalian DNA ligases. Mutat. Res. 407, 1–9. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302, 575–581. Uziel, T., Lerenthal, Y., Moyal, L., Andegeko, Y., Mittelman, L., and Shiloh, Y. (2003). Requirement of the MRN complex for ATM activation by DNA damage. EMBO J. 22, 5612–5621. Uziel, T., Savitsky, K., Platzer, M., Ziv, Y., Helbitz, T., Nehls, M., Boehm, T., Rosenthal, A., Shiloh, Y., and Rotman, G. (1996). Genomic Organization of the ATM gene. Genomics 33, 317–320. van Engelen, B. G., Hiel, J. A., Gabreels, F. J., van den Heuvel, L. P., van Gent, D. C., and Weemaes, C. M. (2001). Decreased immunoglobulin class switching in Nijmegen Breakage syndrome due to the DNA repair defect. Hum. Immunol. 62, 1324–1327. Vanasse, G. J., Concannon, P., and Willerford, D. M. (1999). Regulated genomic instability and neoplasia in the lymphoid lineage. Blood 94, 3997–4010. Varon, R., Vissinga, C., Platzer, M., Cerosaletti, K. M., Chrzanowska, K. H., Saar, K., Beckmann, G., Seemanova, E., Cooper, P. R., Nowak, N. J., Stumm, M., Weemaes, C. M., Gatti, R. A.,

294

pat ri ck r ev y

ET AL.

Wilson, R. K., Digweed, M., Rosenthal, A., Sperling, K., Concannon, P., and Reis, A. (1998). Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 93, 467–476. Vora, K. A., Tumas-Brundage, K. M., Lentz, V. M., Cranston, A., Fishel, R., and Manser, T. (1999). Severe attenuation of the B cell immune response in Msh2-deficient mice. J. Exp. Med. 189, 471–482. Walworth, N. C. (2001). DNA damage: Chk1 and Cdc25, more than meets the eye. Curr. Opin. Genet. Dev. 11, 78–82. Wang, B., Matsuoka, S., Carpenter, P. B., and Elledge, S. J. (2002). 53BP1, a mediator of the DNA damage checkpoint. Science 298, 1435–1438. Wang, Z., Fast, W., Valentine, A. M., and Benkovic, S. J. (1999). Metallo-beta-lactamase: Structure and mechanism. Curr. Opin. Chem. Biol. 3, 614–622. Ward, I. M., and Chen, J. (2001). Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 276, 47759–47762. Ward, I. M., Minn, K., Jorda, K. G., and Chen, J. (2003). Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J. Biol. Chem. 278, 19579–19582. Ward, I. M., Reina-San-Martin, B., Olaru, A., Minn, K., Tamada, K., Lau, J. S., Cascalho, M., Chen, L., Nussenzweig, A., Livak, F., Nussenzweig, M. C., and Chen, J. (2004). 53BP1 is required for class switch recombination. J. Cell Biol. 165, 459–464. Weemaes, C. M., Hustinx, T. W., Scheres, J. M., van Munster, P. J., Bakkeren, J. A., and Taalman, R. D. (1981). A new chromosomal instability disorder: The Nijmegen breakage syndrome. Acta Paediatr. Scand. 70, 557–564. Weller, S., Braun, M. C., Tan, B. K., Rosenwald, A., Cordier, C., Conley, M. E., Plebani, A., Kumararatne, D. S., Bonnet, D., Tournilhac, O., Tchernia, G., Steiniger, B., Staudt, L. M., Casanova, J. L., Reynaud, C. A., and Weill, J. C. (2004). Human blood IgM ‘‘memory’’ B cells are circulating splenic marginal zone B cells harboring a pre-diversified immunoglobulin repertoire. Blood 104, 3647–3654. Weller, S., Faili, A., Garcia, C., Braun, M. C., Le Deist, F. F., de Saint Basile, G. G., Hermine, O., Fischer, A., Reynaud, C. A., and Weill, J. C. (2001). CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proc. Natl. Acad. Sci. USA 98, 1166–1170. Wiesendanger, M., Kneitz, B., Edelmann, W., and Scharff, M. D. (2000). Somatic hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern. J. Exp. Med. 191, 579–584. Williams, B. R., Mirzoeva, O. K., Morgan, W. F., Lin, J., Dunnick, W., and Petrini, J. H. (2002). A murine model of Nijmegen breakage syndrome. Curr. Biol. 12, 648–653. Winter, D. B., Phung, Q. H., Umar, A., Baker, S. M., Tarone, R. E., Tanaka, K., Liskay, R. M., Kunkel, T. A., Bohr, V. A., and Gearhart, P. J. (1998). Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2. Proc. Natl. Acad. Sci. USA 95, 6953–6958. Woods, C. G., and Taylor, A. M. (1992). Ataxia telangiectasia in the British Isles: The clinical and laboratory features of 70 affected individuals. Q. J. Med. 82, 169–179. Wuerffel, R. A., Du, J., Thompson, R. J., and Kenter, A. L. (1997). Ig Sgamma3 DNA-specifc double strand breaks are induced in mitogen-activated B cells and are implicated in switch recombination. J. Immunol. 159, 4139–4144.

d na r e pa i r i n t h e i m m un e sy s t e m

295

Xiao, Z., Chen, Z., Gunasekera, A. H., Sowin, T. J., Rosenberg, S. H., Fesik, S., and Zhang, H. (2003). Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNAdamaging agents. J. Biol. Chem. 278, 21767–21773. Xu, X., and Stern, D. F. (2003). NFBD1/MDC1 regulates ionizing radiation-induced focus formation by DNA checkpoint signaling and repair factors. Faseb. J 17, 1842–1848. Yamane, K., Wu, X., and Chen, J. (2002). A DNA damage-regulated BRCT-containing protein, TopBP1, is required for cell survival. Mol. Cell. Biol. 22, 555–566. Yavuz, S., Yavuz, A. S., Kraemer, K. H., and Lipsky, P. E. (2002). The role of polymerase eta in somatic hypermutation determined by analysis of mutations in a patient with xeroderma pigmentosum variant. J. Immunol. 169, 3825–3830. Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E., and Lieber, M. R. (2003). R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442–451. Zan, H., Komori, A., Li, Z., Cerutti, A., Schaffer, A., Flajnik, M. F., Diaz, M., and Casali, P. (2001). The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation. Immunity 14, 643–653. Zan, H., Wu, X., Komori, A., Holloman, W. K., and Casali, P. (2003). AID-dependent generation of resected double-strand DNA breaks and recruitment of Rad52/Rad51 in somatic hypermutation. Immunity 18, 727–738. Zeng, X., Negrete, G. A., Kasmer, C., Yang, W. W., and Gearhart, P. J. (2004). Absence of DNA polymerase {eta} reveals targeting of C mutations on the nontranscribed strand in immunoglobulin switch regions. J. Exp. Med. 199, 917–924. Zeng, X., Winter, D. B., Kasmer, C., Kraemer, K. H., Lehmann, A. R., and Gearhart, P. J. (2001). DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes. Nat. Immunol. 2, 537–541. Zhu, C., Mills, K. D., Ferguson, D. O., Lee, C., Manis, J., Fleming, J., Gao, Y., Morton, C. C., and Alt, F. W. (2002). Unrepaired DNA breaks in P53-deficient cells lead to oncogenic gene amplification subsequent to translocation. Cell 109, 811–821. Zhu, J., Petersen, S., Tessarollo, L., and Nussenzweig, A. (2001). Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice. Curr. Biol. 11, 105–109. Zhu, Y., Nonoyama, S., Morio, T., Muramatsu, M., Honjo, T., and Mizutani, S. (2003). Type two hyper-IgM syndrome caused by mutation in activation-induced cytidine deaminase. J. Med. Dent. Sci. 50, 41–46.

Antibody Class Switch Recombination: Roles for Switch Sequences and Mismatch Repair Proteins Irene M. Min* and Erik Selsing{ *Genetics Program and {Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111

1. 2. 3. 4. 5.

Abstract............................................................................................................. Introduction ....................................................................................................... Class Switch Recombination.................................................................................. Targeting of the CSR Mechanism ........................................................................... Proteins Involved in CSR...................................................................................... Concluding Remarks............................................................................................ References .........................................................................................................

297 297 298 300 305 319 320

Abstract Mechanisms and targeting of antibody class switch DNA recombination are reviewed. Particular emphasis is on the roles for the DNA sequences comprising switch (S) regions, including the S-region tandem repeats, and on the roles of proteins that are involved in both DNA mismatch repair and in class switch recombination.

1. Introduction The class switch recombination (CSR) mechanism diversifies antibody effector functions by changing the constant regions of antibodies. CSR is mediated by DNA recombination events that juxtapose recombined H-chain variable-diversity-joining (V[D]J) segments with different downstream constant region (CH) gene segments (Honjo and Kataoka, 1978; Iwasato et al., 1990; Matsuoka et al., 1990; von Schwedler et al., 1990). CSR takes place between S regions that are located 50 to each CH region gene; S regions contain unusual tandem-repeat sequences that are important for CSR (Kataoka et al., 1981). The mechanisms, targeting, and regulation of CSR are not yet clear, although there is considerable information to support proposed models for the process. Many proteins that play roles in various DNA repair and recombination mechanisms appear to be actively involved in the CSR mechanism.

297 advances in immunology, vol. 87 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)87008-7

298

ir e ne m . m i n an d e ri k s el si n g

2. Class Switch Recombination 2.1. What Is CSR? Upon antigen challenge, mature B cells expressing IgM and/or IgD can differentiate to express IgG, IgA, or IgE by the process of isotype switching (Honjo et al., 2002; Stavnezer, 1996). This process occurs by class switch DNA recombination (CSR) events that juxtapose a recombined V(D)J region gene segment with one of several downstream constant (CH) region segments within the same chromosome, using a mechanism that loops out and deletes the intervening DNA sequences (Fig. 1) (Honjo et al., 2002; Stavnezer, 1996). The circular DNAs containing the intervening DNA sequences that were deleted during the switching process can be found in cells undergoing switching, supporting the model that switch recombination occurs by a looping out and deletion event (Iwasato et al., 1990; Matsuoka et al., 1990; von Schwedler

Figure 1 Antibody class switch recombination mechanism in mature B cells. Class switch recombination (CSR) is a process by which mature B cells can diversify the antibody effector function. The diagram depicts the CSR process whereby the expressed antibody class is changed from IgM to express IgE. CSR occurs by DNA recombination events that juxtapose a recombined variable (V) gene segment with one of several downstream constant (CH) region segments within the same chromosome. The switch (S) region, an intronic region that is located 50 of each CH gene, has been thought to be involved in mediating the class switch DNA recombination.

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

299

et al., 1990). CSR allows a change in the effector functions of antibodies without changing the antigen specificity. There are eight heavy chain constant region genes in the mouse IgH locus; this locus is organized as 50 -VDJ-Cm-Cd-Cg3-Cg1-Cg2b-Cg2a-C-Ca-30 . CSR occurs between intronic regions located upstream of each CH gene; these intronic regions have been termed switch (S) regions. A number of studies indicate that CSR generally occurs between Sm and one of several downstream S regions (Dunnick et al., 1993). 2.2. The Microenvironments of CSR The majority of B-cell CSR events occur in cells that have been stimulated by antigens within germinal centers (Honjo et al., 2002). These stimulated B cells also undergo somatic hypermutation (SHM), a process that introduces a high level of point mutations into the V(D)J region. Even though SHM and CSR occur in B cells at a similar stage of development and in the same microenvironment, cases have been reported where somatic mutations were found in the V(D)J segments of IgM antibodies (Mantovani et al., 1993; Sohn et al., 1993) or where unmutated IgG antibodies appeared during a primary immune response (Apel and Berek, 1990). Both SHM and CSR require the B-cell-specific protein activation-induced cytidine deaminase (AID) (discussed later in Section 4.1.1); however, mutations in different domains of AID affected SHM and CSR mechanism in an uncoupled manner (Shinkura et al., 2004). These observations indicate that SHM and CSR are independent processes. 2.3. S-Region Transcription Induced by Cytokines Plays a Crucial Role in CSR It has been shown that transcription of unrearranged CH genes precedes isotype switching (Lutzker et al., 1988; Rothman et al., 1988; StavnezerNordgren and Sirlin, 1986; Stavnezer et al., 1988). This transcription initiates from the intronic (I) promoter that is located 50 of every switch region that undergoes CSR. These transcripts include the I exon, S region, and C exon in the primary transcript. However these transcripts, termed germline transcripts, do not appear to code for any functional protein (Alt et al., 1982; Lennon and Perry, 1985). Stimulation of B cells in culture using mitogens and cytokines can induce or suppress germline transcription of specific CH genes and subsequently direct isotype switching to the same isotype. For example, when LPS is added to B-cell cultures, B cells preferentially switch to IgG3 or

300

ir e ne m . m i n an d e ri k s el si n g

IgG2b (Kearney and Lawton, 1975). In contrast, treatment of B cells with LPS and IL-4, a cytokine that is secreted by Th2 cells, leads B cells to switch to IgG1 and IgE isotypes but inhibits switching to IgG3 and IgG2b (Kearney et al., 1976). Germline transcription is indispensable for switching, and the frequency of isotype switching appears to correlate with the rate of germline transcription at the S region (Honjo et al., 2002; Lee et al., 2001). Both deletion of the Ig1 exon with splice donor site and disruption of the Ig2b exon sequences (including the splice site) impaired germline transcription and subsequent switching to g1 or g2b, respectively (Jung et al., 1993; Seidl et al., 1998; Zhang et al., 1993). Replacing I promoter segments with exogenous promoter/enhancer elements (such as the Em enhancer/VH promoter) produced abundant amounts of transcript at each targeted region, yet switching to the targeted allele was significantly reduced (Bottaro et al., 1994). Thus, although germline transcription is needed for switching, it does not appear that transcription per se is sufficient to induce switching. RNA splicing of S region transcripts also appears to be important for isotype switching (Lorenz et al., 1995). Germline transcription has been postulated to open the chromatin around S regions to allow access to the switching mechanism and/or to induce unique S region DNA secondary structures that provide single-stranded DNA segments that are a target for enzymes involved in switching. Similar to CSR, transcription of the V(D)J region is required for SHM targeted to the V(D)J region (Betz et al., 1994), and the rate of mutation is roughly proportional to the transcription rate (Fukita et al., 1998; Jacobs et al., 1999). In addition, modification of the chromatin structure of the V(D)J region is correlated with SHM at the V(D)J region (Woo et al., 2003). In contrast to CSR, the targeting mechanism of SHM appears to be orchestrated by transcription regulatory elements rather than by specific sequences within the V(D)J region, and the position of the Ig promoter is critical for targeting the SHM process (Storb et al., 1998). Placing an Ig promoter just upstream of the Ck gene caused mutations within the normally unmutated Ck region at the same frequency and distribution as the V region (Peters and Storb, 1996). 3. Targeting of the CSR Mechanism 3.1. S-Region Tandem-Repeat Sequences In mammals, S regions are characterized by tandem-repeat sequences that are often G-rich on the nontranscribed DNA strand. In mice, each switch region has a unique G-rich tandem repeat sequence, although some sequence features are shared between all S regions. For example, the Sm region exhibits about 3 kb of tandem repeats that predominantly contain GAGCT

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

301

and GGGGT pentamer sequences, which are also commonly found within S and Sa region, whereas Sg1, Sg3, and Sg2b regions are composed of 49 bp G-rich tandem repeats that also exhibit some GAGCT/GGGGT sequences. The degree of homology of Sm pentamer motifs within different Sg regions is correlated with the gene order in the chromosome; Sg3 > Sg1 > Sg2b > Sg2a. In contrast to mammalian S regions, Xenopus S regions are AT rich; Sm region sequences contain about 35% GC nucleotides (Kitao, 2000, Mussmann et al., 1997 #539). Chicken S regions are relatively GC rich with over 54% GC content (Kitao et al., 2000). Despite varying degree of base compositions of S regions among different species, all S regions feature tandemly repeated sequences containing AGCT motifs. Unlike V(D)J recombination, there are no consensus DNA sequences found at individual switch recombination sites, and switch junctions appear to be found at most positions within tandemly repeated Sg, S, and Sa regions (Dunnick et al., 1993). However, the Sm region may be an exception with regard to the distribution of recombination sites. Location analyses of the junction sites from B-cell hybridomas and myelomas have revealed that about 60% of recombination sites fall within Sm tandem-repeats, and the rest of the recombinations occur within sequences flanking the SmTR region (mostly within the 50 flanking sequences) (Dunnick et al., 1993). In a later study, Sm recombination sites were also found within, but also outside of, the Sm tandem repeats (SmTR) region when assessed by PCR amplification assay using in vitro stimulated splenic B cells (Lee et al., 1998). The unusual tandem-repeat sequences of S regions have suggested that they may play a role in CSR. However, analyses of mice lacking Sm tandem repeats (SmTR-/-) showed that the isotype switching frequency was reduced only by about twofold (Luby et al., 2001a). Therefore, the role of tandem repeats of S regions still remains unclear. Other mice, with a larger deletion of most sequences within the Im-Cm intron including the SmTR, exhibited significant reductions in switching, but switching was not abolished (Khamlichi et al., 2004). About 2–5-fold decreases in isotype switching to IgG2a and IgG2b and approximately 5–20-fold reductions in switching to g1, g3, and a classes are observed in these mice with a larger deletion of the Im-Cm intron (Khamlichi et al., 2004). Neither of the two Sm deletions affected Sm germline transcription (Khamlichi et al., 2004; Luby et al., 2001b). The mice with the largest Sm deletion lacked all of the Sm pentamer motifs, supporting the idea that these motifs are not required for switching and indicating that other sequences can serve as less efficient targets in the switching process. The mice carrying two different deletions of Sm exhibit differences in the targeting of switch recombination events. Switch sites in mice that lack

302

ir e ne m . m i n an d e ri k s el si n g

only the SmTR focus on sequences that are located, in wild-type mice, just downstream of the SmTR (Luby et al., 2001a; Min et al., 2003), whereas the switch sites in mice having the larger Sm deletion focus in the region near the upstream Im promoter (Khamlichi et al., 2004). Comparing switch sites between wild-type mice and the two mutant Sm mice suggests that the ability of Sm sequences to promote switching might reflect not only the underlying DNA sequence but also the location of the sequence relative to the Im promoter. In contrast to results with mice lacking the Sm region, mice with a similar deletion within the Ig1-Cg1 region, including the entire Sg1 tandem-repeat region, showed an almost complete lack of switching to g1 (Shinkura et al., 2003). These mice also exhibited wild-type levels of g1 germline transcription (Shinkura et al., 2003). These results clearly show that some sequence elements within S regions are required for targeting the recombinational mechanism. To study the targeting mechanism of CSR, a number of studies have analyzed recombination of DNA constructs designed to be switch substrates that were transfected into B-cell lines (Chen et al., 2001; Kenter et al., 2004; Kinoshita et al., 1998). Due to various concerns, including low substrate switching efficiencies, there has been controversy regarding the ability of substrate systems to accurately reflect the normal CSR mechanism. In general, substrate studies have shown that two switch regions are required for substrate CSR, that substrate recombination sites are not localized to any particular sequence motif, and that a single repeat unit of Sg1 or Sg3 is sufficient to support substrate CSR (Chen et al., 2001; Kenter et al., 2004; Kinoshita et al., 1998). In addition, analyses of substrate recombinations in different cell lines have suggested that different protein factors are needed for recombinations at different S regions (Kenter et al., 2004). 3.2. S-Region Secondary Structure Several structural features of S regions have been postulated to be involved in targeting of the switching mechanism. One possible structural feature that is associated with the switching mechanism is RNA/DNA hybrid (R-loop) formation at the S region. Mammalian S regions produce predominantly G-rich RNA transcripts, and these RNAs appear to stably associate with the transcribed strand of the S region (Daniels and Lieber, 1995; Reaban and Griffin, 1990; Reaban et al., 1994). In vitro transcription of S regions in their physiological, but not in nonphysiological, orientation induced the formation of stable R-loops (Tian and Alt, 2000). Stable R-loops, which are susceptible to RNaseH and resistant to RNaseA, have been detected in the S regions of stimulated B cells in vivo, and the length of these R-loops can be as long as

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

303

1 kb (Yu et al., 2003). R-loops transcribed in vitro appear to form large aggregates when they are visualized by atomic force microscopy (Mizuta et al., 2003). Inversion of S regions would be expected to generate C-rich, rather than G-rich, transcripts. Supporting the notion of G-rich transcripts as a major factor in stable R-loops formation, extrachomosomal substrates with an inversion of the S-region sequences do not form R-loops (Daniels and Lieber, 1995) or large aggregates (Mizuta et al., 2003) under in vitro transcription conditions. Interestingly, when gene targeting was used to invert Sg1 region sequences in mice, isotype switching to IgG1 was impaired (Shinkura et al., 2003). This finding supports the hypothesis that generation of G-rich transcripts is important to generate R-loops that can serve as targets for the switching mechanism (Shinkura et al., 2003). Unlike mammalian S regions, the Xenopus Sm region is less likely to form R-loop structures due to its relatively AT-rich sequences. However, in mutant mice where the Sg1 region was replaced with the Xenopus Sm region in either the physiological or in nonphysiological orientations, isotype switching was relatively efficient in stimulated B cells (Zarrin et al., 2004). AGCT motifs are prevalent in the Xenopus Sm region and correlate with the locations of switch sites in the mutant mice; these findings suggest that S regions can be targeted for switch recombination by an alternative mechanism that is independent of the ability to form R-loops, and that the presence of AGCT motif may be important to mediate this alternative CSR targeting mechanism (Zarrin et al., 2004). The palindromic nature of pentamer motifs within S regions has also led to the hypothesis that these tandem-repeats of pentamer motifs may form stem-loop structures and that these stem-loop structures of S regions may be a preferential target of the switching mechanism (Honjo et al., 2002; Mussmann et al., 1997; Stavnezer, 1996; Tashiro et al., 2001). Switch junctions in several different species have been predominantly found near the base or neck of the hairpin structures within S-region sequences that are predicted by DNA folding calculations (Mussmann et al., 1997; Tashiro et al., 2001). In addition, two g3-specific double-stranded DNA breaks that were identified by ligationmediated PCR were surrounded by palindromic sequences that can form a stem-loop structure (Wuerffel et al., 1997). Alternately, G-rich DNA sequences within S regions have been proposed to form a G-quartet structure by G-G Hoogsteen bonding, and this structure has been suggested to trigger CSR (Dempsey et al., 1999). However, some studies do not show preferential targeting of switch junctions to stem-loop structures (Min et al., 2003; Pan-Hammarstrom et al., 2004), and no studies have directly assessed the need for either stem-loops or G quartets as targeting motifs for CSR. In the case of RNA/DNA hybrids, nucleotide excision repair molecules, such as XPF and XPG, have been found to cleave DNA strands of the R-loops

304

ir e ne m . m i n an d e ri k s el si n g

formed in the S regions in vitro (Tian and Alt, 2000). As discussed later, cleavage of S regions may require single-stranded DNAs, and this mode of action could explain the importance of transcription and secondary structures of S regions in CSR. However, switching appeared to be unaffected in mice lacking either XPF or XPG (Tian et al., 2004a,b). Other structure-specific enzymes that might target stem-loop structures within the S region are not known. How S regions are targeted in the switching mechanism and whether the secondary structure of the S region, if any, is directly involved in CSR is still poorly understood. 3.3. S-Region Chromatin Structure Induction of germline transcription by cytokines and B-cell mitogens is a critical process for targeting switching to a specific CH gene (StavnezerNordgren and Sirlin, 1986; Stavnezer et al., 1988). It is believed that an increase in transcriptional activity is correlated with increased chromatin accessibility to the CSR machinery. Analyses of hybridomas that secrete various antibody classes indicated that most B cells that have undergone switching exhibit switch recombination events on both parental chromosomes carrying H-chain gene loci and that frequently these events involve the same CH gene on each chromosome (Bottaro et al., 1998; Hummel et al., 1987; Phung et al., 1998; Radbruch et al., 1986; Schultz et al., 1990; Winter et al., 1987). When cells are stimulated to switch to a particular isotype, the two alleles of CH gene that are on the functional and nonfunctional IgH chromosomes both exhibit germline transcription (Delpy et al., 2003). These findings suggest that both alleles of the same isotype are induced into an open configuration that allows CSR. DNase I hypersensitivity sites that reflect an open chromatin structure were first identified in the Sa region when cells were stimulated to switch to IgA (Ono et al., 2000). More recently, the histone acetylation of I and S region chromatin, as assessed by chromatin immunoprecipitation (ChIP) experiments, has been shown to be correlated with germline transcription (and subsequent switching) of a particular isotype in stimulated B cells (Nambu et al., 2003). This study also showed that, in B cells that were stimulated to switch, AID physically interacted with RNA polymerase II, and that AID was associated with acetylated chromatins of the I and S regions. However, causing constitutive histone acetylation of an S region by the use of a histone deacetylase inhibitor concomitant with inhibiting germline transcription of the same S region by the use of a specific cytokine was not sufficient to allow class switching in cells that had been stimulated to undergo isotype switching (Nambu et al., 2003). These data may point to

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

305

germline transcription, rather than histone acetylation, as a critical factor in inducing isotype switching. 3.4. The Characteristics of Class Switch Recombination Sites As mentioned previously in Section 3.1, recombinations associated with CSR can occur anywhere within the Sm tandem repeat region and even in sequences outside of the Sm tandem-repeat region (Dunnick et al., 1993; Lee et al., 1998). In contrast, almost all switch junctions in downstream Sg, S, or Sa regions are found within the tandem-repeat region (Dunnick et al., 1993). One characteristic of many CSR junction sites is a short microhomology (one to three nucleotides) between the Sm germline sequences and the counterpart S region germline sequences (Dunnick et al., 1993). Switch junctions often exhibit point mutations, insertions, deletions, and, occasionally, inversions near the site of recombination (Dunnick et al., 1993). Mutations are clustered around switch junctions and tend to decrease with distance from the CSR junction. Mutations associated with CSR show a very similar pattern to those seen in V region SHM. Both SHM mutations and mutations associated with CSR have a strong bias to occur at G/C nucleotides and at the SHM hotspot motifs, RGYW/WRCY (Martin and Scharff, 2002). These features suggest that both CSR and SHM might be initiated at a DNA level by a similar mechanism. Widespread mutations have also been found in germline Sm regions in B cells that were stimulated to undergo switching (Nagaoka et al., 2002; Petersen et al., 2001; Schrader et al., 2003). Mutations in the germline Sm region exhibit similar sequence specificities that are comparable to the specificities of mutations in recombined Sm/Sg1 segments (Nagaoka et al., 2002; Petersen et al., 2001) but not in recombined Sm/Sg3 segments (Schrader et al., 2003). Despite possible differences in mutational patterns, it appears that mutations in the germline Sm region are dependent on the AID enzyme, which is important for initiating CSR (Nagaoka et al., 2002; Petersen et al., 2001). 4. Proteins Involved in CSR The factors, sequences, and mechanisms involved in targeting of CSR are not clear, although a number of possible models have been proposed. On the other hand, various studies have shown that certain proteins or sequences are either required or important for the switch recombination process. Models for the CSR targeting mechanism must be able to accommodate roles for these factors; this section summarizes features for those proteins that are important

306

ir e ne m . m i n an d e ri k s el si n g

for switching and appear most likely to have roles in the switch targeting process. 4.1. AID May Be Involved in Cleavage of the S Region 4.1.1. What Is AID? Activation induced cytidine deaminase (AID) was isolated by cloning mRNAs that were expressed in switch-induced B-cell lines but not in noninduced B-cell lines using the cDNA subtraction method (Muramatsu et al., 1999). Subsequently, it was found that AID is specifically expressed in germinal center B cells (Muramatsu et al., 1999). Genetic deficiencies of AID in mice and humans completely blocked isotype switching and abrogated V(D)J region SHM (Muramatsu et al., 2000; Revy et al., 2000). Furthermore, ectopic expression of AID in fibroblasts (Okazaki et al., 2002; Yoshikawa et al., 2002), in centroblast-like cell lines (Martin et al., 2002), or in a chicken B-cell line (Arakawa et al., 2002; Harris et al., 2002) induced CSR, SHM, or antibody somatic gene hyperconversion (SHC), respectively. These findings support the concept that AID is the only B-cell specific protein that is necessary for all three of these processes. 4.1.2. The Function and Enzymatic Activities of AID Numerous studies have suggested that AID has a cytidine deaminase activity that directly acts on DNA. Overexpression of AID in E. coli resulted in a mutator phenotype with a pattern of mutation that was shifted to transition mutations at dC/dG residues (Petersen-Mahrt et al., 2002). AID expression in E. coli mutants lacking uracil-DNA glycosylase (UNG) yielded a much higher frequency of mutations than the sum of the mutation frequencies that were observed in E. coli either expressing AID or lacking UNG (Petersen-Mahrt et al., 2002). UNG can remove uracil residues in DNA by a base-excision repair pathway (Krokan et al., 2002), suggesting that AID deaminates cytidines in DNA and mutations are accumulated at dC/dG residues in the absence of UNG (Petersen-Mahrt et al., 2002). Deficiency of UNG in chicken B-cell lines and in mice altered SHM patterns and inhibited isotype switching (Di Noia and Neuberger, 2002; Rada et al., 2002). Biochemical studies show that AID can deaminate cytidines on single-stranded DNAs (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Pham et al., 2003; Ramiro et al., 2003), supporting the hypothesis that DNA deamination is important for SHM and CSR. AID has homology to APOBEC-1, which is a mammalian RNA editing enzyme that converts C to U at a specific residue of ApoB mRNAs to generate two different proteins, ApoB100 and ApoB48, with different biological

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

307

functions (Smith and Sowden, 1996). This finding led to suggestions that AID might edit precursor mRNAs to encode proteins needed for CSR and SHM. This RNA-editing hypothesis was further supported by data showing the level of CSR, presumably mediated by AID, was decreased in the presence of the protein synthesis inhibitors (Doi et al., 2003). A recent study has shown that the role of UNG in isotype switching may involve activities other than removal of uracil residues and induction of S-region DNA breaks (Begum et al., 2004). This finding could also support the concept that AID might play an indirect role in the switching mechanism by generating a separate protein rather than having a direct role in switch region DNA cleavage though dC deamination. Regardless of the controversies surrounding whether AID is a DNA or an RNA-editing enzyme, AID is a critical protein mediating CSR, SHM, and SHC processes (Arakawa et al., 2002; Harris et al., 2002; Martin et al., 2002; Muramatsu et al., 2000; Revy et al., 2000). Overexpression of AID appears to be sufficient in inducing CSR, SHM, and SHC processes in various cell lines that are normally unable to undergo these Ig gene diversification processes (Arakawa et al., 2002; Harris et al., 2002; Martin et al., 2002; Okazaki et al., 2002; Yoshikawa et al., 2002). In addition, in the case of SHM, the mutations of the target nucleotides of AID-induced SHM were biased toward GC base pairs and were targeted toward sequences within the RGYW/WRCY SHM hotspot motifs in fibroblasts (Yoshikawa et al., 2002), hybridomas (Martin et al., 2002), and in vitro when aided by replication protein A (RPA) (Chaudhuri et al., 2004). Biochemical studies show that AID can deaminate cytidines in DNA, but not in RNA or RNA/DNA hybrids, also suggesting that AID mediates SHM and CSR by direct deamination of DNA. When double-stranded DNAs were subjected to transcription by exogenous promoters in E. coli or in vitro, AID preferentially mutated dC residues located within WRC sequences (Beale et al., 2004; Pham et al., 2003; Yu et al., 2004); this WRC motif is similar to the SHM hotspot motif, WRCY. The WRC sequence is also part of the GAGCT pentamer motif found in most S regions, suggesting that the WRC motif may also have relevance for CSR. Interestingly, the ‘‘hottest’’ of hotspot sequences for SHM, WGCW, comprises overlapping WRC motifs on opposite strands (Beale et al., 2004; Yu et al., 2004). Thus, AID cytidine deaminase activity on the WGCW motifs on the opposing strands of DNA may lead to double-stranded DNA breaks with short extensions. This type of DNA break has been proposed to be the intermediate that is resolved by nonhomologous end joining (NHEJ) during CSR (Lieber et al., 2003; Rooney et al., 2004). It has been shown that AID is required for the formation of nuclear foci at IgH loci when B cells are stimulated to undergo switching (Petersen et al., 2001). These nuclear foci contain the Nijmegen breakage syndrome protein (Nbs1) and the phosphorylated form of H2AX; both of these proteins are known to facilitate

308

ir e ne m . m i n an d e ri k s el si n g

DNA double-strand break repair. The involvement of the NHEJ pathway in the CSR mechanism indicates that DNA double-strand breaks are involved in the mechanism of CSR. Blunt ended and staggered DNA double strand breaks associated with CSR have been detected recently by ligation-mediated PCR assays (Catalan et al., 2003; Rush et al., 2004). AID appears to play a crucial role in the induction of DNA breakage during CSR, because these double-strand DNA breaks are dependent on AID (Catalan et al., 2003; Rush et al., 2004). The processes by which single-stranded nicks generated by the activities of AID and UNG might be converted to double-stranded CSR breaks are not yet known. R-loop formation on the transcribed C-rich strand of the S region is suggested to inhibit the AID deaminase activity on cytidines in the transcribed DNA strand. Indeed, AID proteins are unable to deaminate the cytidines on RNA/DNA hybrids or double-stranded DNAs (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Pham et al., 2003; Ramiro et al., 2003). One hypothesis suggests that the removal of R-loops can lead to the misalignment between switch repeats that are located on the opposite stands of DNAs (Yu and Lieber, 2003). When R-loops formed at S-region chromosomal DNAs were removed by RNaseH, short stretches of singlestranded DNAs were found on both DNA strands (Yu et al., 2003). This finding suggests that single-stranded DNAs may be present on both strands of S-region DNAs. Misalignment of switch repeats between opposite DNA strands might generate single-stranded loops on both strands of the S-region DNAs, with the exposed single-stranded DNA loops targeted by AID to yield double-stranded DNA breaks (Yu and Lieber, 2003). An alternative mechanism for AID targeting has been proposed for SHM where the targeted V(D)J exon sequences are unlikely to form R-loops due to their normal level of G-rich sequences. During SHM, V(D)J exons are under active transcription, and transcription bubbles are likely to provide small segments of single-stranded DNAs on both DNA strands. Replication protein A (RPA) protein has been shown to interact with AID on in vitro SHM substrates, and this complex formation correlated with an increased level of AID deaminase activity preferentially on the nontranscribed DNA strand (Chaudhuri et al., 2004). These results may reflect increased stabilization of single-stranded DNAs and enhanced targeting efficiency of AID on DNA strands of V(D)J exons. Whether the RPA and AID protein complexes might play a similar role in CSR is not yet known. 4.1.3. AID Is a Central Enzyme to Both CSR and SHM As mentioned previously, mice and humans lacking AID enzyme are deficient in both SHM and CSR. This suggests that DNA elements of both V(D)J and S segments may be targeted by AID, and that AID may initiate events in both

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

309

regions that lead to DNA breaks associated with SHM and CSR. It still remains unknown how AID is specifically targeted to the V(D)J and the S region for SHM and CSR mechanisms, respectively. Mutations in the C-terminal domain of AID disrupt class switching efficiency despite retaining the cytidine deaminase activity and SHM activity (Ito et al., 2004). In addition, some mutations in the N-terminal region of AID (that partially overlap with the nuclear localization signal) disrupt the SHM activity but do not disrupt the CSR activity (Ito et al., 2004). These studies indicate that there are separate domains of AID that are required for SHM and CSR. It may be possible that the mechanisms of SHM and CSR are distinguished by the interaction of AID with specific cofactors that are recruited to different substrates such as the V(D)J region for SHM and the S region for CSR. Although the initiation phases of SHM and CSR may share all or most of the same enzyme activities, it appears that the joining phases of the two mechanisms are different. Unlike SHM, CSR involves the recombination of two S regions that can be about 175 kb apart. Thus, it is likely that CSR requires proteins that can target the S regions and synapse these regions before the religation step of the switch mechanism. In addition, it is well documented that CSR requires at least some proteins involved in NHEJ (see later discussion) and, therefore, that DNA breaks in CSR may be religated by the NHEJ pathway. In contrast, DNA strand break repair in SHM seems less likely to be resolved by the NHEJ pathway, because SHM is not affected in mice deficient in DNA-PKcs or Ku complexes (Bemark et al., 2000; Sale et al., 2001). 4.2. NHEJ May Be Involved in Joining of Two S Regions 4.2.1. CSR in NHEJ-Defective Mice Studies have shown that animals or cells that lack proteins such as Ku80, Ku70, or DNA-PKcs have major defects in isotype switching, indicating that class switching involves NHEJ DNA repair activity (Casellas et al., 1998; Manis et al., 1998, 2002; Rolink et al., 1996). In the classical pathway of NHEJ, Ku70/Ku80 heterodimers bind to broken double-stranded DNA ends and recruit DNA-PKcs, which has a kinase activity that can self-phosphorylate and phosphorylate Ku70/Ku80 (Lieber et al., 2003). Presumably, NHEJ activity is involved in rejoining the double-stranded DNA breaks that must accompany CSR. However, analyses of mice with mutations in DNA-PKcs revealed a somewhat complicated picture of the involvement of this protein in CSR. Despite

310

ir e ne m . m i n an d e ri k s el si n g

the finding that CSR to most isotypes was severely defective in DNA-PKcsdeficient mice, switching to IgG1 was unaffected (Manis et al., 2002). In addition, SCID mice, which have a mutation in DNA-PKcs that affects only the kinase activity of the protein, could undergo switching with almost the same efficiency as wild-type mice (Bosma et al., 2002). These data suggest that the kinase activity of the DNA–PKcs protein may not be directly involved in the final joining step of CSR. Effects on the CSR mechanism in mice with a null or a partial mutation of DNA-PKcs parallel the extent of the V(D)J recombination defects in the same mice. DNA-PKcs-null mice exhibit severe defects in both coding join and RS signal join formations (Fukumura et al., 2000; Gao et al., 1998), whereas the SCID mutation affects only the coding joint formation (Lieber et al., 1988; Malynn et al., 1988). Effects of NHEJdeficiencies on CSR have been studied in mice carrying gene-targeted IgH and IgL alleles that have rearranged V(D)J genes to circumvent the requirement for NHEJ proteins in V(D)J recombination and B-lymphocyte development. Both Ku70- and Ku80-deficient B cells were unable to undergo isotype switching when stimulated in vitro and in vivo (Gu et al., 1997; Ouyang et al., 1997; Zhu et al., 1996). However, cells deficient in either Ku70 or Ku80 have considerable defects in cellular proliferation and development (Gu et al., 1997; Ouyang et al., 1997; Vogel et al., 1999; Zhu et al., 1996). Thus, it is unclear whether the reductions of CSR in these cells are entirely due to the defects in the CSR mechanism or might also reflect cellular proliferation defects. Kudeficient mice have a wide range of defects in mouse development, indicating that Ku proteins play many roles other than those involved in CSR (Gu et al., 1997; Ouyang et al., 1997; Vogel et al., 1999; Zhu et al., 1996). The assessment of the effect of CSR in cells lacking XRCC4 or DNA ligase IV, which is exclusively involved in the NHEJ pathway (Rooney et al., 2004), might help to address the direct involvement of the NHEJ pathway in CSR. 4.2.2. NHEJ Proteins Involved in CSR May Be Different from Those in V(D)J Recombination The kinase activities of the Artemis and the DNA-PKcs proteins appear to be unimportant in the CSR mechanism (Bosma et al., 2002; Rooney et al., 2004). The activities of both Artemis and the DNA-PKcs proteins have been suggested to be important for opening the hairpins of the coding joins that are generated after Rag introduces double-stranded DNA breaks during the V(D)J recombination process (Ma et al., 2002). These findings suggest that broken DNA ends associated with CSR might, at least in some cases, be repaired by NHEJ pathways that are slightly different from those that take part in the V(D)J recombination process. Furthermore, deficiencies in any

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

311

of the DNA damage response proteins including H2AX, 53BP1, ATM, and Mre11 lead to more profound defects in CSR than in V(D)J recombination (Lahdesmaki et al., 2004; Manis et al., 2004; Pan-Hammarstrom et al., 2003; Petersen et al., 2001). It has recently been shown that B cells lacking H2AX have impaired switch recombination but have normal internal switch region deletions (Reina-San-Martin et al., 2003). It has been suggested that H2AX may facilitate synapsis of the Sm with downstream S regions. The 53BP1 protein may also facilitate S region synapses (Manis et al., 2004). Mutations in the germline Sm region that have been suggested to be introduced via the mechanism involved in CSR do not require the presence of the H2AX protein (Petersen et al., 2001), suggesting that H2AX acts downstream of AID in the CSR process.

4.3. Mismatch Repair Proteins Play a Role in CSR 4.3.1. MMR Mechanism in CSR Mice deficient in mismatch repair (MMR) proteins have diminished efficiencies in isotype switching (Ehrenstein and Neuberger, 1999; Ehrenstein et al., 2001; Schrader et al., 1999), indicating that MMR proteins participate in the CSR mechanism. However, mice lacking different components of the MMR pathway exhibit somewhat different phenotypes in terms of the extent of switch reduction (Ehrenstein and Neuberger, 1999; Ehrenstein et al., 2001; Schrader et al., 1999; Vora et al., 1999), switch junction structure (Ehrenstein and Neuberger, 1999; Ehrenstein et al., 2001; Schrader et al., 2002, 2003b), and mutations surrounding the switch junctions (Schrader et al., 2003a). Each MMR family protein can function in a different way (Evans and Alani, 2000; Kolodner and Marsischky, 1999) and pleiotropic phenotypes in each MMR-defective mouse (Baker et al., 1995; de Wind et al., 1995; Edelmann et al., 1996; Lipkin et al., 2002; Reitmair et al., 1995) have complicated the analyses of MMR protein roles in CSR. 4.3.2. What is MMR? The mismatch repair pathway is critical in guarding the integrity of the genome. MMR targets and corrects base-base mismatches and insertion/ deletion loops that have been generated by DNA damage, errors in DNA replication, and errors in homologous recombination (Kolodner and Marsischky, 1999). In addition, proteins involved in MMR are important in inhibiting recombination between divergent DNA sequences (Datta et al., 1996; Matic et al., 1995) or processing the nonhomologous DNA ends during homologous recombination in yeast (Sugawara et al., 1997). Some MMR

312

ir e ne m . m i n an d e ri k s el si n g

proteins are required for crossover recombination during meiosis (Schofield and Hsieh, 2003). 4.3.3. Generalized Pathway of MMR and Proteins that Interact with MMR Various MMR proteins in eukaryotes are classified as MutS or MutL homologs, and eukaryotic MMR proteins function as heterodimers (this section is covered in detail in these reviews [Evans and Alani, 2000; Kolodner and Marsischky, 1999; Schofield and Hsieh, 2003]). In yeast, there are six mutS homologs (MSH1-MSH6) and four mutL homologs (MLH1, MLH2, MLH3, and PMS1 [PMS2 in humans]). Genetic and biochemical studies demonstrate that Msh2/Msh6 heterodimers repair base-base mismatches, that Msh2/Msh6 and Msh2/Msh3 heterodimers repair 1 bp insertion/deletion loops, and that Msh2/Msh3 heterodimers play a major role in repairing 2-8 bp insertion/ deletion loops. MutS homologs bind to DNA strands with mismatches in an ATP-dependent manner. This binding recruits MutL homologs that act as regulators of MutS homologs. Mlh1/Pms1 heterodimers can bind to Msh2/ Msh3 heterodimers and presumably to Msh2/Msh6 heterodimers. These complexes are important primarily in repairing postreplication errors. Other MutL heterodimers appear to have more specialized roles in MMR; Mlh1/ Mlh2 heterodimers appear to suppress frameshift mutations (Flores-Rozas and Kolodner, 1998), and Mlh1/Mlh3 heterodimers are known to function in meiosis (Lipkin et al., 2002). Additional proteins can be recruited to MMR heterodimers and participate in MMR pathways. Proteins with exonuclease activity, such as exonuclease 1 (Rad2), FEN1 (Rad27), DNA polymerase d, and DNA polymerase, can bind to MMR proteins. Exonuclease 1 and FEN1 have a preference for degrading double-stranded DNA, and it has been proposed that both of these proteins provide processing activities in MMR pathways. A number of DNA replication factors, including DNA polymerase d, RPA, RFC, and PCNA, have also been implicated in the MMR pathway (Dzantiev et al., 2004). The DNA replication factors appear to be required for DNA synthesis during MMR activity. In addition, interactions between MMR complexes and DNA replication factors have been suggested to help distinguish parental strands and daughter strands during the MMR process. 4.3.4. CSR in Msh2-Deficient Mice Msh2 deficiency in mice leads to twofold to fivefold reductions in switching frequencies for the IgG, IgE, and IgA isotypes (Ehrenstein and Neuberger, 1999; Schrader et al., 1999; Vora et al., 1999). In addition, Sm switch junction sites in msh2-/- mice show a skewed distribution that is focused on consensus

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

313

pentamer motifs, unlike the scattered junction site locations that are observed in wild-type mice (Ehrenstein and Neuberger, 1999). One study of msh2-/- mice showed an increased level of apoptosis in germinal center B cells, suggesting that programmed cell death might play a role in the observed class switching reductions (Vora et al., 1999). This suggestion seems less likely, however, because B cells expressing an Msh2 mutant that disrupts apoptosis signaling while retaining normal ATPase activity, still exhibited reductions in switching efficiency similar to msh2-/- mutant (Aldrich et al., 2003). Exonuclease 1, which interacts with Msh2 and Mlh1 proteins in yeast and mammalian cells (Schmutte et al., 1998; Tishkoff et al., 1997; Tran et al., 2001), also appears to participate in the same pathway as Msh2 in the CSR mechanism. Exo1-/- B cells exhibit a reduced class switching efficiency that is similar to the level in msh2-/- B cells, and both msh2-/- and exo1-/- B cells display reduced levels of microhomologies at switch junctions relative to wild-type cells (Bardwell et al., 2004). Recent studies have suggested that Msh2/Msh6, but not Msh2/Msh3 heterodimers, are likely to be the major protein complexes that are involved in the switching mechanism (Li et al., 2004; Martomo, Yang and Gearhart, 2004). Msh2-/- and msh6-/- mice, but not msh3-/- mice, display increased focus of mutations at S regions to WGCW motifs, C/G base pairs, and RGYW/WRCY SHM motifs. Mice deficient in error-prone DNA polymerase Z (eta) also exhibit switch junction mutations similar to those in Msh2- and Msh6-deficient mouse strains (Faili et al., 2004). Overall, these findings indicate that Msh2/Msh6 heterodimers, exo1, and pol Z may participate directly in the CSR mechanism. 4.3.5. CSR in Mlh1- or Pms2-Deficient Mice Mice lacking either the Mlh1 or Pms2 MMR proteins also exhibit reductions in isotype switching that are similar to msh2-/- or exo1-/- mice (Ehrenstein et al., 2001; Schrader et al., 1999). However, mlh1-/- or pms2-/- mice have an increased donor/acceptor homology at switch junctions in contrast to Msh2 or Exo1-deficient mice (Ehrenstein et al., 2001; Schrader et al., 2002). The switching frequency in Msh2/Mlh1 double-deficient mice was reduced to a level similar to that of single-deficient mice, but switch junctions from Msh2/Mlh1 double-deficient mice resemble those of Mlh1-deficient mice. Taken together, these data suggest that Mlh1 and Msh2 might play somewhat different roles in the switching mechanism. 4.3.6. MMR Mechanism May Play a Role in Both CSR and SHM Mice deficient in some components involved in MMR pathways exhibit decreases in the levels of both isotype switching and SHM (Cascalho et al., 1998; Ehrenstein and Neuberger, 1999; Ehrenstein et al., 2001; Kim

314

ir e ne m . m i n an d e ri k s el si n g

et al., 1999; Phung et al., 1998, 1999; Rada et al., 1998; Schrader et al., 1999; Vora et al., 1999; Wiesendanger et al., 2000; Winter et al., 1998). Despite the involvement of MMR complexes in a common DNA repair process, the phenotypes associated with different MMR proteins in CSR and SHM show some differences. SHM patterns in msh2-/- mice were altered to a strongly GC-biased mutation pattern, and mutation frequencies were decreased (Ehrenstein and Neuberger, 1999; Phung et al., 1998; Rada et al., 1998; Wiesendanger et al., 2000). The mutational analyses of mlh1-/- or pms2-/- mice in different reports showed either normal numbers of somatic mutations or decreased somatic mutations (Cascalho et al., 1998; Ehrenstein et al., 2001; Kim et al., 1999; Phung et al., 1999; Schrader et al., 2002; Winter et al., 1998). However, unlike msh2-/- mice, both Mlh1- and Pms2-deficient mice retained a normal pattern of mutations. These differences, in the extent and/or the pattern of SHM in msh2-/-, mlh1-/-, and pms2-/- mice, correlate with differences that are also observed in these three types of MMR-deficient mice in CSR (see Sections 4.3.4 and 4.3.5). Thus, in both SHM and CSR, Msh2 appears to play a somewhat different role than Mlh1 or Pms2. 4.3.7. MSH2 and UNG Activities May Provide Parallel Pathways for Switch Recombination DNA Cleavage Previous analyses of SHM in mice deficient in Msh2 have shown that frequencies of mutations at V(D)J segments are lower (Bertocci et al., 1998; Frey et al., 1998; Rada et al., 1998) or normal (Frey et al., 1998; Jacobs et al., 1998; Phung et al., 1998). Based on some studies reporting lower rates of SHM in msh2-/mice and on the mismatch repair activity of Msh2, it has been previously proposed that Msh2 recognizes and repairs the G:U mismatches that have been caused by the action of AID, and that MMR repair of these residues might be involved in SHM (Di Noia and Neuberger, 2002; Martin and Scharff, 2002). Msh2 deficiency in mice also leads to decreases in isotype switching (Ehrenstein and Neuberger, 1999; Schrader et al., 1999), thus it is possible that Msh2 might also be involved in repairing AID-induced G:U mismatches within S regions (Fig. 2). This hypothesis suggests that the uracil residues in G:U mismatches are subject to two parallel repair pathways that can lead to CSR. The UNG enzyme is likely to be one mechanism for recognizing the G:U mismatches and removing the uracil residues (Di Noia and Neuberger, 2002; Krokan et al., 2002; Rada et al., 2002). This removal will lead to abasic sites through the base excision repair pathway, and further activities by apurine/ apyrimide exonuclease can convert these abasic sites into single-stranded DNA nicks. Analogous to the action of UNG, Msh2 has also been proposed

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

315

Figure 2 The MMR protein, Msh2, and UNG may act in parallel pathways to introduce DNA breaks during CSR. AID enzymatic activity is suggested to deaminate cytidines, giving rise to G:U mismatches in single‐stranded DNAs of S regions. Uracil residues in G:U mismatches may be resolved by two parallel pathways to generate DNA nicks. UNG and subsequent base excision repair proteins, including apyrimidine/apurine endonuclease (APE), are likely to provide the major activity, and Msh2/Msh6 heterodimers with recruitment of other proteins, such as Mlh1, Pms2, Exo1, and PolZ, are also likely to play a role for removing the uracil and subsequently generating CSR‐associated DNA breaks. Msh2 might also have a secondary role in processing staggered DNA breaks to generate blunt double‐stranded DNA breaks. These blunt DNA ends are likely to be rejoined with downstream S‐region DNA breaks by proteins in the nonhomologous end joining (NHEJ) repair pathway.

to recognize the G:U mismatches that are produced by AID and lead to mismatch repair-directed DNA digestion and end processing (Fig. 2) (Di Noia and Neuberger, 2002; Martin and Scharff, 2002). Mice that lack the UNG protein exhibit substantial reductions in isotype switching (Rada et al., 2002), suggesting that G:U mismatches in S regions are predominantly processed through the UNG pathway. If Msh2 is involved in recognizing some G:U mismatches in S regions, it is not clear how these mismatches will be processed. The methyl-CpG binding domain 4 (Mbd4)

316

ir e ne m . m i n an d e ri k s el si n g

glycosylase has been shown to interact with the mismatch repair protein, Mlh1, and it has been suggested that Mbd4 glycosylase activity is involved in repairing G:T and G:U mismatches as a component of the MMR system (Bellacosa et al., 1999). However, the level of isotype switching was unaffected in mice lacking Mbd4 (Bardwell et al., 2003), indicating that the Mbd4 glycosylase is unlikely to be involved in the introduction of DNA breaks through the presumed Msh2 activities of recognizing the G:U mismatch and provoking nicks during CSR. Mice that lack both UNG and Msh2 proteins exhibit very low levels of serum IgG, IgE, or IgA, and isotype switching is almost abrogated in stimulated B cells from these mice (Rada et al., 2004). Furthermore, SHM in ung-/-: msh2-/- mice resulted predominantly in transition mutations at G or C nucleotides, and this mutation pattern appears to reflect a synergistic combination of SHM patterns in mice singly deficient in UNG or Msh2. These findings support the hypothesis that Msh2/Msh6 heterodimers can play a role in generating DNA cleavages at the G:U mismatches introduced by AID during the switching process (Fig. 1). 4.3.8. Msh2 May End-Process Flap DNA Ends Generated During Switch Recombination UNG-deficient mice exhibit about 10-fold reductions in switching relative to wild-type mice (Rada et al., 2002) and, as discussed previously, studies of ung-/-:msh2-/- mice indicate that Msh2 is important for the minor pathway of switching that remains in UNG-deficient animals. However, Msh2-deficient mice show fairly large reductions in CSR (twofold to threefold), suggesting that Msh2 may play additional roles in the switching mechanism beyond a minor contribution to DNA cleavage events. Mice deficient for both the Sm tandem-repeat element (SmTR) and the Msh2 (MMR) protein exhibit severe defects in immunoglobulin antibody class switch recombination (5–10% of wild-type mice) (Min et al., 2003). In comparison, SmTR-/- and msh2-/- mice show a reduced level of isotype switching of about only twofold to threefold relative to wild-type mice (Luby et al., 2001a; Min et al., 2003; Rada et al., 1998; Schrader et al., 1999). These data suggest that the SmTR element is crucial for isotype switching when Msh2 protein is deficient in mice and that Msh2 is important for most CSR using sequences outside of the SmTR but not for sequences within the SmTR. If Msh2 plays an important role in mediating CSR for sequences flanking the SmTR as hypothesized previously, the switch recombination events utilizing these sequences would be expected to be significantly reduced in mice lacking Msh2. Analyses of the distribution of switch site frequencies within

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

317

different segments of the Im-Cm region indicated that the majority of the switch junctions in msh2-/- mice were shifted to the DNA region within the SmTR element, and that switch sites in sequences outside of the SmTR element were significantly reduced (Min and Selsing, unpublished results). These findings support the hypothesis that Msh2 is more important for mediating recombinations for sequences flanking the SmTR than those within the SmTR element. A previous report also showed that, within a small subregion of Sm, Msh2 deficiency in mice caused switch junction locations to focus on the pentamer motifs, mostly within the SmTR region, in contrast to the more random locations found in wild-type mice (Ehrenstein and Neuberger, 1999). Why might Msh2 deficiency affect switch recombinations in sequences outside of the SmTR more than within the SmTR itself? The role of Msh2 in S-region DNA cleavage that is discussed previously does not seem likely to provide an explanation because it is not clear why effects on cleavage should predominantly focus on sequences outside of the SmTR, and because, as discussed previously, the role of Msh2 in cleavage does not seem sufficiently large to account for the substantial switching reductions that are observed in Msh2-deficient mice. We have proposed that the ability of Msh2 to recruit exonuclease activities (Schmutte et al., 1998; Tishkoff et al., 1997) may be involved in processing S-region DNA breaks that are generated by the switching mechanism and that have extended flap ends (Fig. 3) (Min et al., 2003). Msh2-induced processing could then provide additional blunt or almost blunt S-region DNA ends that can be joined by NHEJ proteins. In the absence of Msh2, DNA flap ends would not be processed and would not be available for the joining mechanism, resulting in reductions in successful switch recombinations. To account for the greater effect of Msh2 deficiency on CSR in regions flanking the SmTR, we have also proposed that DNA breaks with flap ends predominate in these flanking sequences whereas DNA breaks that are blunt or almost blunt predominate within the SmTR. These differences in DNA breaks between sequences within or outside of the SmTR could reflect the densities of potential cleavage sites (each of which can provide single-stranded cleavage) in the two regions (Fig. 3). The high site-density within the SmTR can provide mostly ‘‘blunt’’ ends that do not require Msh2 processing, whereas the low site-density in flanking regions results in mostly flap ends that cannot complete recombination unless processed through Msh2. This model for Msh2 DNA end-processing function in switching appears to correlate with the distributions of specific sequence motifs within the Im-Cm region that may be targeted by AID activity. As mentioned previously, the WGCW motif represents two AID target sites positioned on opposite strands of DNA (Beale et al., 2004; Pham et al., 2003; Yu et al., 2004) and elicits the

318

ir e ne m . m i n an d e ri k s el si n g

Figure 3 The Msh2 MMR protein may play an important role for processing DNA ends with long extensions in the S region during CSR. The Sm tandem repeat (SmTR) region is proposed to contain a high density of CSR‐associated DNA cleavage sites. In contrast to the SmTR region, DNA segments flanking the SmTR region are likely to be composed of a fewer number of DNA cleavage sites. Because the densities of AID target sites in two regions are disparate, these two regions are predicted to generate different types of DNA ends when both strands of DNAs are targeted by AID. The SmTR region is likely to generate predominantly DNA ends with short extensions or blunt DNA double‐stranded breaks, whereas the flanking regions outside of the SmTR are more likely to yield flap DNA ends. We propose that Msh2 is important for processing the flap DNA ends to produce blunt DNA ends or DNA ends with short extensions, which can then be joined with downstream S regions by proteins in the NHEJ-mediated DNA break repair mechanism.

strongest AID cytidine deaminase activity in biochemical and genetic assays (Beale et al., 2004; Yu et al., 2004). Perhaps the WGCW motif within the S region represents a predominant target site for AID activity during CSR and, due to cleavage of the sites on opposing strands, provides almost blunt DNA breaks that can undergo recombinational joining via NHEJ proteins. The frequencies of WGCW motifs are high within the SmTR region and very low in the flanking DNA regions just outside of the SmTR, correlating with the frequency of switch site locations when Msh2 activity is absent. In contrast,

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

319

WRC sequences, which might be expected to provide only single-stranded nicks after AID deamination, are more evenly distributed throughout the JH-Cm region. AID activity at WRC sites that are on opposing strands but separated by a distance would lead to DNA breaks with flap ends that, in the presence of Msh2, can be processed to undergo recombinational joining. Although these types of ends are likely to be generated both within and outside of the SmTR region, the low levels of WGCW sites in flanking regions outside of the SmTR implies that DNA breaks in the flanking region would mainly exhibit flap ends and require Msh2-initiated processing. In determining the roles of MMR proteins in switch recombination, studies of the Msh2 protein have progressed the furthest, suggesting that Msh2 is involved in both DNA cleavage of switch regions and in processing the cleaved DNA ends to allow recombinational joining. The roles of other MMR proteins (Msh6, Mlh1, Pms2, Exo1) are less clear. Mice deficient for these proteins show some shared features and some features that differ. Additional genetic studies of mice with combinations of deficiencies for various proteins or DNA sequences will indicate whether various MMR proteins are affecting similar steps in the switch mechanism pathways and may provide insights into the roles of each protein in the recombinational process. 5. Concluding Remarks The CSR mechanism diversifies antibody effector functions by changing the constant regions of antibodies. The class switch mechanism is mediated by DNA recombination events that juxtapose H-chain V(D)J segments with different downstream CH gene segments (Honjo and Kataoka, 1978; Iwasato et al., 1990; Matsuoka et al., 1990; von Schwedler et al., 1990). CSR takes place between S regions 50 to each constant region gene, and all S regions contain tandem-repeat sequences (Kataoka et al., 1981). The discovery of AID as a main mediator of CSR (Muramatsu et al., 2000; Revy et al., 2000) has opened a gate for more findings that have subsequently indicated that AID and basic excision repair proteins are important for generating DNA breaks in the S regions during the switching mechanism (Bransteitter et al., 2003; Chaudhuri et al., 2003; Pham et al., 2003; Ramiro et al., 2003). AID affects somatic hypermutation, suggesting that SHM and CSR mechanisms might be linked (Muramatsu et al., 2000; Revy et al., 2000). S regions are important for switching because they provide high concentrations of sequence motifs that appear to preferred sites for AID activity, and they may also delineate (through an unusual structure or other feature) a region that is accessible for switch recombination. Mismatch repair proteins are important for the highest efficiencies in class switch recombination and appear to be

320

ir e ne m . m i n an d e ri k s el si n g

involved in both cleaving S-region DNA and processing the cleaved DNA ends to allow recombinational joining. References Aldrich, M. B., Chen, W., Blackburn, M. R., Martinez-Valdez, H., Datta, S. K., and Kellems, R. E. (2003). Impaired germinal center maturation in adenosine deaminase deficiency. J. Immunol. 171, 5562–5570. Alt, F. W., Rosenberg, N., Casanova, R. J., Thomas, E., and Baltimore, D. (1982). Immunoglobulin heavy-chain expression and class switching in a murine leukaemia cell line. Nature 296, 325–331. Apel, M., and Berek, C. (1990). Somatic mutations in antibodies expressed by germinal centre B cells early after primary immunization. Int. Immunol. 2, 813–819. Arakawa, H., Hauschild, J., and Buerstedde, J. M. (2002). Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295, 1301–1306. Baker, S. M., Bronner, C. E., Zhang, L., Plug, A. W., Robarzek, M., Warren, G., Elliott, E. A., Yu, J., Ashley, T., Arnheim, N., Flavell, R. A., and Liskay, R. M. (1995). Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis. Cell 82, 309–319. Bardwell, P. D., Martin, A., Wong, E., Li, Z., Edelmann, W., and Scharff, M. D. (2003). Cutting edge: The G-U mismatch glycosylase methyl-CpG binding domain 4 is dispensable for somatic hypermutation and class switch recombination. J. Immunol. 170, 1620–1624. Bardwell, P. D., Woo, C. J., Wei, K., Li, Z., Martin, A., Sack, S. Z., Parris, T., Edelmann, W., and Scharff, M. D. (2004). Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice. Nat. Immunol. 5, 224–229. Beale, R. C., Petersen-Mahrt, S. K., Watt, I. N., Harris, R. S., Rada, C., and Neuberger, M. S. (2004). Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: Correlation with mutation spectra in vivo. J. Mol. Biol. 337, 585–596. Begum, N. A., Kinoshita, K., Kakazu, N., Muramatsu, M., Nagaoka, H., Shinkura, R., Biniszkiewicz, D., Boyer, L. A., Jaenisch, R., and Honjo, T. (2004). Uracil DNA glycosylase activity is dispensable for immunoglobulin class switch. Science 305, 1160–1163. Bellacosa, A., Cicchillitti, L., Schepis, F., Riccio, A., Yeung, A. T., Matsumoto, Y., Golemis, E. A., Genuardi, M., and Neri, G. (1999). MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1. Proc. Natl. Acad. Sci. USA 96, 3969–3974. Bemark, M., Sale, J. E., Kim, H. J., Berek, C., Cosgrove, R. A., and Neuberger, M. S. (2000). Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) or recombination-activating gene (RAG)1 activity. J. Exp. Med. 192, 1509–1514. Bertocci, B., Quint, L., Delbos, F., Garcia, C., Reynaud, C. A., and Weill, J. C. (1998). Probing immunoglobulin gene hypermutation with microsatellites suggests a nonreplicative short patch DNA synthesis process. Immunity 9, 257–265. Betz, A. G., Milstein, C., Gonzalez-Fernandez, A., Pannell, R., Larson, T., and Neuberger, M. S. (1994). Elements regulating somatic hypermutation of an immunoglobulin kappa gene: Critical role for the intron enhancer/matrix attachment region. Cell 77, 239–248. Bosma, G. C., Kim, J., Urich, T., Fath, D. M., Cotticelli, M. G., Ruetsch, N. R., Radic, M. Z., and Bosma, M. J. (2002). DNA-dependent protein kinase activity is not required for immunoglobulin class switching. J. Exp. Med. 196, 1483–1495.

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

321

Bottaro, A., Lansford, R., Xu, L., Zhang, J., Rothman, P., and Alt, F. W. (1994). S region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process. EMBO J. 13, 665–674. Bottaro, A., Young, F., Chen, J., Serwe, M., Sablitzky, F., and Alt, F. W. (1998). Deletion of the IgH intronic enhancer and associated matrix-attachment regions decreases, but does not abolish, class switching at the mu locus. Int. Immunol. 10, 799–806. Bransteitter, R., Pham, P., Scharff, M. D., and Goodman, M. F. (2003). Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100, 4102–4107. Cascalho, M., Wong, J., Steinberg, C., and Wabl, M. (1998). Mismatch repair co-opted by hypermutation. Science 279, 1207–1210. Casellas, R., Nussenzweig, A., Wuerffel, R., Pelanda, R., Reichlin, A., Suh, H., Qin, X. F., Besmer, E., Kenter, A., Rajewsky, K., and Nussenzweig, M. C. (1998). Ku80 is required for immunoglobulin isotype switching. EMBO J. 17, 2404–2411. Catalan, N., Selz, F., Imai, K., Revy, P., Fischer, A., and Durandy, A. (2003). The block in immunoglobulin class switch recombination caused by activation-induced cytidine deaminase deficiency occurs prior to the generation of DNA double strand breaks in switch mu region. J. Immunol. 171, 2504–2509. Chaudhuri, J., Khuong, C., and Alt, F. W. (2004). Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature 430, 992–998. Chaudhuri, J., Tian, M., Khuong, C., Chua, K., Pinaud, E., and Alt, F. W. (2003). Transcriptiontargeted DNA deamination by the AID antibody diversification enzyme. Nature 422, 726–730. Chen, X., Kinoshita, K., and Honjo, T. (2001). Variable deletion and duplication at recombination junction ends: Implication for staggered double-strand cleavage in class-switch recombination. Proc. Natl. Acad. Sci. USA 98, 13860–13865. Daniels, G. A., and Lieber, M. R. (1995). RNA:DNA complex formation upon transcription of immunoglobulin switch regions: Implications for the mechanism and regulation of class switch recombination. Nucleic Acids Res. 23, 5006–5011. Datta, A., Adjiri, A., New, L., Crouse, G. F., and Jinks Robertson, S. (1996). Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae. Mol. Cell. Biol. 16, 1085–1093. de Wind, N., Dekker, M., Berns, A., Radman, M., and te Riele, H. (1995). Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer. Cell 82, 321–330. Delpy, L., Le Bert, M., Cogne, M., and Khamlichi, A. A. (2003). Germ-line transcription occurs on both the functional and the non-functional alleles of immunoglobulin constant heavy chain genes. Eur. J. Immunol. 33, 2108–2113. Dempsey, L. A., Sun, H., Hanakahi, L. A., and Maizels, N. (1999). G4 DNA binding by LR1 and its subunits, nucleolin and hnRNP D, A role for G-G pairing in immunoglobulin switch recombination. J. Biol. Chem. 274, 1066–1071. Di Noia, J., and Neuberger, M. S. (2002). Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419, 43–48. Dickerson, S. K., Market, E., Besmer, E., and Papavasiliou, F. N. (2003). AID mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197, 1291–1296. Doi, T., Kinoshita, K., Ikegawa, M., Muramatsu, M., and Honjo, T. (2003). De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination. Proc. Natl. Acad. Sci. USA 100, 2634–2638. Dunnick, W., Hertz, G. Z., Scappino, L., and Gritzmacher, C. (1993). DNA sequences at immunoglobulin switch region recombination sites. Nucleic Acids Res. 21, 365–372.

322

ir e ne m . m i n an d e ri k s el si n g

Dzantiev, L., Constantin, N., Genschel, J., Iyer, R. R., Burgers, P. M., and Modrich, P. (2004). A defined human system that supports bidirectional mismatch-provoked excision. Mol. Cell 15, 31–41. Edelmann, W., Cohen, P. E., Kane, M., Lau, K., Morrow, B., Bennett, S., Umar, A., Kunkel, T., Cattoretti, G., Chaganti, R., Pollard, J. W., Kolodner, R. D., and Kucherlapati, R. (1996). Meiotic pachytene arrest in MLH1-deficient mice. Cell 85, 1125–1134. Ehrenstein, M. R., and Neuberger, M. S. (1999). Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: Parallels with somatic hypermutation. EMBO J. 18, 3484–3490. Ehrenstein, M. R., Rada, C., Jones, A. M., Milstein, C., and Neuberger, M. S. (2001). Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination. Proc. Natl. Acad. Sci. USA 98, 14553–14558. Evans, E., and Alani, E. (2000). Roles for mismatch repair factors in regulating genetic recombination. Mol. Cell. Biol. 20, 7839–7844. Faili, A., Aoufouchi, S., Weller, S., Vuillier, F., Stary, A., Sarasin, A., Reynaud, C. A., and Weill, J. C. (2004). DNA polymerase eta is involved in hypermutation occurring during immunoglobulin class switch recombination. J. Exp. Med. 199, 265–270. Flores-Rozas, H., and Kolodner, R. D. (1998). The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations. Proc. Natl. Acad. Sci. USA 95, 12404–12409. Frey, S., Bertocci, B., Delbos, F., Quint, L., Weill, J. C., and Reynaud, C. A. (1998). Mismatch repair deficiency interferes with the accumulation of mutations in chronically stimulated B cells and not with the hypermutation process. Immunity 9, 127–134. Fukita, Y., Jacobs, H., and Rajewsky, K. (1998). Somatic hypermutation in the heavy chain locus correlates with transcription. Immunity 9, 105–114. Fukumura, R., Araki, R., Fujimori, A., Tsutsumi, Y., Kurimasa, A., Li, G. C., Chen, D. J., Tatsumi, K., and Abe, M. (2000). Signal joint formation is also impaired in DNA-dependent protein kinase catalytic subunit knockout cells. J. Immunol. 165, 3883–3889. Gao, Y., Chaudhuri, J., Zhu, C., Davidson, L., Weaver, D. T., and Alt, F. W. (1998). A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination. Immunity 9, 367–376. Gu, Y., Seidl, K. J., Rathbun, G. A., Zhu, C., Manis, J. P., van der Stoep, N., Davidson, L., Cheng, H. L., Sekiguchi, J. M., Frank, K., Stanhope-Baker, P., Schlissel, M. S., Roth, D. B., and Alt, F. W. (1997). Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity 7, 653–665. Harris, R. S., Sale, J. E., Petersen-Mahrt, S. K., and Neuberger, M. S. (2002). AID is essential for immunoglobulin V gene conversion in a cultured B cell line. Curr. Biol. 12, 435–438. Honjo, T., and Kataoka, T. (1978). Organization of immunoglobulin heavy chain genes and allelic deletion model. Proc. Natl. Acad. Sci. USA 75, 2140–2144. Honjo, T., Kinoshita, K., and Muramatsu, M. (2002). Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annu. Rev. Immunol. 20, 165–196. Hummel, M., Berry, J. K., and Dunnick, W. (1987). Switch region content of hybridomas: The two spleen cell Igh loci tend to rearrange to the same isotype. J. Immunol. 138, 3539–3548. Ito, S., Nagaoka, H., Shinkura, R., Begum, N., Muramatsu, M., Nakata, M., and Honjo, T. (2004). Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1. Proc. Natl. Acad. Sci. USA 101, 1975–1980. Iwasato, T., Shimizu, A., Honjo, T., and Yamagishi, H. (1990). Circular DNA is excised by immunoglobulin class switch recombination. Cell 62, 143–149.

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

323

Jacobs, H., Fukita, Y., van der Horst, G. T., de Boer, J., Weeda, G., Essers, J., de Wind, N., Engelward, B. P., Samson, L., Verbeek, S., de Murcia, J. M., de Murcia, G., te Riele, H., and Rajewsky, K. (1998). Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice. J. Exp. Med. 187, 1735–1743. Jacobs, H., Puglisi, A., Rajewsky, K., and Fukita, Y. (1999). Tuning somatic hypermutation by transcription. Curr. Top Microbiol. Immunol. 246, 149–158. Jung, S., Rajewsky, K., and Radbruch, A. (1993). Shutdown of class switch recombination by deletion of as witch region control element. Science 259, 984–987. Kataoka, T., Miyata, T., and Honjo, T. (1981). Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes. Cell 23, 357–368. Kearney, J. F., Cooper, M. D., and Lawton, A. R. (1976). B cell differentiation induced by lipopolysaccharide. IV. Development of immunoglobulin class restriction in precursors of IgG-synthesizing cells. J. Immunol. 117, 1567–1572. Kearney, J. F., and Lawton, A. R. (1975). B lymphocyte differentiation induced by lipopolysaccharide. I. Generation of cells synthesizing four major immunoglobulin classes. J. Immunol. 115, 671–676. Kenter, A. L., Wuerffel, R., Dominguez, C., Shanmugam, A., and Zhang, H. (2004). Mapping of a functional recombination motif that defines isotype specificity for mu–>gamma3 switch recombination implicates NF-kappaB p50 as the isotype-specific switching factor. J. Exp. Med. 199, 617–627. Khamlichi, A. A., Glaudet, F., Oruc, Z., Denis, V., Le Bert, M., and Cogne, M. (2004). Immunoglobulin class-switch recombination in mice devoid of any S mu tandem repeat. Blood 103, 3828–3836. Kim, N., Bozek, G., Lo, J. C., and Storb, U. (1999). Different mismatch repair deficiencies all have the same effects on somatic hypermutation: Intact primary mechanism accompanied by secondary modifications. J. Exp. Med. 190, 21–30. Kinoshita, K., Tashiro, J., Tomita, S., Lee, C.-G., and Honjo, T. (1998). Target specificity of immunoglobulin class switch recombination is not determined by nucleotide sequences of S regions. Immunity 9, 849–858. Kitao, H., Arakawa, H., Kuma, K., Yamagishi, H., Nakamura, N., Furusawa, S., Matsuda, H., Yasuda, M., Ekino, S., and Shimizu, A. (2000). Class switch recombination of the chicken IgH chain genes: Implications for the primordial switch region repeats. Int. Immunol. 12, 959–968. Kolodner, R. D., and Marsischky, G. T. (1999). Eukaryotic DNA mismatch repair. Curr. Opin. Genet. Dev. 9, 89–96. Krokan, H. E., Drablos, F., and Slupphaug, G. (2002). Uracil in DNA–occurrence, consequences and repair. Oncogene 21, 8935–8948. Lahdesmaki, A., Taylor, A. M., Chrzanowska, K. H., and Pan-Hammarstrom, Q. (2004). Delineation of the role of the Mre11 complex in class switch recombination. J. Biol. Chem. 279, 16479–16487. Lee, C., Kondo, S., and Honjo, T. (1998). Separation and identification of microcystins in cyanobacteria by frit-fast atom bombardment liquid chromatography/mass spectrometry. Current Biology 8, 227–237. Lee, C. G., Kinoshita, K., Arudchandran, A., Cerritelli, S. M., Crouch, R. J., and Honjo, T. (2001). Quantitative regulation of class switch recombination by switch region transcription. J. Exp. Med. 194, 365–374. Lennon, G. G., and Perry, R. P. (1985). C mu-containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 50 -nontranslatable exon. Nature 318, 475–478. Li, Z., Scherer, S. J., Ronai, D., Iglesias-Ussel, M. D., Peled, J. U., Bardwell, P. D., Zhuang, M., Lee, K., Martin, A., Edelmann, W., and Scharff, M. D. (2004). Examination of Msh6- and

324

ir e ne m . m i n an d e ri k s el si n g

Msh3-deficient mice in class switching reveals overlapping and distinct roles of MutS homologues in antibody diversification. J. Exp. Med. 200, 47–59. Lieber, M. R., Hesse, J. E., Lewis, S., Bosma, G. C., Rosenberg, N., Mizuuchi, K., Bosma, M. J., and Gellert, M. (1988). The defect in murine severe combined immune deficiency: Joining of signal sequences but not coding segments in V(D)J recombination. Cell 55, 7–16. Lieber, M. R., Ma, Y., Pannicke, U., and Schwarz, K. (2003). Mechanism and regulation of human non-homologous DNA end-joining. Nat. Rev. Mol. Cell. Biol. 4, 712–720. Lipkin, S. M., Moens, P. B., Wang, V., Lenzi, M., Shanmugarajah, D., Gilgeous, A., Thomas, J., Cheng, J., Touchman, J. W., Green, E. D., Schwartzberg, P., Collins, F. S., and Cohen, P. E. (2002). Meiotic arrest and aneuploidy in MLH3-deficient mice. Nat. Genet. 31, 385–390. Lorenz, M., Jung, S., and Radbruch, A. (1995). Switch transcripts in immunoglobulin class switching. Science 267, 1825–1828. Luby, T. M., Schrader, C. E., Stavnezer, J., and Selsing, E. (2001a). The mu switch region tandem repeats are important, but not required, for antibody class switch recombination. J. Exp. Med. 193, 159–198. Luby, T. M., Sigurdardottir, D., Berger, E. D., and Selsing, E. (2001b). Sequences associated with the mouse Smu switch region are important for immunoglobulin heavy chain transgene expression in B cell development. Eur. J. Immunol. 31, 2866–2875. Lutzker, S., Rothman, P., Pollock, R., Coffman, R., and Alt, F. W. (1988). Mitogen- and IL-4regulated expression of germ-line Ig gamma 2b transcripts: Evidence for directed heavy chain class switching. Cell 53, 177–184. Ma, Y., Pannicke, U., Schwarz, K., and Lieber, M. R. (2002). Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108, 781–794. Malynn, B. A., Blackwell, T. K., Fulop, G. M., Rathbun, G. A., Furley, A. J., Ferrier, P., Heinke, L. B., Phillips, R. A., Yancopoulos, G. D., and Alt, F. W. (1988). The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism. Cell 54, 453–460. Manis, J. P., Dudley, D., Kaylor, L., and Alt, F. W. (2002). IgH class switch recombination to IgG1 in DNA-PKcs-deficient B cells. Immunity 16, 607–617. Manis, J. P., Gu, Y., Lansford, R., Sonoda, E., Ferrini, R., Davidson, L., Rajewsky, K., and Alt, F. W. (1998). Ku70 is required for late B cell development and immunoglobulin heavy chain class switching. J. Exp. Med. 187, 2081–2089. Manis, J. P., Morales, J. C., Xia, Z., Kutok, J. L., Alt, F. W., and Carpenter, P. B. (2004). 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination. Nat. Immunol. 5, 481–487. Mantovani, L., Wilder, R. L., and Casali, P. (1993). Human rheumatoid B-1a (CD5þ B) cells make somatically hypermutated high affinity IgM rheumatoid factors. J. Immunol. 151, 473–488. Martin, A., Bardwell, P. D., Woo, C. J., Fan, M., Shulman, M. J., and Scharff, M. D. (2002). Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas. Nature 415, 802–806. Martin, A., and Scharff, M. D. (2002). AID and mismatch repair in antibody diversification. Nat. Rev. Immunol. 2, 605–614. Martomo, S. A., Yang, W. W., and Gearhart, P. J. (2004). A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination. J. Exp. Med. 200, 61–68. Matic, I., Rayssiguier, C., and Radman, M. (1995). Interspecies gene exchange in bacteria: The role of SOS and mismatch repair systems in evolution of species. Cell 80, 507–515. Matsuoka, M., Yoshida, K., Maeda, T., Usuda, S., and Sakano, H. (1990). Switch circular DNA formed in cytokine-treated mouse splenocytes: Evidence for intramolecular DNA deletion in immunoglobulin class switching. Cell 62, 135–142.

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

325

Min, I. M., Schrader, C. E., Vardo, J., Luby, T. M., D’Avirro, N., Stavnezer, J., and Selsing, E. (2003). The Smu tandem repeat region is critical for Ig isotype switching in the absence of Msh2. Immunity 19, 515–524. Mizuta, R., Iwai, K., Shigeno, M., Mizuta, M., Uemura, T., Ushiki, T., and Kitamura, D. (2003). Molecular visualization of immunoglobulin switch region RNA/DNA complex by atomic force microscope. J. Biol. Chem. 278, 4431–4434. Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563. Muramatsu, M., Sankaranand, V. S., Anant, S., Sugai, M., Kinoshita, K., Davidson, N. O., and Honjo, T. (1999). Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J. Biol. Chem. 274, 18470–18476. Mussmann, R., Courtet, M., Schwager, J., and Du Pasquier, L. (1997). Microsites for immunoglobulin switch recombination breakpoints from Xenopus to mammals. Eur. J. Immunol. 27, 2610–2619. Nagaoka, H., Muramatsu, M., Yamamura, N., Kinoshita, K., and Honjo, T. (2002). Activationinduced deaminase (AID)-directed hypermutation in the immunoglobulin Smu region: implication of AID involvement in a common step of class switch recombination and somatic hypermutation. J. Exp. Med. 195, 529–534. Nambu, Y., Sugai, M., Gonda, H., Lee, C. G., Katakai, T., Agata, Y., Yokota, Y., and Shimizu, A. (2003). Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 302, 2137–2140. Okazaki, I. M., Kinoshita, K., Muramatsu, M., Yoshikawa, K., and Honjo, T. (2002). The AID enzyme induces class switch recombination in fibroblasts. Nature 416, 340–345. Ono, S. J., Zhou, G., Tai, A. K., Inaba, M., Kinoshita, K., and Honjo, T. (2000). Identification of a stimulus-dependent DNase I hypersensitive site between the Ialpha and Calpha exons during immunoglobulin heavy chain class switch recombination. FEBS Lett. 467, 268–272. Ouyang, H., Nussenzweig, A., Kurimasa, A., Soares, V. C., Li, X., Cordon-Cardo, C., Li, W., Cheong, N., Nussenzweig, M., Iliakis, G., Chen, D. J., and Li, G. C. (1997). Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination in vivo. J. Exp. Med. 186, 921–929. Pan-Hammarstrom, Q., Dai, S., Zhao, Y., van Dijk-Hard, I. F., Gatti, R. A., Borresen-Dale, A. L., and Hammarstrom, L. (2003). ATM is not required in somatic hypermutation of VH, but is involved in the introduction of mutations in the switch mu region. J. Immunol. 170, 3707–3716. Pan-Hammarstrom, Q., Zhao, Y., and Hammarstrom, L. (2004). Lack of association between human switch recombination breakpoints and the secondary structure of targeted DNA regions. J. Immunol. 172, 2727. Peters, A., and Storb, U. (1996). Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4, 57–65. Petersen, S., Casellas, R., Reina-San-Martin, B., Chen, H. T., Difilippantonio, M. J., Wilson, P. C., Hanitsch, L., Celeste, A., Muramatsu, M., Pilch, D. R., Redon, C., Ried, T., Bonner, W. M., Honjo, T., Nussenzweig, M. C., and Nussenzweig, A. (2001). AID is required to initiate Nbs1/ gamma-H2AX focus formation and mutations at sites of class switching. Nature 414, 660–665. Petersen-Mahrt, S. K., Harris, R. S., and Neuberger, M. S. (2002). AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–103. Pham, P., Bransteitter, R., Petruska, J., and Goodman, M. F. (2003). Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424, 103–107.

326

ir e ne m . m i n an d e ri k s el si n g

Phung, Q. H., Winter, D. B., Alrefai, R., and Gearhart, P. J. (1999). Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein. J. Immunol. 162, 3121–3124. Phung, Q. H., Winter, D. B., Cranston, A., Tarone, R. E., Bohr, V. A., Fishel, R., and Gearhart, P. J. (1998). Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein. J. Exp. Med. 187, 1745–1751. Rada, C., Di Noia, J. M., and Neuberger, M. S. (2004). Mismatch recognition and uracil excision provide complementary paths to both Ig switching and the A/T-focused phase of somatic mutation. Mol. Cell 16, 163–171. Rada, C., Ehrenstein, M. R., Neuberger, M. S., and Milstein, C. (1998). Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting. Immunity 9, 135–141. Rada, C., Williams, G. T., Nilsen, H., Barnes, D. E., Lindahl, T., and Neuberger, M. S. (2002). Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755. Radbruch, A., Muller, W., and Rajewsky, K. (1986). Class switch recombination is IgG1 specific on active and inactive IgH loci of IgG1-secreting B-cell blasts. Proc. Natl. Acad. Sci. USA 83, 3954–3957. Ramiro, A. R., Stavropoulos, P., Jankovic, M., and Nussenzweig, M. C. (2003). Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nat. Immunol. 4, 452–456. Reaban, M. E., and Griffin, J. A. (1990). Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region. Nature 348, 342–344. Reaban, M. E., Lebowitz, J., and Griffin, J. A. (1994). Transcription induces the formation of a stable RNA.DNA hybrid in the immunoglobulin alpha switch region. J. Biol. Chem. 269, 21850–21857. Reina-San-Martin, B., Difilippantonio, S., Hanitsch, L., Masilamani, R. F., Nussenzweig, A., and Nussenzweig, M. C. (2003). H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation. J. Exp. Med. 197, 1767–1778. Reitmair, A. H.Schmits, R.Ewel, A.Bapat, B.Redston, M.Mitri, A.Waterhouse, P.Mittrucker, H. W. Wakeham, A.Liu, B., et al. (1995). MSH2 deficient mice are viable and susceptible to lymphoid tumours. Nat. Genet. 11, 64–70. Revy, P., Muto, T., Levy, Y., Geissmann, F., Plebani, A., Sanal, O., Catalan, N., Forveille, M., Dufourcq-Labelouse, R., Gennery, A., Tezcan, I., Ersoy, F., Kayserili, H., Ugazio, A. G., Brousse, N., Muramatsu, M., Notarangelo, L. D., Kinoshita, K., Honjo, T., Fischer, A., and Durandy, A. (2000). Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575. Rolink, A., Melchers, F., and Andersson, J. (1996). The SCID but not the RAG-2 gene product is required for S mu-S epsilon heavy chain class switching. Immunity 5, 319–330. Rooney, S., Chaudhuri, J., and Alt, F. W. (2004). The role of the non-homologous end-joining pathway in lymphocyte development. Immunol. Rev. 200, 115–131. Rothman, P., Lutzker, S., Cook, W., Coffman, R., and Alt, F. W. (1988). Mitogen plus interleukin 4 induction of C epsilon transcripts in B lymphoid cells. J. Exp. Med. 168, 2385–2389. Rush, J. S., Fugmann, S. D., and Schatz, D. G. (2004). Staggered AID-dependent DNA double strand breaks are the predominant DNA lesions targeted to S mu in Ig class switch recombination. Int. Immunol. 16, 549–557. Sale, J. E., Calandrini, D. M., Takata, M., Takeda, S., and Neuberger, M. S. (2001). Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412, 921–926.

a nt i bo dy cl a s s s w i t c h r e c om bi nat i on

327

Schmutte, C., Marinescu, R. C., Sadoff, M. M., Guerrette, S., Overhauser, J., and Fishel, R. (1998). Human exonuclease I interacts with the mismatch repair protein hMSH2. Cancer Res. 58, 4537–4542. Schofield, M. J., and Hsieh, P. (2003). DNA mismatch repair: Molecular mechanisms and biological function. Annu. Rev. Microbiol. 57, 579–608. Schrader, C., Edelmann, W., Kucherlapati, R., and Stavnezer, J. (1999). Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes. J. Exp. Med. 190, 323–330. Schrader, C. E., Bradley, S. P., Vardo, J., Mochegova, S. N., Flanagan, E., and Stavnezer, J. (2003a). Mutations occur in the Ig Smu region but rarely in Sgamma regions prior to class switch recombination. EMBO J. 22, 5893–5903. Schrader, C. E., Vardo, J., and Stavnezer, J. (2002). Role for mismatch repair proteins Msh2, Mlh1, and Pms2 in immunoglobulin class switching shown by sequence analysis of recombination junctions. J. Exp. Med. 195, 367–373. Schrader, C. E., Vardo, J., and Stavnezer, J. (2003b). Mlh1 can function in antibody class switch recombination independently of Msh2. J. Exp. Med. 197, 1377–1383. Schultz, C., Petrini, J., Collins, J., Claflin, J. L., Denis, K. A., Gearhart, P., Gritzmacher, C., Manser, T., Shulman, M., and Dunnick, W. (1990). Patterns and extent of isotype-specificity in the murine H chain switch DNA rearrangement. J. Immunol. 144, 363–370. Seidl, K. J., Bottaro, A., Vo, A., Zhang, J., Davidson, L., and Alt, F. W. (1998). An expressed neo(r) cassette provides required functions of the 1gamma2b exon for class switching. Int. Immunol. 10, 1683–1692. Shinkura, R., Ito, S., Begum, N. A., Nagaoka, H., Muramatsu, M., Kinoshita, K., Sakakibara, Y., Hijikata, H., and Honjo, T. (2004). Separate domains of AID are required for somatic hypermutation and class-switch recombination. Nat. Immunol. 5, 707–712. Shinkura, R., Tian, M., Smith, M., Chua, K., Fujiwara, Y., and Alt, F. W. (2003). The influence of transcriptional orientation on endogenous switch region function. Nat. Immunol. 4, 435–441. Smith, H. C., and Sowden, M. P. (1996). Base-modification mRNA editing through deamination– the good, the bad and the unregulated. Trends Genet. 12, 418–424. Sohn, J., Gerstein, R. M., Hsieh, C. L., Lemer, M., and Selsing, E. (1993). Somatic hypermutation of an immunoglobulin mu heavy chain transgene. J. Exp. Med. 177, 493–504. Stavnezer, J. (1996). In ‘‘Advances in Immunology,’’ p. 79. Academic Press, Inc. Stavnezer, J., Radcliffe, G., Lin, Y. C., Nietupski, J., Berggren, L., Sitia, R., and Severinson, E. (1988). Immunoglobulin heavy-chain switching may be directed by prior induction of transcripts from constant-region genes. Proc. Natl. Acad. Sci. USA 85, 7704–7708. Stavnezer-Nordgren, J., and Sirlin, S. (1986). Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching. EMBO J. 5, 95–102. Storb, U., Peters, A., Klotz, E., Kim, N., Shen, H. M., Kage, K., Rogerson, B., and Martin, T. E. (1998). Somatic hypermutation of immunoglobulin genes is linked to transcription. Curr. Top Microbiol. Immunol. 229, 11–19. Sugawara, N., Paques, F., Colaiacovo, M., and Haber, J. E. (1997). Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination. Proc. Natl. Acad. Sci. USA 94, 9214–9219. Tashiro, J., Kinoshita, K., and Honjo, T. (2001). Palindromic but not G-rich sequences are targets of class switch recombination. Int. Immunol. 13, 495–505. Tian, M., and Alt, F. W. (2000). Transcription-induced cleavage of immunoglobulin switch regions by nucleotide excision repair nucleases in vitro. J. Biol. Chem. 275, 24163–24172. Tian, M., Jones, D. A., Smith, M., Shinkura, R., and Alt, F. W. (2004). Deficiency in the nuclease activity of xeroderma pigmentosum G in mice leads to hypersensitivity to UV irradiation. Mol. Cell. Biol. 24, 2237–2242.

328

ir e ne m . m i n an d e ri k s el si n g

Tian, M., Shinkura, R., Shinkura, N., and Alt, F. W. (2004). Growth retardation, early death, and DNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF. Mol. Cell. Biol. 24, 1200–1205. Tishkoff, D. X., Boerger, A. L., Bertrand, P., Filosi, N., Gaida, G. M., Kane, M. F., and Kolodner, R. D. (1997). Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2. Proc. Natl. Acad. Sci. USA 94, 7487–7492. Tran, P. T., Simon, J. A., and Liskay, R. M. (2001). Interactions of Exo1p with components of MutLalpha in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 98, 9760–9765. Vogel, H., Lim, D. S., Karsenty, G., Finegold, M., and Hasty, P. (1999). Deletion of Ku86 causes early onset of senescence in mice. Proc. Natl. Acad. Sci. USA 96, 10770–10775. von Schwedler, U., Jack, H. M., and Wabl, M. (1990). Circular DNA is a product of the immunoglobulin class switch rearrangement. Nature 345, 452–456. Vora, K. A., Tumas-Brundage, K. M., Lentz, V. M., Cranston, A., Fishel, R., and Manser, T. (1999). Severe attenuation of the B cell immune response in Msh2-deficient mice. J. Exp. Med. 189, 471–482. Wiesendanger, M., Kneitz, B., Edelmann, W., and Scharff, M. D. (2000). Somatic hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern. J. Exp. Med. 191, 579–584. Winter, D. B., Phung, Q. H., Umar, A., Baker, S. M., Tarone, R. E., Tanaka, K., Liskay, R. M., Kunkel, T. A., Bohr, V. A., and Gearhart, P. J. (1998). Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2. Proc. Natl. Acad. Sci. USA 95, 6953–6958. Winter, E., Krawinkel, U., and Radbruch, A. (1987). Directed Ig class switch recombination in activated murine B cells. EMBO J. 6, 1663–1671. Woo, C. J., Martin, A., and Scharff, M. D. (2003). Induction of somatic hypermutation is associated with modifications in immunoglobulin variable region chromatin. Immunity 19, 479–489. Wuerffel, R. A., Du, J., Thompson, R. J., and Kenter, A. L. (1997). Ig Sgamma3 DNA-specific double strand breaks are induced in mitogen-activated B cells and are implicated in switch recombination. J. Immunol. 159, 4139–4144. Yoshikawa, K., Okazaki, I. M., Eto, T., Kinoshita, K., Muramatsu, M., Nagaoka, H., and Honjo, T. (2002). AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts. Science 296, 2033–2036. Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E., and Lieber, M. R. (2003). R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442–451. Yu, K., Huang, F. T., and Lieber, M. R. (2004). DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine. J. Biol. Chem. 279, 6496–6500. Yu, K., and Lieber, M. R. (2003). Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism. DNA Repair (Amst) 2, 1163–1174. Zarrin, A. A., Alt, F. W., Chaudhuri, J., Stokes, N., Kaushal, D., Du Pasquirer, L., and Tian, M. (2004). An evolutionarily conserved target motif for immunoglobulin class-switch recombination. Nat. Immunol. 5, 1275–1281. Zhang, J., Bottaro, A., Li, S., Stewart, V., and Alt, F. W. (1993). A selective defect in IgG2b switching as a result of targeted mutation of the I gamma 2b promoter and exon. EMBO J. 12, 3529–3537. Zhu, C., Bogue, M. A., Lim, D. S., Hasty, P., and Roth, D. B. (1996). Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 86, 379–389.

Index

A A-T-like deficiency (ATLD), 253 A-T. See Ataxia-telangiectasia ABC. See Activated B-cell-like Activated B-cell-like (ABC), 163 Activation induced cytidine deaminase (AID), 237, 238 alternative targeting mechanisms of, 308 CSR and, 249–251 as CSR enzyme, 308–309 definition of, 306 enzymatic activities of, 306–308 function of, 306–308 S region cleavage and, 306–309 as SHM enzyme, 308–309 Acute lymphoblastic T-cell leukemia (T-ALL), 275 Adaptive immune receptors, VLRs as, 216–221 Adaptive/innate receptors, leukocyte regulatory receptor with, 226–228 Adaptive receptors v. innate receptors, 211–212 relative complexity in, 211–212 Adhesion, 45 immunointegration by, 44–46 AFP. See Alpha-fetoprotein AID. See Activation induced cytidine deaminase Alpha-fetoprotein (AFP), 272 Antibody class switch recombination CSR and, 298 CSR targeting and, 300–305 Antibody class switch recombination mechanism, in B cells, 298 Antigen-presenting cells (APCs), 61 Antigens for CD8 T-cell clones, 146–149

329

for pathogenic T-cell clones, 140–149 APCs. See Antigen-presenting cells APE. See Apyrimidinic endonuclease Apyrimidinic endonuclease (APE), 251 Artemis, 258–260 genomic organization of, 245–248 hypomorphic mutations of, 259 identification of, 258–259 structure of, 248 Artemis mutations, in RS-SCID patients, 258– 260 Ataxia-telangiectasia (A-T), 269, 270 cellular characteristics of, 272–273 Ataxia-telangiectasia-like disorder (ATLD), 269 Ataxia-telangiectasia mutated (ATM) protein, 268, 269, 270–271 genetics of, 271–272 ATLD. See Ataxia-telangiectasia-like disorder ATM protein. See Ataxia-telangiectasia mutated protein Autoimmune diabetes, pathogenic T-cell clones in, 123–153 B B-cell(s), antibody class switch recombination mechanism in, 298 B-cell differentiation, regulatory biology of, 167–172 B-cell receptors (BCRs), 210 diversification mechanisms of, 210 B-cell stimulation through antigen receptor (BCR), 249 B-NHL. See Non-Hodgkin’s B-cell lymphomas Barbara Davis Center (BDC) panel, 126 of CD4 T-cell clones, 126–129, 130 Base excision repair (BER), 240, 255

330 BCR. See B-cell stimulation through antigen receptor BCRs. See B-cell receptors BDC panel. See Barbara Davis Center panel BER. See Base excision repair C b–CASP, 247 CD4 T-cell clones BDC panel of, 126–129, 130 with defined antigen specificity, 140–143 homing of, 133–134 pathogenic, 130–131 peptide mimotopes for, 144–146 with undefined antigen specificity, 143–144 CD4 T-cell clones, pathogenic, tetramers for, 150–152 CD8 T-cell clones antigens for, 146–149 homing of, 134–135 4.4 T-cell receptor transgenic (TCR-Tg) mice from, 139 CD8 T-cell clones, pathogenic, tetramers for, 149–150 CDR-type variation. See Complementarity determining region-type variation Cell-mediated immune responses, T cells and, 27–51 Chemokines, 44 immunointegration via, 42–45 Chinese hamster ovary (CHO), 244 CHO. See Chinese hamster ovary Chromatin immunoprecipitation (ChIP), 304 CIS. See Cytokine-inducible SH2 domaincontaining protein Class switch recombination (CSR), 237, 248– 249 AID and, 249–251 antibody class switch recombination and, 298 definition of, 298–299 lymphoid specific DNA cleavage/repair mechanisms and, 248–254 mechanism of, 297 microenvironments of, 299 MMR in, 311 in Msh1-deficient mice, 313 in Msh2-deficient mice, 312–313 in NHEJ-defective mice, 309–310

i nd e x in Pms2-deficient mice, 313 proteins involved in, 305–319 SHM defects and, 266–268 targeting of, 300–302 Class switch recombination (CSR) sites, characteristics of, 305 Class switch recombination specific DNA-dsb (CSR-DNA-dsb), 249–251 Class switch recombination (CSR) specific DNA-dsb resolution, error prone DNA polymerases and, 252–253 Class switch recombination specific DNA-dsb (CSR-DNA-dsb) resolution, 251–254 HR system and, 251–252 MMR and, 252 NHEJ and, 252 Class switch recombination (CSR) targeting, antibody class switch recombination and, 300–305 Cleavage and polyadenylation specificity factor (CPSF), 247 Costimulatory molecules, immunointegration by, 44–46 Complementarity determining region (CDR)type variation, 224 COOH-terminal tyrosine, 1 CPSF. See Cleavage and polyadenylation specificity factor CSR-DNA-dsb resolution. See Class switch recombination specific DNA-dsb resolution CSR-DNA-dsb. See Class switch recombination specific DNA-dsb CSR. See Class switch recombination Cytokine-inducible SH2 domain-containing protein (CIS), 68 Cytokine-inducible SH2 domain-containing protein-knockout (CIS-KO) mice, 68 Cytokine signalings, 65–64 negative regulation of, 61–103 pathway of, 63 by SOCS, 61–103 SOCS protein regulation of, 68–82 Cytokines, 43–44 immunointegration via, 41–42 S region transcription by, 299–300 Cytolysis, immune integration via, 33–40 Cytolytic gd T cell(s) immune integration through, 34–40

index targeting by, 40–40 Cytotoxicity, 36 D DC8 T-Cell clones, 131–132 DCs. See Dendritic cells Defective cell cycle control following DNA damage, human primary immunodeficiency disorders with, 268– 274 Defective DNA repair, in immune system, 274–278 Defective lymphoid specific DNA repair, human primary immunodeficiency disorders and, 257–268 Defined antigen specificity, CD4 T-cell clones with, 140–143 Dendritic cells (DCs), 61–62 SOCS-1 and, 101–102 Diabetes. See Autoimmune diabetes; Type 1 diabetes; Type II diabetes Diffuse large B-cell lymphoma (DLBCL), 163 diseases within, 165–167 gene expression-based survival predictors for, 178–179 germinal center B-cell signature of, 179–181 lymph node signature of, 180, 182–183 MHC class II signature of, 180, 183–184 proliferation signature of, 180, 184–185 Diffuse large B-cell lymphoma (DLBCL) subgroups, 167–172 clinical differences between, 176–177 oncogenic pathways and, 175–176 DLBCL. See Diffuse large B-cell lymphoma DNA damage in human primary immunodeficiency disorders, 268–270 in immune system, 238–240 initial reaction to, 242–243 recognition of, 242 repair of, 237–279 DNA-dependent protein kinase (DNA-PK), 244 DNA-dependent protein kinase catalytic subunit (DNA-PKcs), 244 DNA double strand break (DNA-dsb) cellular response of, 269 introduction of, 242–243 repair of, 247, 250

331 DNA-dsb. See DNA double strand break DNA ends processing, Msh2-deficient mice and, 316–319 DNA ligase IV defect, 260–265 clinical/cellular characteristics of, 261–263 molecular basis of, 263–265 mutation analysis of, 263–265 phenotypic characteristics of, 262 V(D)J recombination in, 263 DNA microarrays, 164 DNA-PK. See DNA-dependent protein kinase DNA-PKcs. See DNA-dependent protein kinase catalytic subunit DNA repair factors, alternative, 253–254 DNA repair mechanisms, 240 V(D)J recombination and, 240–242 Dystrophia myotonica kinase (MTK), 149 E Effector function, T-cell clones and, 126–132 Embryonic stem cells (ES), 248 Error prone DNA polymerases CSR specific DNA-dsb resolution and, 252– 253 SHM process and, 257 ES. See Embryonic stem cells EST screens, 216, 217 F Fibrinogen-related proteins (FREPs), 209 genes of, 222 IgSFs and, 221–222 VCBPs and, 224 Follicular lymphoma, gene expression-based survival predictor for, 191–197 FREPs. See Fibrinogen-related proteins G GCG. See Germinal center B-cell-like Gene expression-based survival predictors for DLBCL, 178–179 for follicular lymphoma, 191–197 of human lymphoid malignancies, 177–197 for mantle cell lymphoma, 185–191 Gene expression profiling, 163 human lymphoid malignancies and, 163–198 Gene mutation, human cancer and, 164 Gene protein structures, of immune recognition mechanisms, 218

332 Genetics, T-cell integration and, 49–51 Genome integrity, in human primary immunodeficiency disorders, 268–270 Genomic caretakers, 277–278 Germinal center B-cell-like (GCG), 163 Germinal center B-cell (GCG) signature, of DLBCL, 179–181 H H2AX, 253, 254 High mobility group (HMG) proteins, 243 HIGM. See Hyper IgM syndrome HMG proteins. See High mobility group proteins Hodgkin lymphoma, PMBL and, 172–174 Homeostasis, 94–95 Homing of CD4 T-cell clones, 133–134 of CD8 T-cell clones, 134–135 Homologous recombination (HR), 240 NHEJ and, 255–257 Homologous recombination (HR) system, 251– 252 Host defenses, 93–94 HR. See Homologous recombination Human cancer, gene mutation and, 164 Human genome sequence, 164 Human lymphoid malignancies gene expression-based survival predictors of, 177–197 gene expression profiling and, 163–198 molecular analysis of, 165–167 Human primary immunodeficiency disorders with defective cell cycle control following DNA damage, 268–274 defective lymphoid specific DNA repair and, 257–268 DNA damages in, 268–270 genome integrity in, 268–270 lessons from, 237–279 Hyper IgM syndrome (HIGM), 250 I IgCSR. See Immunoglobulin class switch recombination IgSF. See Immunoglobulin gene superfamily Immune diseases, SOCS-1 and, 75 Immune integration, via cytolysis, 33–40 Immune receptor diversification, 209–231

i nd e x Immune recognition mechanisms, of gene protein structures, 218 Immune responses public good and, 28–29 TLRs and, 212 Immune system defective DNA repair in, 274–278 development/maturation of, 239 DNA damage in, 238–240 malignancies in, 274–278 maturation modifications of, 237–279 Immunity, 61–62 Immunodeficiency disorders. See Human primary immunodeficiency disorders Immunoglobulin class switch recombination (IgCSR), model for, 249 Immunoglobulin gene superfamily (IgSF), 209 FREPs and, 221–222 LRRs and, 230 rearrangement/configuration of, 241 Immunoglobulin gene superfamily (IgSF)/ Fibrinogen-related proteins (FREPs), 222 Immunoglobulin gene superfamily (IgSF)-type immune receptors, invertebrate variations in, 221–223 Immunointegration by adhesion, 44–46 by costimulatory molecules, 44–46 via chemokines, 42–45 via cytokines, 41–42 Immunomodulation, 39 Immunoreceptor tyrosine-based inhibitory motifs (ITIMs), 226 Immunoregulation, clues to, 46–47 Innate immune receptors evolutionary diversification of, 212–215 large diversified multigene families and, 215–216 Innate receptors v. adaptive receptors, 211–212 relative complexity in, 211–212 variation in, 211–216 Intracellular negative regulators, TLRmediated signal pathways and, 98 Invertebrate variations, in IgSF-type immune receptors, 221–223 IRAK-M, TLR-mediated signaling and, 96 ITIMs. See Immunoreceptor tyrosine-based inhibitory motifs

index

J

JAK kinases, 71–72 K Killer cell Ig-like receptors (KIRs), 211, 215 KIRs. See Killer cell Ig-like receptors L b-lactam ring, 246 Large diversified multigene families, innate immune receptors and, 215–216 Lat / mutant mice ab T-cell development in, 5–6 ab T-cell development in, 7 LAT. See Linker for activation of T cells LAT signalosome, 2–5 schematic representation of, 3 Lat Y7/8/9F mice, in gd T-cell(s), 12–13 Lat Y7/8/9f mutant mice, ab T-cell development in, 5–6 Lat Y136F mutant mice cell types of, 11 ab T-cell development in, 6–7 LatY6/7/8/9f mutant mice, ab T-cell development in, 5–6 LatY7/8/9F mice, T-cell development in, 20 LatY136F CD4 T cells in TCR signaling responses, 11 TCR signaling responses in, 11 LatY136F/LatY7/8/9F, Th2-type immunity in, 19 LatY136F mice positive/negative selection in, 13–15 T-cell development in, 20 ab T-cell(s) in, 9–10 LatY136F mutation, cell type operation of, 11 Leucine-rich repeat(s) (LRRs), 212, 214 Leucine-rich repeat (LRR) proteins, 209 Leucine-rich repeat (LRR) receptors, IgSF and, 230 Leucine-rich repeat (LRR) type innate receptors, VLRs and, 219 Leukocyte regulatory receptor, with adaptive/ innate receptors, 226–228 Ligands for TLR2, 86 for TLR3, 86 for TLR4, 83–85 for TLR5, 86 for TLR7, 86 for TLR9, 86

333 for TLRs, 83 Linker for activation of T cells (LAT), 1, 2 negative regulatory role of, 9–13 pre-TCR and, 5–7 signal termination mechanisms by, 18–19 T cell development role of, 1–22 gd T-cell development and, 7–9 T cells and, 3, 4 Linker for activation of T cells (LAT) fishing line, 15, 16 Linker for activation of T cells (LAT) signalosome, cooperative assemblies within, 15–17 Linker for activation of T cells (LAT) signalosome pathology, 19–21 Linker for activation of T cells (LAT) tyrosines, redundancy among, 17–18 Louis Bar syndrome, 270 LRR proteins. See Leucine-rich repeat proteins LRRs. See Leucine-rich repeat(s) Lymph node signature, of DLBCL, 180, 182– 183 Lymphoid malignancies. See Human lymphoid malignancies; Diffuse large B-cell lymphoma Lymphoid specific DNA cleavage/repair mechanisms artemis and, 245–248 CSR and, 248–254 fundamental mechanisms of, 240–257 identifying/resealing the break in, 243–244 SHM and, 254–257 Lymphomas/leukemias, from illegitimate V(D) J/CSR recombination, 275–276 M Major histocompatibility complex (MHC), 123 Mantle cell lymphoma, gene expression-based survival predictor for, 185–191, 192 MAPK. See Mitogen-activated protein kinase MEFs. See Murine embryonic fibroblasts Mesappariement, 257 Metallo-b-lactamases, 246, 247 MHC class II signature, of DLBCL, 180, 183– 184 MHC. See Major histocompatibility complex MHC tetramers, pathogenic T-cell clones with, 149–152

334

i nd e x

Migration, of pathogenic T-cell clones, 133– 135 Mismatch repair proteins (MMR) in CSR, 311 CSR specific DNA-dsb resolution and, 252 definition of, 311–312 generalized pathway of, 312 mutations and, 257 protein interaction with, 312 structure of, 315 Mismatch repair proteins (MMR) mechanism, in CSR/SHM, 313–316 Mitogen-activated protein kinase (MAPK), 89 MMR. See Mismatch repair proteins Molecular analysis, of human lymphoid malignancies, 165–167 Msh1-deficient mice, CSR in, 313 Msh2-deficient mice CSR in, 312–313 DNA ends processing and, 316–319 Msh2 gene, UNG activities and, 314–316 MTK. See Dystrophia myotonica kinase Murine embryonic fibroblasts (MEFs), 248 Murine scid, 244 MyD88. See Myeloid differentiation factor 88 Myeloid differentiation factor 88 (MyD88), 88 TLR-mediated signaling and, 97 Myeloid differentiation factor 88 (MyD88)mediated pathway, 89–90

Nbs1 protein and, 273–274 Nijmegen breakage syndrome 1 (NBS1) protein, 273–274 NITRs. See Novel immune-type receptors NK cells. See Natural killer cells NOD mice. See Nonobese diabetic mice NOD2, TLR-mediated signaling and, 97–99 Non-Hodgkin’s B-cell lymphomas (B-NHL), 275 Nonhomologous end joining (NHEJ), 240 additional defects of, 265 CSR specific DNA-dsb resolution and, 252 factors of, 244 HR and, 255–257 pathways of, 243 repair defects of, 257–258 S region and, 309–311 of V(D)J recombination, 277–278 Nonhomologous end joining (NHEJ)-defective mice, CSR in, 309–310 Nonhomologous end joining (NHEJ) proteins, in CSR, 309–310 Nonobese diabetic (NOD) mice, 123–124 Novel immune-type receptor (NITR) genes, 227 Novel immune-type receptors (NITRs), 209 types of, 226–228 Nuclear export signal (NES), 250 Nucleotide excision repair (NER), 240

N Natural killer (NK) cells, 215 unconventional T cells and, 48 NBS. See Nijmegen breakage syndrome NBS1 protein. See Nijmegen breakage syndrome 1 protein Negative regulation of cytokine signalings, 64–68 of SOCS-1 cytokine signaling, 71–72 of SOCS-3 cytokine signaling, 77–78 of TLR cytokine signaling, 100 of TLR signaling, 96 NER. See Nucleotide excision repair NES. See Nuclear export signal NHEJ. See Nonhomologous end joining Nijmegen breakage syndrome (NBS), 269 cellular characteristics of, 274 clinical characteristics of, 272 genetics of, 274

O Oncogenic pathways, in DLBCL subgroups, 175–176 Ontogenetic progression, of T-cell development, 50 P PAMPs. See Pathogen-associated molecular patterns Pathogen-associated molecular patterns (PAMPs), 212 Pathogenic CD4 T-cell clones, tetramers for, 150–152 Pathogenic CD8 T-cell clones, tetramers for, 149–150 Pathogenic T-cell clones antigens for, 140–149 in autoimmune diabetes, 123–153 with MHC tetramers, 149–152

index migration of, 133–135 Peptide mimotopes, for CD4 T-cell clones, 144–146 Phosphoinositide 3 kinase (PI3K), 253 Phylogeny, of species/taxonomic groups, 213 PI3K. See Phosphoinositide 3 kinase PIAS. See Protein inhibitors of activated STATs PMBL. See Primary mediastinal B-cell lymphoma Pms2-deficient mice, CSR in, 313 Pre-TCR, components of, 5–7 Primary mediastinal B-cell lymphoma (PMBL), 163 definition of, 172 Hodgkin lymphoma and, 172–174 Proliferation signature, of DLBCL, 180, 184– 185 Protein inhibitors of activated STATs (PIAS), 66 Protein tyrosine phosphorylation events (PTKs), 4 PTKs. See Protein tyrosine phosphorylation events R RAD51/52 proteins, 255 Radiosensitive severe combined immunodeficiencies (RS-SCID), 245 artemis mutations in, 258–260 clinical/cellular characteristics of, 258 patients with, 258–259 RAG1. See Recombination activating gene 1 RAG2. See Recombination activating gene 2 RDA. See Representational Difference Analysis Recombination activating gene 1 (RAG1), 242, 243, 276 Recombination activating gene 2 (RAG2), 242, 243, 276 (V(D)J) recombination. See Variable Diversity Junction recombination Recombination Specific Sequences (RSS), 242 Regulatory biology, of B-cell differentiation, 167–172 Representational Difference Analysis (RDA), 33 Retrogenic TCR mice, 139–140 RIP3, TLR-mediated signaling and, 97 RS-SCID. See Radiosensitive severe combined immunodeficiencies

335 RSS. See Recombination Specific Sequences S S regions. See Switch region(s) SAGE. See Serial Analysis of Gene Expression SCID. See Severe combined immunodeficiencies Serial Analysis of Gene Expression (SAGE), 33 Severe combined immunodeficiencies (SCID), 257 SHP-1, 64–66 SIGIRR. See Single Ig IL-1R-related molecule Signal termination mechanisms, by LAT, 18–19 Signal transduction pathways, through TLRs, 88–89 Signaling complexes, through LAT in T cells, 3, 4 Single Ig IL-1R-related molecule (SIGIRR), TLR-mediated signaling and, 97 SOCS. See Suppressor of cytokine signalings Somatic hypermutation (SHM), 238, 248 AID and, 308–309 CSR and, 266–268 lymphoid specific DNA cleavage/repair mechanisms and, 254–257 models of, 256 Somatic hypermutation (SHM) process, error prone DNA polymerases and, 257 ST2, TLR-mediated signaling and, 97–99 STAT5, 68 Suppressor of cytokine signalings (SOCS) cytokine signalings by, 61–103 protein family functions of, 69–70 protein family of, 67–68 TLR-mediated signaling and, 97 Suppressor of cytokine signalings-1 (SOCS-1), 71–76 acquired immunity and, 74 DCs and, 101–102 immune diseases and, 75 physiological function of, 72–74 TLR and, 101 as tumor suppressor, 75–76 Suppressor of cytokine signalings-1 (SOCS-1) cytokine signaling, negative regulation of, 71–72 Suppressor of cytokine signalings-2 (SOCS-2), 76–77

336 Suppressor of cytokine signalings-3 (SOCS-3), 77–81 physiological roles of, 79–81 TLR signal modulation and, 102 Suppressor of cytokine signalings-5 (SOCS-5), 81 Suppressor of cytokine signalings-6 (SOCS-6), 81–82 Suppressor of cytokine signalings-7 (SOCS-7), 82 Suppressor of cytokine signalings (SOCS-3) cytokine signaling, negative regulation of, 77–78 Suppressor of cytokine signalings (SOCS) protein regulation, of cytokine signalings, 68–82 Switch (S) region(s), 297 joining of, 309–311 NHEJ and, 309–311 Switch (S) region chromatin structure, 304–305 Switch (S) region cleavage, AID and, 306–309 Switch (S) region secondary structure, 302–304 Switch (S) region tandem-repeat sequences, 300–302 Switch (S) region transcription, by cytokines, 299–300 T T-ALL. See Acute lymphoblastic T-cell leukemia T cell(s), 2 cell-mediated immune responses and, 27–51 integration of, 27–51 LAT and, 3, 4 T-cell antigen receptor (TCR), 2 diversification mechanisms of, 210 rearrangement/expression of, 241 T-cell antigen receptor (TCR) gene rearrangement, 210 T-cell antigen receptor (TCR) signaling responses, in LatY136F CD4 T cells, 11 T-cell clones effector function and, 126–132 in TCR-Tg mice, 135–140 T-cell clones, pathogenic, 126–132 antigens for, 140–149 in autoimmune diabetes, 123–153 migration of, 133–135 T cell(s), conventional, 28

i nd e x T-cell development LAT role in, 1–22 in LatY7/8/9F mice, 20 in LatY136F mice, 20 ontogenetic progression of, 50 T-cell integration developmental program of, 48–49 disease and, 49–51 evidence for, 29–33 genetics and, 49–51 T-cell receptor transgenic (TCR-Tg) mice, 123 T-cell clones in, 135–140 2.5 T-cell receptor transgenic (TCR-Tg) mice, 135–137 4.1 T-cell receptor transgenic (TCR-Tg) mice, 137–138 4.4 T-cell receptor transgenic (TCR-Tg) mice, from CD8 T-cell clones, 139 6.9 T-cell receptor transgenic (TCR-Tg) mice, 138 T cell(s), unconventional, 28 action mechanisms of, 33 NK cells and, 48 spectrum of, 47–48 ab T-cell(s), in LatY136F mice, 9–10 ab T-cell development in Lat / mutant mice, 5–6, 7 in Lat Y6/7/8/9F mutant mice, 5–6 in Lat Y7/8/9F mutant mice, 5–6 in Lat Y136F mutant mice, 6–7 gd T-cell(s) cytolysis of, 34–40 in Lat Y7/8/9F mice, 12–13 gd T-cell development, LAT role in, 7–9 T1D. See Type 1 diabetes T2D. See Type II diabetes TCR. See T-cell antigen receptor TCR-Tg mice. See T-cell receptor transgenic mice Tetramers for pathogenic CD4 T-cell clones, 150–152 for pathogenic CD8 T-cell clones, 149–150 Th2 differentiation, LAT role in, 1–22 Th2-type immunity, in LatY136F/LatY7/8/9F, 19 TIR domain-containing adapter inducing IFNb (TRIF), 88 TIR domain-containing adapter inducing IFNb-mediated pathway, 90–92

index TIR domain-containing adapter inducing IFNb-related adaptor molecule (TRAM), 88 TIR. See Toll IL-1 receptor TIR8. See Toll IL-1 receptor 8 TIRAP. See Toll IL-1 receptor domaincontaining adapter protein Tissue immunosurveillance conventional model of, 32 integrated model of, 32 TLR genes, 215–216 TLR2. See Toll-like receptor 2 TLR3. See Toll-like receptor 3 TLR4. See Toll-like receptor 4 TLR7. See Toll-like receptor 7 TLR9. See Toll-like receptor 9 TLR11. See Toll-like receptor 11 TLRs. See Toll-like receptor(s) Toll IL-1 receptor (TIR), 88, 212 Toll IL-1 receptor 8 (TIR8), TLR-mediated signaling and, 97 Toll IL-1 receptor domain-containing adapter protein (TIRAP), 88 Toll-like receptor(s) (TLRs), 61 immune responses and, 212 ligands for, 83 signal transduction pathways through, 88–89 SOCS-1 and, 101 Toll-like receptor (TLR)-mediated cell activation, 92–93 Toll-like receptor (TLR)-mediated pathways, 83–88 Toll-like receptor (TLR)-mediated signal pathways intracellular negative regulators and, 98 pathophysiological roles for, 93–94 Toll-like receptor (TLR)-mediated signaling, 62 Toll-like receptor (TLR)/MyD88-mediated pathways, in disease, 95 Toll-like receptor 2 (TLR2), ligands for, 86 Toll-like receptor 3 (TLR3), ligands for, 86 Toll-like receptor 4 (TLR4), ligands for, 83–85 Toll-like receptor 5 (TLR5), ligands for, 86 Toll-like receptor 7 (TLR7), ligands for, 86 Toll-like receptor 9 (TLR9), ligands for, 86 Toll-like receptor 11 (TLR11), 87 Toll-like receptor (TLR) cytokine signaling, negative regulation of, 101 Toll-like receptor (TLR) domain-containing adapter inducing IFN-b (TRIF), 88

337 Toll-like receptor (TLR) expression, on various cell types, 87–88 Toll-like receptor (TLR) ligands, 62 Toll-like receptor (TLR) signal modulation, SOCS-3 and, 102 Toll-like receptor (TLR) signaling, negative regulation of, 96 Toll-like receptor (TLR) SOCS signaling, regulation of, 101 TRAIL. See Tumor necrosis factor-related apoptosis inducing ligand TRAM. See TIR domain-containing adapter inducing IFN-b-related adaptor molecule TRIF. See TIR domain-containing adapter inducing IFN-b Tumor necrosis factor-related apoptosis inducing ligand (TRAIL), 35 Tumor suppressor, SOCS-1 as, 75–76 Type 1 diabetes (T1D), 123 Type II diabetes (T2D), 124 U Undefined antigen specificity, CD4 T-cell clones with, 143–144 UNG-deficient mice. See Uracil N glycosylasedeficient mice UNG. See Uracil N glycosylase Uracil N glycosylase (UNG), 251 Uracil N glycosylase (UNG)-deficient mice, 314 Msh2 and, 314–316 Uridine residues, 250, 251 V Variable Diversity Junction (V(D)J)/CSR recombination, lymphomas/leukemias from, 275–276 Variable Diversity Junction (V(D)J) recombination, 237, 238 in DNA ligase IV defect, 263 DNA repair mechanisms and, 240–242 reaction initiation of, 242–243 rearrangement configuration of, 240, 241 repair steps in, 242 Variable lymphocyte receptors (VLRs), 209, 217 as adaptive immune receptors, 216–221 genomic structure of, 219 LRR type innate receptors and, 219

338 Variable region-containing chitin-binding proteins (VCBPs), 209 expression of, 225 and FREPs, 224 sequence/annotation of, 225 variability of, 225 VCBPs. See Variable region-containing chitinbinding proteins V(D)J recombination, NHEJ of, 277–278 VLRs. See Variable lymphocyte receptors

i nd e x

X X-Ray Repair Cross Complementing (XRCC) proteins, 244 XRCC proteins. See X-Ray Repair Cross Complementing proteins Z ZAP-70 protein tyrosine kinase, 1

Contents of Recent Volumes

Anthony J. Coyle, and Jose-Carlos Gutierrez-Ramos

Volume 77 The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction S. Celeste Posey Morley and Barbara E. Bierer

Selected Comparison of Immune and Nervous System Development Jerold Chun Index

Raft Membrane Domains and Immunoreceptor Functions Thomas Harder

Volume 78

Human Basophils: Mediator Release and Cytokine Production John T. Schroeder, Donald W. MacGlashan, Jr., and Lawrence M. Lichtenstein

Toll-like Receptors and Innate Immunity Shizuo Akira Chemokines in Immunity Osamu Yoshie, Toshio Imai, and Hisayuki Nomiyama

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

Attractions and Migrations of Lymphoid Cells in the Organization of Humoral Immune Responses Christoph Schaniel, Antonius G. Rolink, and Fritz Melchers Factors and Forces Controlling V(D)J Recombination David G. T. Hesslein and David G. Schatz

The Antiviral Activity of Antibodies in Vitro and in Vivo Paul W. H. I. Parren and Dennis R. Burton

T Cell Effector Subsets: Extending the Th1/Th2 Paradigm Tatyana Chtanova and Charles R. Mackay

Mouse Models of Allergic Airway Disease Clare M. Lloyd, Jose-Angel Gonzalo,

339

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

co n t e nt s o f re c e nt vo l um es 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-DumA˚nil, Edda Fiebiger, CA˚cile LagaudriA˚re-Gesbert, and Hidde L. Ploegh Cytokine Memory of T Helper Lymphocytes Max Lo«hning, Anne Richter, and Andreas Radbruch Ig Gene Hypermutation: A Mechanism Is Due Jean-Claude Weill, Barbara Bertocci, Ahmad Faili, Said Aoufouchi, StA˚phane Frey, Annie De Smet, SA˚bastian Storck, Auriel Dahan, FrA˚dA˚ric Delbos, Sandra Weller, Eric Flatter, and Claude-AgnA˚s Reynaud Generalization of Single Immunological Experiences by Idiotypically Mediated Clonal Connections Hilmar Lemke and Hans Lange The Aging of the Immune System B. Grubeck-Loebenstein and G. Wick Index

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

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

341 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 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

Volume 82 Transcriptional Regulation in Neutrophils: Teaching Old Cells New Tricks Patrick P. McDonald

Volume 83 Lineage Commitment and Developmental Plasticity in Early Lymphoid Progenitor Subsets David Traver and Koichi Akashi

342

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

The CD4/CD8 Lineage Choice: New Insights into Epigenetic Regulation during T Cell Development Ichiro Taniuchi, Wilfried Ellmeier, and Dan R. Littman

Customized Antigens for Desensitizing Allergic Patients Fa¨tima Ferreira, Michael Wallner, and Josef Thalhamer

CD4/CD8 Coreceptors in Thymocyte Development, Selection, and Lineage Commitment: Analysis of the CD4/CD8 Lineage Decision Alfred Singer and Remy Bosselut

Immune Response Against Dying Tumor Cells Laurence Zitvogel, Noelia Casares, Marie O. Pe¨quignot, Nathalie Chaput, Mathew L. Albert, and Guido Kroemer

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 CD8þ Effector Cells Pierre A. Henkart and Marta Catalfamo An Integrated Model of Immunoregulation Mediated by Regulatory T Cell Subsets Hong Jiang and Leonard Chess Index

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 Index

Volume 85 Cumulative Subject Index Volumes 66–82

Volume 84 Interactions Between NK Cells and B Lymphocytes Dorothy Yuan Multitasking of Helix-Loop-Helix Proteins in Lymphopoiesis Xiao-Hong Sun

Volume 86 Adenosine Deaminase Deficiency: Metabolic Basis of Immune Deficiency and Pulmonary Inflammation Michael R. Blackburn and Rodney E. Kellems

c o nt e n t s of re c e n t vo l u m es Mechanism and Control of V(D)J Recombination Versus Class Switch Recombination: Similarities and Differences Darryll D. Dudley, Jayanta Chaudhuri, Craig H. Bassing, and Frederick W. Alt Isoforms of Terminal Deoxynucleotidyltransferase: Developmental Aspects and Function To-Ha Thai and John F. Kearney Innate Autoimmunity Michael C. Carroll and V. Michael Holers

343 Interleukin-2, Interleukin-15, and Their Roles in Human Natural Killer Cells Brian Becknell and Michael A. Caligiuri Regulation of Antigen Presentation and Cross-Presentation in the Dendritic Cell Network: Facts, Hypothesis, and Immunological Implications Nicholas S. Wilson and Jose A. Villadangos Index

Formation of Bradykinin: A Major Contributor to the Innate Inflammatory Response Kusumam Joseph and Allen P. Kaplan